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Doctoral Thesis

Amphiphilic nucleic acid conjugates for transfection ofdifferentiated intestinal cells

Author(s): Moroz, Elena V.

Publication Date: 2016

Permanent Link: https://doi.org/10.3929/ethz-a-010735196

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DISS. ETH NO. 23768

AMPHIPHILIC NUCLEIC ACID CONJUGATES FOR

TRANSFECTION OF DIFFERENTIATED INTESTINAL CELLS

A thesis submitted to attain the degree of

DOCTOR OF SCIENCES of ETH ZURICH

(Dr. sc. ETH Zurich)

presented by

ELENA VIKTOROVNA MOROZ

MSc in Chemistry, Lomonosov Moscow State University

born on 26.01.1990

citizen of Russian Federation

accepted on the recommendation of

Prof. Dr Jean-Christophe Leroux, examiner

Prof. Dr Jonathan Hall, co-examiner

2016

I

Abstract

Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal (GI) diseases

that currently lack appropriate treatments. The delivery of oligonucleotides (ONs) directly to the

GI mucosa allows for high local concentrations with minimal systemic exposure and subsequent

side-effects. Indeed, several locally administered ONs are currently progressing through clinical

trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of free

ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would

be highly desirable. The aim of the doctoral work presented herein was to explore the silencing

properties of highly resistant ONs modified with a hydrophobic alkyl chain on differentiated

intestinal epithelial cells in conditions mimicking those of the GI tract.

A variety of innovative nucleic acid delivery approaches ranging from single-molecule-based

conjugates to designer viruses are currently available, some of which are being tested in clinical

trials. These strategies are discussed in Chapter 1 of this thesis. The issues related to the oral

delivery of biomacromolecules, such as nucleic acids, and the approaches to overcome these

hurdles are reviewed and discussed in Chapter 2 with a focus on clinical applications. Chapter 3

presents the synthesis and screening of a series of lipid-ON conjugates for the silencing of a model

Bcl-2 mRNA. Among the conjugates tested, the ON modified with 2′-deoxy-2′-fluoro-

arabinonucleic acid bearing a docosanoyl moiety (L-FANA) was the most potent candidate with

the lowest toxicity. The efficacy of L-FANA conjugate was confirmed in two different colon

carcinoma cell lines and was preserved in an inverted transfection setup, in contrast to a

conventional particulate lipoplex-based delivery carrier. The conjugation of the lipid was essential

for efficient target silencing, since FANA without the lipid group failed to significantly

downregulate the Bcl-2 mRNA. In Chapter 4, the efficacy of the L-FANA conjugate was tested

in simulated intestinal fluids and in an oil-in-water emulsion. Given the resistance of L-FANA to

digestion, its silencing efficacy remained intact after its interaction with the oil phase of the oil-in-

water emulsion was disrupted with pancreatin-derived lipases. Remarkably, L-FANA conjugate was

able to downregulate the target gene expression at both mRNA and protein levels in difficult-to-

ABSTRACT

II

transfect polarized epithelial cell monolayers in the absence of complexation, delivery devices, and

membrane-disturbing agents. These findings indicate that lipid-ON conjugates are promising

therapeutics for the treatment of intestinal diseases, as well as valuable tools for the discovery of

new therapeutic targets. Chapter 5 summarizes the findings of the thesis and presents future

perspectives of the work.

Schematic representation of the main questions addressed in this thesis.

III

Zusammenfassung

Die Behandlung von Erkrankungen des Gastrointestinal-Trakts (GIT) ist heutzutage immer

noch schwierig, da es an geeigneten Therapien fehlt. Der Einsatz von Nukleinsäuren zur lokalen

Behandlung von GIT-Erkrankungen könnte eine vorteilhafte Option sein. Mittels peroraler

Verabreichung können Oligonukleotide (ON) direkt auf der Magen-Darm-Schleimhaut zur

Wirkung kommen, wobei die systemische Aufnahme und die damit verbundenen Nebenwirkungen

sehr gering bleiben. Mehrere laufende klinische Studien mit lokal verabreichten ON unterstreichen

die Bedeutung dieser Therapieform zur Behandlung chronisch-entzündlicher Darmerkrankungen.

ON werden jedoch nur in geringem Masse in die Zellen der Darmschleimhaut aufgenommen,

weshalb derzeitige Anstrengungen darauf hinzielen, die Wirkstärke oral verabreichter ON zu

erhöhen. Ziel dieser Dissertation war die Untersuchung der Gen-Stilllegungseigenschaften («gene

silencing») von stabilen, mit hydrophoben Alkylketten modifizierten ON-Derivaten an

differenzierten intestinalen Epithelzellen. Die Untersuchungsbedingungen imitierten dabei jene des

Darmtrakts.

Bereits heute gibt es eine Vielzahl von neuartigen Strategien zur Verabreichung von

Nukleinsäuren, angefangen von Konjugaten einzelner Moleküle bis hin zu biotechnisch

veränderten Viren. Erste Therapien basierend auf derartigen Konzepten befinden sich bereits in

klinischen Studien. Solche Nukleinsäure-Delivery Strategien werden in Kapitel 1 der Dissertation

behandelt. Kapitel 2 betrachtet und diskutiert Probleme, die sich aus der oralen Verabreichung

von Biomakromolekülen, wie etwa Nukleinsäuren, ergeben, sowie Anstrengungen dieselben zu

überwinden, mit besonderem Augenmerk auf klinische Anwendungen. Kapitel 3 beschreibt die

Synthese und das Screening einer Reihe von Lipid-ON-Konjugaten, die zum Gen-Silencing eines

Bcl-2 mRNA Modells dienen sollen. Das wirkungsstärkste und am wenigsten toxische ON-

Konjugat war mit Docosanoyl enthaltender 2‘-Deoxy-2‘-fluoro-arabinonukleinsäure (L-FANA)

modifiziert. Die Wirksamkeit dieses L-FANA Konjugates wurde in zwei unterschiedlichen

Darmkrebszelllinien nachgewiesen und blieb auch mit den invertiert angeordneten Zellen erhalten,

im Gegensatz zu einem konventionellen partikulären lipoplex-basierten Transfektionsvehikel. Die

ZUSAMMENFASSUNG

IV

Konjugation des Lipides erwies sich als essentiell, um die Zielsequenz in ausreichendem Masse

abzuschalten, da FANA ohne Lipidgruppe zu einer statistisch signifikant geringeren

Herunterregulierung der Bcl-2 mRNA führte. In Kapitel 4 wurde die Wirksamkeit des L-FANA-

Konjugats in künstlichem Darmsaft und einer Öl-in-Wasser (O/W) Emulsion überprüft. Der

silencing Effekt blieb erhalten, nachdem die Interaktion von L-FANA mit der Ölphase der O/W-

Emulsion durch Pankreatin-Lipasen aufgehoben worden war; dies bestätigte die

Widerstandsfähigkeit von L-FANA gegenüber Verdauungsprozessen. Das L-FANA Konjugat

konnte die Expression des Modellgens sowohl auf mRNA-Ebene als auch auf Proteinebene

herunterregulieren. Bemerkenswerterweise gelang dies ohne den Zusatz von Komplexbildnern,

Abgabevehikeln oder zellmembranschädigenden Substanzen in einschichtigen, polarisierten

Epithelzellen, die gewöhnlich schwierig zu transfizieren sind. Diese Erkenntnisse zeigen, dass

Lipid-ON-Konjugate vielversprechende Therapeutika zur Behandlung von Darmerkrankungen

darstellen und nützliche Werkzeuge für die Aufdeckung neuer therapeutischer Angriffsziele sind.

Kapitel 5 fasst die bisherigen Erkenntnisse zusammen und zeigt zukünftige Forschungsbereiche

im Zusammenhang mit dieser Arbeit auf.

Schematische Darstellung der in dieser Dissertation untersuchten zentralen Fragestellungen

V

Table of Contents

Abstract ................................................................................................................................................... I

Zusammenfassung ............................................................................................................................... III

1 Background and Purpose .............................................................................................................. 1

1.1 Nucleic acid drugs ................................................................................................................. 1

1.2 Enhancing the stability of nucleic acids ................................................................................ 4

1.3 Nucleic acids delivery strategies ........................................................................................... 6

1.3.1 Particle-based delivery approaches 8

1.3.2 Single-molecule conjugate delivery approaches 11

1.4 Delivery of nucleic acids to the gastrointestinal (GI) tract .................................................. 17

1.5 Scope of this thesis .............................................................................................................. 24

2 Oral delivery of biomacromolecules .......................................................................................... 27

2.1 Introduction ......................................................................................................................... 27

2.2 Local delivery ...................................................................................................................... 29

2.2.1 Peptides 31

2.2.2 Antibodies 34

2.2.3 Oral enzyme therapy 35

2.2.4 Cytoprotective/anti-inflammatory proteins 36

2.2.5 Nucleic acids 37

2.3 Systemic delivery ................................................................................................................ 38

2.3.1 Peptides 40

2.3.2 Proteins 46

2.3.3 Nucleic acids 49

2.4 Safety of bioavailability-increasing excipients ................................................................... 50

2.4.1 Protease inhibitors 51

2.4.2 Permeation enhancers 52

2.5 Conclusion ........................................................................................................................... 53

3 Amphiphilic nucleic acid conjugates .......................................................................................... 55

3.1 Introduction ......................................................................................................................... 55

3.2 Materials and Methods ........................................................................................................ 57

3.2.1 Materials 57

3.2.2 Synthesis of ONs and their derivatives 58

3.2.3 Cell culture 58

3.2.4 Cytotoxicity assay 59

3.2.5 Screening of Bcl-2 silencing efficiencies of various L-ONs 59

3.2.6 Target Bcl-2 mRNA silencing study with different incubation

times 60

TABLE OF CONTENTS

VI

3.2.7 Comparison of transfection efficiencies in upright and inverted

transfection setups 60

3.2.8 Statistical analysis 60

3.3 Results ................................................................................................................................. 61

3.3.1 L-ON library screening for Bcl-2 mRNA knockdown efficacy 61

3.3.2 Bcl-2 mRNA silencing by L-FANA conjugate in two colon

carcinoma cell lines 66

3.3.3 Bcl-2 mRNA silencing in upright and inverted transfection setups 66

3.3.4 Bcl-2 mRNA silencing with different incubation times 67

3.3.5 Kinetics of Bcl-2 mRNA silencing by L-FANA or siRNA/LF

lipoplex 68

3.4 Discussion ........................................................................................................................... 69

4 Effect of intestinal conditions on transfection ........................................................................... 71

4.1 Introduction ......................................................................................................................... 71

4.2 Materials and Methods ........................................................................................................ 72

4.2.1 Materials 72

4.2.2 Cell culture 73

4.2.3 Cytotoxicity assay 74

4.2.4 Interaction of FANA and L-FANA with oil 74

4.2.5 L-FANA conjugates stability in simulated intestinal fluid (SIF) and

soybean oil emulsion 74

4.2.6 Transfection of L-FANA pre-incubated with SIF and soybean oil

emulsion 75

4.2.7 Transfection of differentiated Caco-2 cell monolayers 76

4.2.8 Statistical analysis 77

4.3 Results ................................................................................................................................. 77

4.3.1 Stability and silencing properties of L-FANA in SIF 77

4.3.2 The effect of oil on L-FANA silencing properties 79

4.3.3 L-FANA silencing properties in differentiated intestinal monolayer 80

4.4 Discussion ........................................................................................................................... 84

5 Conclusions and Outlook ............................................................................................................ 87

6 Appendix....................................................................................................................................... 91

6.1 Synthesis of L-MOE and L-siRNA conjugates ................................................................... 91

6.2 Synthesis of L-DNA, L-FANA, negative control L-FANA conjugates, and FANA .......... 92

6.3 Characterization of ONs ...................................................................................................... 93

References ............................................................................................................................................ 99

List of Abbreviations ......................................................................................................................... 125

Curriculum Vitae .............................................................................................................................. 129

Scientific Contributions .................................................................................................................... 131

Acknowledgements ............................................................................................................................ 133

1

1 Background and Purpose

1.1 Nucleic acid drugs

Nucleic acids are biomacromolecules consisting of tens to millions of nucleotide-based

monomer units which perform the most essential functions in living cells, such as carrying the

genetic information and regulating gene expression and protein synthesis. Therefore, altering some

of these functions by delivering synthetic nucleic acids to cells not only represents an extremely

valuable research tool, but also possesses a tremendous therapeutic potential. Nucleic acid drug

candidates comprise a wide class of compounds with distinct mechanisms of action capable of

targeting genomic DNA, messenger RNA (mRNA), proteins, and endogenous gene regulators

such as long non-coding RNA (lncRNA) and microRNA (miRNA) (Scheme 1.1). Nucleic acid

therapeutics can be divided in two major classes based on their size: oligonucleotides (ONs) and

large nucleic acids. These groups require specific delivery approaches due to differences in stability,

size, and cell uptake efficacy.

ONs comprise a large family of molecules including antisense ONs (AONs), RNA

interference (RNAi)-based ONs (small interfering RNAs (siRNAs) and miRNAs), miRNA-binding

ONs (antimiRs), triplex-forming ONs (TFOs), decoy ONs, DNA/ribozymes, immunostimulatory

ONs, and aptamers. They are typically single- or double-stranded sequences of nucleotides (usually

15 – 35 bases long) with relatively high molecular weight (> 5000 Da) and consequently low cell

permeability. The key appeal of AON, RNAi, ribozyme, and antimiR therapies is the ability to

rationally design them to modulate the expression of any target gene of interest with high specificity

based on the Watson-Crick pairing rules. AON's and siRNA's binding to target mRNA can

potentially result in splicing correction, endonuclease-mediated degradation, and/or translation

repression depending on the chemical structure and degree of complementarity (1–3).

Alternatively, antimiRs bind endogenously expressed complementary miRNAs thereby inhibiting

their function (4, 5). Ribozymes are ONs with nuclease activity capable of mRNA digestion in a

sequence-specific fashion. Notably, DNAzymes and chemically-modified XNAzymes with higher

stability than ribozymes have been developed (6).

CHAPTER 1: BACKGROUND AND PURPOSE

2

Scheme 1.1. Types of nucleic acid drugs and their mechanisms of action. mRNA – messenger

RNA, pDNA – plasmid DNA, AON – antisense ON, shRNA – short hairpin RNA, TFO – triplex-

forming ON, TF – transcription factor, TFD – TF decoy, gRNA – guiding RNA, RISC – RNA-

induced silencing complex. (i) A group of nucleic acid drugs acting directly on the genomic DNA.

TFD and TFO modulate expression of the target gene by direct binding or sequestration of the

corresponding TF. Co-expression of Cas9 and gRNA from a pDNA leads to targeted genome

editing. (ii) A group of nucleic acid drugs exerting their effects on mRNA or regulatory RNAs.

Ribozymes and their analogs (e.g., DNAzymes) degrade the target mRNA in a sequence-specific

manner. shRNA is processed by the endonuclease Dicer to miRNA or siRNA, leading to the target

mRNA degradation via RISC and/or translation repression. AONs lead to knockdown of target

protein via RNAse H-mediated degradation or inhibition of translation. Additionally, they may be

used for splicing correction. AntimiRs can bind and inactivate target miRNAs. (iii) A group of

nucleic acids acting on protein level. Transfection of mRNA or pDNA could lead to

overexpression of target protein. Aptamers can recognize cell surface receptor leading to its

(in)activation or endocytosis. CpG-containing ONs can activate/modulate the immune response.


3

In addition to RNA-binding ONs, DNA-based ONs containing unmethylated CpG motif

stimulate the Toll-like receptor (TLR) 9 expressed primarily by immune cells, leading to modulation

of the immune response. For example, DNA-based ON CPG 7909 (Pfizer Inc.) was tested as a

vaccine adjuvant for the prevention and treatment of sepsis (7), and BL-7040 (BioLineRx) is being

developed for the treatment of inflammatory bowel diseases (IBDs) (8). Moreover, the ability of

ONs to form rigid 3D structures can be exploited to obtain specific protein binders, known as

aptamers, with remarkable binding affinities comparable to those of antibodies (9, 10). ON therapy

constitutes a highly promising approach for the treatment of several genetic, (auto)immune,

cancerous, or infectious diseases where conventional therapy is ineffective or unavailable (e.g.,

Duchenne muscular dystrophy (11), rheumatoid arthritis (12), cancer (13), or hepatitis (14)).

Currently, only a few ONs have reached the market (15): Kynamro®, an AON for the treatment of

hypercholesterolemia, Macugen®, an aptamer approved for the treatment of age-related macular

degeneration (AMD), and Vitravene™, an antisense drug approved for the treatment of

cytomegalovirus retinitis in AIDS patients but discontinued in 2002 due to commercial reasons.

Larger nucleic acid drugs primarily consist in mRNA and plasmid DNA (pDNA) encoding

functional protein or short hairpin RNA (shRNA) for target gene silencing. In comparison to

pDNA, mRNA has some advantages since there is no risk of integration into the genome and

nuclear entry is not required for its activity (16). Currently, several gene therapies based on viral

delivery of DNA have been approved: Gendicine™ (Shenzhen Sibiono GeneTech Co.),

Glybera® (UniQure), Imlygic® (Amgen Inc.), and Strimvelis™ (GlaxoSmithKline). Gendicine™,

the first gene therapy approved for marketing, carries the wild-type p53 gene which aims at

restoring the ability of cancer cells to undergo apoptosis, and it received authorization in China in

2003 for the treatment of head and neck squamous cell carcinoma (17). Glybera®, a therapy for

familial lipoprotein lipase (LPL) deficiency, relies on the expression of an enhanced variant of LPL

upon intramuscular injection (18), and has been approved in Europe in 2012. The first FDA-

approved (in 2015) oncolytic virus Imlygic™ was engineered from herpes simplex virus 1 to carry

the gene encoding human granulocyte-macrophage colony-stimulating factor for the treatment of

inoperable melanoma via intratumoral injection (19). Strimvelis™ involves the autologous

transplantation of bone marrow cells transduced ex-vivo with a gene encoding for adenosine

deaminase (ADA) (20). It was approved in Europe in 2016 for the treatment of severe combined

immunodeficiency due to ADA deficiency. Due to the high costs of product manufacturing and

clinical development and low number of patients, currently approved gene therapies are extremely

expensive (e.g., Glybera® costs about $1 million), making their marketing even more difficult.


4

Another application of pDNA and mRNA under investigation is the development of vaccines

against cancer and viral or bacterial infections (21, 22). Delivered nucleic acid vaccines express the

desired immunogenic protein and do not impose stable integration into the genome. The recent

discovery of the bacterial defense system CRISPR-Cas9, capable of permanent gene editing with

unprecedented accuracy in a desired location of the genome, has opened the possibility of

performing gene therapy not only to transiently alleviate symptoms but rather to cure genetic and

ageing-related diseases (23).

While the advances in understanding the molecular pathways of nucleic acids are tremendous

and promise a plethora of novel therapeutic strategies for the treatment of multiple devastating

diseases, achieving their safe and targeted delivery constitutes a bottleneck. Therefore, current

research in the field of nucleic acid therapeutics is largely focused on improving their cellular uptake

and specific tissue targeting.

1.2 Enhancing the stability of nucleic acids

Nucleases, extra- and intracellular enzymes catalyzing RNA and DNA cleavage, perform

crucial functions, such as DNA replication, cell death, RNA maturation, or defense against viral

and bacterial infection, and are thus ubiquitously expressed in our body (24). Therefore, to

successfully develop nucleic acid-based therapeutics, it is crucial to overcome the stability issues

posed by nucleases. Introducing unnatural chemical bonds that cannot be easily recognized and

cleaved by these nucleases is one of the strategies employed to address the stability-related

limitations of nucleic acid delivery (25) (Scheme 1.2).

First-generation modified ONs include phosphorothioate (PS) substituents on their

backbone, resulting in increased nuclease resistance and higher cellular uptake (26). Further

improvements in the nuclease resistance, and in some cases potency, have been achieved by

combining PS backbone with chemically modified sugar rings. These second-generation ONs most

commonly include 2′-O-methyl- (OMe), 2′-O-(2-methoxyethyl)- (MOE), 2′-F-RNA, or locked

nucleic acids (LNA) modifications (Scheme 1.2). For certain ONs (e.g., AONs or siRNAs), their

mechanism of action involving enzymatic recognition and cleavage of target mRNA imposes

certain limitations on the type and location of chemical alterations that can be incorporated.

Typically, AONs (short DNA oligomers) rely on RNAse H-mediated recognition and cleavage of

the formed DNA:mRNA heteroduplex (27); therefore, an unmodified DNA stretch of 8-10 bases

should be included in the structure of antisense ONs (28, 29). RNA-like modifications (e.g., OMe,


5

MOE) generally do not support RNAse H binding and therefore can be incorporated only at the

termini of the antisense ON (“gapmer” design). A recently discovered 2′-deoxy-2′-fluoro-

arabinonucleic acid (FANA) modification was shown to preserve DNA-like conformation upon

binding to the complementary mRNA, thereby allowing for RNAse H recognition (30). FANA can

be incorporated also in the central DNA stretch previously unavailable to 2′-O-modified

ribonucleotides, which may further improve resistance to endonucleases (“altimer” design).

Contrary to antisense ONs, the chemical modification of an siRNA generally leads to

reduction of its potency despite improving nuclease resistance (31). To retain siRNA-mediated

gene silencing, the free 5′ end of its guide strand should be preserved (31). PS backbone

modification is the simplest approach to imbue siRNA with nuclease resistance (32), and selective

placement of sugar modifications (e.g., OMe) was shown to reduce off-target activation of TLRs

and incorporation of the incorrect strand into the RNA-induced silencing complex (RISC) (31).

Complete substitution of the sugar and phosphodiester moieties with an unnatural backbone

(e.g., morpholino (PMO) or peptide nucleic acids (PNA)) is the basis for obtaining extremely robust

third-generation oligomers which are unrecognizable by natural enzymes. AntimiRs, splicing

correcting ONs, steric blocking oligomers, or aptamers do not require enzymatic recognition and

can be entirely composed of such heavily modified bases or of the second-generation

modifications (33, 34).

Scheme 1.2. The most commonly used chemical modifications of nucleic acid sugar rings (A) and

backbone (B).


6

1.3 Nucleic acids delivery strategies

While the advances in ON chemical modifications have tackled the stability barrier for the

development of nucleic acid therapeutics, the pending challenge lies in their high molecular weight

and polyanionic nature which limit their permeation across cellular membranes. Only at high

concentrations (typically, µM) are ONs able to enter cells via various pathways (e.g., adsorptive

endocytosis, receptor-mediated uptake, macropinocytosis; Scheme 1.3), reach the

cytoplasm/nucleus compartment, and induce target gene silencing (35–37). When administered

systemically, modified ONs are distributed to various tissues within hours and can exhibit

prolonged activity (up to several weeks in some cases) (38). The tissue distribution of ONs upon

systemic administration is broad, and their highest levels are typically found in the liver and kidney

followed by the bone marrow, adipocytes, and lymph nodes (38). PS and MOE modifications have

been shown to promote ON binding to serum albumin reducing their glomerular filtration by the

kidneys thereby increasing circulation times and tissue bioavailability (39). In contrast, unmodified

ONs including siRNAs and non-charged oligomers (PMOs) present negligible binding to serum

proteins, and are thus rapidly cleared via renal filtration with predominant uptake by the kidneys

(40, 41). Generally, ONs do not cross the blood-brain barrier, but are taken up by neuronal cells

and can cause a potent gene silencing when injected into the cerebrospinal fluid (42). This delivery

strategy is currently being evaluated in a phase III clinical trial for the treatment of spinal muscular

atrophy (NCT02292537).

Despite their preferential deposition in the liver and kidneys upon systemic administration,

some of the most potent chemically-modified ONs exhibit strong silencing in multiple organs such

as heart, lung, and gall bladder (38). The poor organ-specificity of silencing could potentially lead

to adverse effects. Systemic toxicity may be induced when high doses of ONs are administered

(40). Lethal doses were estimated to be around 1000 mg·kg-1 in mice (41). Adverse effects may

result from off-target gene silencing, unspecific protein binding, induction of immune response via

TLRs signaling or complement activation (41, 43). While some deleterious effects such as

stimulation of TLRs (44) and off-target gene silencing due to incorporation of the incorrect siRNA

strand into RISC (45) can be avoided through careful design of the ONs sequence and modification

strategy, most of them are in fact structure-specific and cannot be predicted . For instance, the only

FDA-approved antisense ON, mipomersen, has to be administered subcutaneously at a high dose

of 200 mg once per week and is often accompanied by injection-site reaction and fever, which

could indicate a mild allergic reaction (46). Moreover, mipomersen has been associated to an

increased risk of hepatic side effects (e.g., elevated liver transaminase and liver fat levels), which are


7

probably caused by the drug's mechanism of action relying on the prevention of the hepatic

excretion of triglycerides (47). In order to improve the uptake in the targeted tissue and decrease

the therapeutic dose required, several physical methods have been tested in preclinical and clinical

studies (48). These methods are based on the disruption of the cell membrane and include

electroporation, hydrodynamic injection, ultrasound-mediated sonoporation, and gene gun.

Despite showing some efficacy for certain diseases, these methods are far from being universally

applicable; therefore, several approaches for nucleic acid delivery encompassing particle-based and

single-molecule-based strategies have been developed as discussed in detail in the following

sections.

Scheme 1.3. Cellular uptake and trafficking of ONs (36). TGN – trans-Golgi network; MVBs –

multivesicular bodies. ONs can enter cells via several pathways, such as macropinocytosis, caveolin-

or clathrin-dependent endocytosis, initially guiding them to early endosomes. After that, ONs can

be recycled back to the cell exterior or trafficked further to late endosomes/MVBs. Eventually,

ONs can be either degraded in the lysosomes or guided by the retromer complex to the TGN.

Even though the intracellular trafficking is highly regulated by multiple proteins, particularly by

Rab family of GTPases, a small portion of ON can leak into the cytosol and the nucleus. The

formation of exosomes within the MVBs is controlled by the ESCRT-I protein complex, which

was recently connected with the efficacy of ONs (49).


8

1.3.1 Particle-based delivery approaches

One of the most commonly exploited strategies to deliver nucleic acids inside the cells is via

nano/microscale delivery vehicles (50). They can accommodate ONs and larger molecules such as

pDNA and mRNA, can be decorated with targeting ligands to ensure cell/tissue-specific uptake,

and can be imbued with stimuli-responsive properties in order to release their cargo under specific

conditions. Ideally, this approach should allow for (i) protection of nucleic acid cargo from

extracellular enzymes, (ii) targeting nucleic acids to a desired tissue/cell type with minimal uptake

by the other tissues, and (iii) delivery to the intracellular compartment of interest (cytosol or

nucleus), bypassing the lysosomal degradation pathway.

Viral and bacterial vectors

Viruses are naturally evolved nucleic acid delivery systems that can be genetically engineered

for therapeutic use (51). They are considered to be the most efficient transfection agents, which is

supported by the four viral-based gene therapies approved worldwide, as mentioned previously.

One of the limitations of viral delivery systems is the maximal size of nucleic acid payload they can

carry (up to 5 – 150 kb DNA, depending on the virus type (52)). Additionally, due to the strict

safety requirements and relatively complex manufacturing process, their production is expensive

(53). Another more important concern is that some viruses (e.g., lentivirus) tend to insert their genes

into the host genome, which could lead to the reading frame shift or to the activation of host's

oncogenic genes (54). Although safer viruses exist that lack this property (e.g., adenovirus, adeno-

associated virus), their capsids still possess immunogenic properties which can lead to serious

adverse events or fast clearance of the viral carriers upon repeated administration (54). Genetically

engineered invasive bacteria have also been tested as gene delivery systems (55, 56). However, due

to the immunogenicity of bacterial products upon systemic exposure, bacteria-derived delivery

systems could find their applications primarily for the local therapy of the gastrointestinal (GI)

diseases (55, 56). Considering the limitations posed by viral and bacterial gene delivery systems, the

development of non-biological vehicles has attracted much attention and raised hopes for bringing

safe, efficient, and inexpensive gene therapies on market.


9

Inorganic particles

As an alternative to biological vectors, inorganic nano/microparticles composed of metal,

calcium phosphate, metal oxides, silica, or carbon nanotubes have been developed (57). Some of

these materials offer unique electrical, optical, and magnetic properties enabling various novel

biomedical applications, such as combination of therapy and diagnostics (theranostics) (57–59).

Inorganic delivery vehicles are generally more stable than biological ones, inexpensive, and easy to

functionalize. Indeed, nucleic acids can be directly grafted onto the surface of the carrier (e.g., gold

nanoparticle via thiol linker (60)), complexed with the cationic-polymer-functionalized particle (e.g.,

poly(amidoamine) (PAMAM)-modified iron oxide (61)), or incorporated during the particle

formation process (e.g., calcium phosphate vehicles (62, 63)). Among the disadvantages of inorganic

delivery systems are low loading efficiency, insufficient cargo protection, and poor endosomal

escape (64). Another major drawback is often the lack of biodegradation and, therefore, potential

toxicity due to accumulation in healthy tissues following repeated dosing (65). While the calcium

phosphate nanoparticles are biocompatible and biodegradable, the issue regarding their stability

represent an obstacles for their clinical translation. The use of stabilizers (e.g., PEGylation) is an

attractive strategy to tackle this problem (63, 66, 67).

Organic complexes (polyplexes and lipoplexes)

Nucleic acids are negatively charged biopolymers that can form nano/microsized complexes

with oppositely charged cationic polymers (e.g., poly(ethylene imine) (PEI) (68)) or lipids (e.g.,

Lipofectamine™ 2000 (LF) (69)), referred to as polyplexes and lipoplexes, respectively. Despite

being less efficient in transfecting cells in comparison to viral carriers, organic complexes are

advantageous due to tunable chemistries, lower risk of immunogenicity, and the possibility of large-

scale production at acceptable costs (70). Generally, the excess of the positive charges on the

particles' surface is a prerequisite for their high transfection efficacy in vitro (70). Such particles

interact with the negatively charged cell membrane and enter cells via endocytic pathways. In order

to release their nucleic acid cargo into the cytoplasm, these carriers must disrupt endosomes via

the proton-sponge effect (71), formation of nanopores (72), or membrane fusion/destabilization

(73). In case of polyplexes, the inherent polydispersity of polymers potentially results in difficult-

to-characterize complexes. The need for the systems with controlled and reproducible structure

has led to the development of molecularly-defined cationic dendrimers (e.g., PAMAM (74)) as gene

delivery vehicles. Nevertheless, the repeated administration of synthetic non-degradable cationic


10

polymers such as PEI and PAMAM have raised some safety concerns (75). Incorporation of

biodegradable bonds into the polymer backbone (e.g., ester (76) or disulfide (77)) may help to

facilitate the polymer's renal clearance after its cleavage in the body. The use of natural

biodegradable polycations (e.g., chitosan (78)) or non-charged biodegradable polymers (e.g.,

poly(lactide-co-glycolide) (PLGA) (79)) are the alternative strategies under investigation. In

comparison to synthetic polymers, phospholipids and some lipids have an advantage of higher

biocompatibility and biodegradability (80), which was confirmed in multiple clinical trials (81).

In vivo, before exerting their function, positively charged polyplexes and lipoplexes are rapidly

coated with serum proteins (i.e., opsonins) followed by their recognition and clearance by

mononuclear phagocyte system (MPS). These processes can cause an immune reaction and liver

toxicity (68, 82). Conferring particles with the stealth effect by PEGylation may reduce not only

the serum protein binding, but also cell uptake of cationic particles (68). Therefore, a combination

of PEGylation and attachment of a targeting ligand for the enhanced uptake by the desired cell

type may help to overcome these issues (68). For example, CALAA-01, a polyplex system that has

entered clinical trial, relies on the uptake of transferrin-modified cationic cyclodextrin/siRNA

complex by cancer cells overexpressing the transferrin receptor (83). Unfortunately, despite being

PEGylated, CALAA-01 has failed phase Ib clinical trial due to liver toxicity and immunogenicity

of the cationic carrier (83). Therefore, potential toxicity of the cationic gene delivery carriers must

be carefully evaluated before their clinical translation. To solve this problem, the surface positive

charges need to be thoroughly masked in the blood stream and exposed only in the vicinity of or

inside the target cell. This could be achieved through the design of responsive polymers and lipids

(e.g., pH-, light-, temperature-sensitive) that can release their cargo upon defined stimuli (70). For

example, the concept of lipids that become charged only in the acidic environment found in tumor

tissues and in the endosomes has been exploited for the liposomal delivery vehicles Smarticles®

(Marina Biothech Inc.) (84) and for the stable-nucleic-acid lipid particles (SNALPs) (Tekmira

Pharmaceuticals) (85). These pH-sensitive lipid-based nucleic acid delivery vehicles are currently

progressing through several clinical trials (e.g., for the treatment of transthyretin-mediated

amyloidosis or lung cancer (81, 86)).

While numerous non-viral particle-based delivery systems have been tested, so far none of

them has been released on the market. One of the major problems of those delivery systems is

their high carrier-to-cargo ratio, resulting in the accumulation of large amount of potentially toxic

material in the body (36). Importantly, due to their size, nanocarriers cannot escape from the

vascular network and reach parenchyma of most of the underlying organs upon intravenous (i.v.)

administration, but they can diffuse primarily into tissues with fenestrated vasculature such as liver,


11

spleen, solid tumors, or sites of inflammation (Scheme 1.4) (36, 87). Therefore, particle-based gene

delivery approaches can be used mainly for the treatment of pathologies of the liver, cancer, and

inflammatory diseases (36). Importantly, in case of cancer treatment, distribution of the particles

to healthy organs, such as liver and spleen, may raise toxicity issues.

1.3.2 Single-molecule conjugate delivery approaches

Due to the obstacles associated with nano/microsized delivery vehicles, single-molecule

systems have received much attention for the development of ON-based therapeutics. They can

easily diffuse through the blood vessel endothelium upon i.v. or subcutaneous administration and

reach organs inaccessible for nanoparticles (Scheme 1.4) (88). Importantly, the reduction in the

amount of administered excipients (e.g., non-biodegradable polymer) may help to avoid some of

the toxicity and immunogenicity issues. However, this comes at a certain cost: the need to stabilize

unprotected ONs via chemical modifications due to exposure to extracellular nucleases. From an

industrial point of view, ON conjugates are attractive because their relatively simple and well-

defined structure allows their preparation by solid-phase synthesis, thus reducing the production,

characterization, and purification costs. The major drawback of single-molecule ON conjugates is

their low potency (typically µM in vitro), which requires administration of higher doses of ONs to

achieve the therapeutic effect in comparison to particulate carriers (89, 90).

Conjugation of a receptor-targeting moiety

The conjugation of a receptor-targeting moiety to the ONs is the main strategy employed to

facilitate their cell uptake (91). When selecting a suitable receptor for targeting, one must consider

its expression levels in target versus non-target tissues, the rate of receptor recycling, and (optionally)

the availability of small-molecule ligands (36). In addition, potential activation/inhibition of

downstream signaling cascades induced by ligand binding to the selected receptor must be carefully

evaluated to avoid potential off-target events especially upon chronic administration (36). Among

the receptors often used for targeting are integrins, G protein-coupled receptors, human tyrosine

kinases, TLRs, scavenger receptors, the asialoglycoprotein receptor (ASGR), the folate receptor,

and some others (36). A range of ligands have been conjugated to ONs for enhancement of their

receptor-mediated uptake and tissue selectivity including small-molecule ligands, aptamers,

peptides, and even larger molecules such as antibodies. Although it is often difficult to find a

suitable small-molecule ligand for a new receptor candidate, biomolecular techniques such as phage


12

display and SELEX (Systematic Evolution of Ligands by Exponential Enrichment) can be

exploited for the search of high-affinity biologics for virtually any target of interest.

The most successful examples of targeted ON delivery systems are those relying on the

ASGR-mediated uptake with the help of N-acetylgalactosamine (GalNAc) ligands. The ASGR is

predominantly expressed on hepatocytes (92), and is a particularly attractive target due to its

presence at a high density on the cell surface, and its rapid internalization, cargo release, and

recycling back to the plasma membrane (92). However, recent findings pointed out the

involvement of ASGR signaling in platelet homeostasis, which could be disturbed by chronic

GalNAc-ON administration (93). Despite this fact, several GalNAc-ON conjugates based on

siRNA and antimiR are currently advancing through clinical trials for the treatment of diseases with

liver-localized targets (e.g., transthyretin-mediated amyloidosis, hypercholesterolemia, hemophilia,

or hepatitis C infection). Integrins constitute another attractive class of targets due to their high

expression levels, recycling, and selective expression in certain tissues/disease states (36). For

instance, αvβ3 integrin is overexpressed in newly formed blood vessels and invasive cancer cells

(94). Cyclic pentapeptide Arg-Gly-Asp-d-Phe-Lys (cRGD), a well-known ligand for the αvβ3

integrin, was conjugated to an siRNA against vascular endothelial growth factor (VEGF), and

achieved significant tumor growth retardation in mice upon systemic administration at a relatively

low dose of 0.75 mg·kg-1 (94). Some other cancer-specific targets including the prostate-specific

membrane antigen (PSMA), B-cell-activating factor receptor (95), and cancer-associated receptor

tyrosine kinases (96) were targeted with the help of aptamers (nucleic acid-based binders). The

main drawback of aptamer-based targeting ligands –i.e. their rapid degradation in the serum– has

been recently addressed by development of polymerases that can operate with chemically modified

nucleobases, such as FANA (34). These polymerases have enabled SELEX-based selection of fully

modified enzymatically stable aptamers (34). Moreover, another recent optimization of SELEX-

based aptamer selection method has helped to identify aptamers that trigger efficient internalization

of the aptamer-ON conjugate by the target cells (97).


13

Scheme 1.4. Schematic biodistribution of nucleic acid delivery systems upon systemic

administration. Singe-molecule-based systems can easily diffuse through the blood vessel

endothelium (gaps of <5.5 nm) and reach organs inaccessible for nanoparticles (except for the

brain). Due to their size, particulate carriers cannot escape from the vascular network and reach

parenchyma of most of the underlying organs upon i.v. administration, but they can diffuse

primarily into tissues with fenestrated vasculature such as liver, spleen, solid tumors, or sites of

inflammation. After reaching the target tissue, nucleic acid delivery carriers can be taken up

unspecifically or via receptor-mediated endocytosis. They should be able to escape the endosomes

to exert their function.

Due to their size (<20 kDa), ON conjugates with small-molecules, short peptides, or

aptamers penetrate well into tissues. It is worth noting that the conjugation of intact antibodies to

ONs (>150 kDa) results in drastically reduced tumor extravasation in vivo (98); therefore, strategies

involving conjugation of antibody fragments or smaller non-antibody scaffolds (e.g., DARPins (99))

may be more promising. However, as mentioned previously, the molecular weight of the ON

conjugate should be tuned in the optimal range, since some of the ONs (e.g., siRNA) are rapidly

excreted via renal filtration before exerting their function. Therefore, the additional conjugation of

macromolecules is a way to achieve long circulation times and significant target binding. Indeed,

PEGylated siRNA conjugated to an aptamer against the PSMA showed prolonged target gene

silencing in a prostate cancer xenograft model in comparison to its non-PEGylated


14

counterpart (100). Conjugation of targeted ONs to a serum protein (e.g., albumin) is another

interesting approach to increase the circulation time, which additionally allows for display of

multiple copies of ONs in a single carrier molecule (up to 15) (101). The resulting conjugates are

small enough (about 12 nm) for efficient tissue penetration, but too large for renal clearance (101).

The potential drawback of the albumin-conjugated ON delivery systems is the absence of

endosome-destabilizing properties. The endosomal escape can be achieved by conjugation of a

pH-responsive polymer (74). For example, the Dynamic Polyconjugate™ (DPC™) delivery system

(Arrowhead Research) relies on the attachment of a targeted (e.g., against tumor-expressed integrins

or ASGR of hepatocytes) endosomolytic polymer to siRNA via a disulfide linker (102). The cationic

charges of the amphiphilic polymer are masked with pH- and protease-labile groups, which are

being cleaved in the endosomal environment thereby promoting the release of siRNA into the

cytoplasm (102). Overall, despite having been in focus only for a few years, a strategy of direct

conjugation of a targeting moiety to ONs has already achieved significant clinical results at least

for liver targeting. The advantages of single-molecule-based systems for clinical development, such

as the well-defined structure and their better diffusion in the tissue parenchyma, render them highly

competitive with the most clinically advanced lipoplex-based delivery systems.

Conjugation of non-specific uptake enhancers

Conjugation of cell-penetrating peptides (CPPs) and lipids to ONs has been shown in vitro

to enhance their uptake in multiple cell types, however, they lack cell selectivity (103). Therefore,

such systems could find their application primarily for the ON delivery to the cells that are broadly

distributed in the body (e.g., muscle cells, virally-infected cells, bacterial cells in the circulation), to

the liver, or locally (103–105).

CPPs are typically positively charged peptides containing hydrophobic amino acid residues

that interact with cell membranes and trigger uptake (104). They can form insoluble complexes

with oppositely charged nucleic acids, similarly to cationic polymers and lipids (103, 104). However,

to obtain structurally defined single-molecule-based conjugates, CPPs are preferably attached to

non-charged ONs such as PMOs and PNAs. Notably, it was recently shown that relatively

hydrophobic CPP-PMO conjugates can self-assemble into nanoparticles and be taken up via

scavenger receptors, a type of pathogen-recognition receptors (106). These conjugates have been

mainly used for correcting the mis-spliced transcript of dystrophin, the protein involved in the

pathology of Duchenne muscular dystrophy (104). Thus, a single i.v. administration of a CPP-


15

PMO conjugate (12.5 mg·kg-1) to mice resulted in a strong dystrophin expression not only in

skeletal muscles, but also in the heart, which is particularly challenging (107). However, the possible

toxicity of the cationic CPPs and their interaction with serum proteins should be carefully

considered when designing an ON delivery system (103). Another concern related to CPP-based

conjugates is their cell-type dependent efficacy (103). In a recent study from our group, a library of

15 different CPPs (previously reported to induce ON-mediated silencing) was conjugated to a

single PNA sequence targeting luciferase and screened for gene silencing in a colon cancer cell line

(108). While most of the CPP-PNA conjugates were trapped in the endosomes as evidenced by

their enhanced activity in the presence of the endosomolytic agent chloroquine, only one CPP was

capable of target gene silencing (108). Apart from targeting genetic diseases, CPP-based ON

conjugates have been investigated as antiviral and antibacterial agents with some success in vivo

(109, 110). However, elimination of the cationic CPP carrier and incorporation of positively

charged piperazine groups directly into the backbone of PMOs resulted in less toxic antiviral drug

candidates, which were well tolerated in phase I clinical trial for the treatment of Marburg virus

infection (111).

Several authors have shown that the conjugation of neutral lipids such as cholesterol and

aliphatic fatty acids to ONs improves their uptake and silencing efficacy both in vitro and in vivo

(112–114). Following systemic administration, lipid-ON conjugates accumulate mainly in the liver

probably due to binding to so-called “in vivo vehicles”, such as low-density lipoproteins (113),

chylomicrons (115), or albumin (112, 116). The vehicle/ON complex can bind to the

corresponding cell surface receptor on hepatocytes (e.g., lipoprotein receptor) and facilitate the

endocytosis of amphiphilic ON conjugates (in case of siRNA conjugates, possibly via a channel for

double stranded RNA, SID-1) (113). In the absence of in vivo vehicle, the uptake and silencing

efficacy of amphiphilic ONs was found to vary greatly depending on the lipophilic moiety and the

cell line tested (112, 117, 118). The cell-line-dependent efficacy of the conjugates could be related

to the different expression of the surface receptors involved in the ON's uptake. For example, an

siRNA conjugated with a double-tailed myristic acid derivative was efficiently incorporated into

the cell membrane and triggered β2-integrin-mediated endocytosis of the conjugate in certain cell

types with high-level expression of β2-integrin (119). The association with the cell membrane seems

to be the first step in the uptake of amphiphilic conjugates (120), followed by adsorptive

endocytosis/pinocytosis or receptor-mediated endocytosis of the ON (117, 119). In the case of

amphiphilic bile acid-siRNA derivatives, uptake was shown to be mediated by a hypothetical

“needle and thread” transport route (121). Due to the limited number of studies, however, the


16

mechanism of cellular uptake of amphiphilic ONs remains unclear and further studies are necessary

to draw reliable conclusions.

Recently, local administration of carrier-free hydrophobically modified ONs has been

exploited for the treatment of several disorders (121–126). For example, intravitreal injection of

bile acid-modified siRNA against Sjo ̈gren syndrome type B antigen lead to moderate target

knockdown without the signs of inflammation in rats (121). Cholesterol- or docosahexaenoyl-

modified chemically-stabilized siRNAs silenced target huntingtin mRNA in mouse brain upon local

injection (122, 123). Another cholesterol-modified stabilized siRNA (Accell® siRNA) (127)

targeting the mutant keratin 6a mRNA for the treatment of pachyonychia congenital showed

enhanced uptake and target silencing in human skin ex-vivo and in a mouse model via local injection

(124, 125). RXi Pharmaceuticals is developing hydrophobized RNAi compounds for topical and

systemic applications (126). The proprietary nucleic acid delivery platform is an OMe- and 2′-F-

modified hybrid between siRNA-like short duplex region and single-stranded PS-modified region

improving nuclease resistance (126). It is conjugated with lipophilic groups, such as sterol,

enhancing the uptake in multiple cell types, i.e., its ‘self-delivery’ (sd-rxRNA®) (126). According to

the company, RXI-109, a lead compound currently in phase II clinical trials for reducing skin

scarring, has an anti-fibrotic action due to the silencing of connective tissue growth factor. It is

additionally being evaluated in AMD patients after intravitreal administration for reduction of

retinal fibrosis that contributes to vision loss.

The major drawback of lipid-ON conjugates is their relatively low potency, which requires

high dose. Co-administration with small molecules or peptides that enhance ON's uptake and

endosomal release is an emerging field of research. Thus, the administration of cholesterol-siRNA

conjugate in combination with DPC™ endosomolytic amphiphilic peptide is currently being

evaluated for the treatment of hepatitis B infection in phase II clinical trial (105). Recently, co-

administration of hydrophobically modified siRNA with FDA-approved small molecule drug

guanabenz (an antihypertensive agent) was shown to enhance its silencing effect in vitro (128). In

another recent in vitro study, high throughput screening helped to reveal 28 compounds that

improved the cholesterol-siRNA silencing by enhancing its cell uptake or release from the

endolysosomal compartments (129), opening new directions in the ON delivery strategies.


17

1.4 Delivery of nucleic acids to the gastrointestinal (GI) tract

Due to the transient nature of mRNA knockdown, ON therapeutics require regular

administration, for which oral delivery would be the most convenient route (130). Several attempts

to systemically deliver nucleic acids via oral intake have been undertaken in animals and humans.

Even when imbued with enzymatic resistance by chemical modification, nucleic acid drugs proved

to be minimally absorbed in the GI tract due to their polyanionic nature and high molecular weight

(>5000 Da). For instance, in comparison to the highly permeable small molecule drug naproxene,

permeability coefficients of 20-mer PS-modified ONs across rat small intestine are approximately

two orders of magnitude smaller (Papp = 1-2×10-4 cm/s vs. 2-8×10-6 cm/s) (131).

Several pathways have been described for ON translocation from the GI tract to the systemic

circulation (Scheme 1.5). Those include: (i) internalization of particulate carriers by dendritic

cells/M cells of intestinal Peyer's patches and consequently by associated lymphoid macrophages

(132, 133), (ii) uptake by the intraepithelial lymphocytes (134), (iii) transcytosis/paracellular

transport through the absorptive epithelium (115, 133–135), which in certain cases leads to (iv)

binding of hydrophobized ON conjugates to chylomicrons in the lymph duct followed by transport

to the systemic circulation (115). These pathways result in the delivery of ONs to MPS cells of

peritoneum, liver, spleen, kidneys, lungs, and liver hepatocytes (115, 132–138).

Scheme 1.5. Putative uptake routes for systemic delivery of nucleic acid therapeutics after oral

administration: (i) M cell/dendritic cell-mediated uptake, (ii) intraepithelial lymphocyte uptake, (iii)

paracellular transport/transcytosis, in certain cases leading to (iv) lymph chylomicron-mediated

transport of hydrophobized nucleic acids (115, 132–138).


18

Employing a permeation enhancer (e.g., sodium caprate) may promote paracellular transport

(the passage between epithelial cells) thus improving ON's absorption (131). However, the systemic

bioavailability of ONs was found to be highly variable when co-administered with sodium caprate

ranging from 0.7% to 27.5% in humans (139), making this strategy less valuable for clinical

applications. The results of clinical trials testing this strategy are discussed in Chapter 2 of the

present thesis.

As an alternative delivery approach, the targeting of intrinsic pathways connecting the

intestinal mucosa with the systemic compartment could be employed, such as the uptake by the

intestinal immune cells and their migration to remote organs or the lipid transport system (Scheme

1.5). Indeed, the pioneer system GeRP (β1,3-D-glucan encapsulated siRNA particles) composed

of baker's yeast-derived shell enveloping PEI/siRNA/tRNA mixed polyplex and the amphiphilic

peptide Endoporter was taken up via receptor-mediated endocytosis by macrophages both in vitro

and in vivo (132). After gavage of GeRP particles to mice, fluorescently labelled yeast shell and

siRNA co-localized with macrophages of peripheral organs such as spleen, liver, lung tissues, and

peritoneum (132). These particles suppressed lipopolysaccharide (LPS)-induced systemic

inflammation by silencing target Map4k4 mRNA in macrophages, reduced TNF serum levels, and

improved survival rate (132). However, the same delivery vehicle loaded with anti-TNF siRNA

failed to significantly downregulate the target mRNA and, consequently, to reduce the severity of

colitis in a mouse model (140). In general, GeRP represents a highly complex delivery system

requiring multi-step preparation, which makes it less attractive for clinical translation.

A simpler particle-based approach relying on the delivery of chitosan/siRNA polyplex

showed the presence of intact unmodified siRNA as detected by PCR analysis in the liver, spleen,

and kidneys 1 and 5 hours after gavage, but not at the later time point (24 h after gavage) (134).

Presumably, this delivery vehicle was either not taken up by the intestinal epithelial and submucosal

cells or it was rapidly disassembled within 45 min, as the fluorescently labelled siRNA was detected

only at the luminal surface of the intestinal epithelium (134). Unfortunately, the bioactivity of this

system remains unknown as it was not further investigated (134).

In subsequent studies, the oral administration of modified chitosan-based polyplexes

containing ONs against TNF led to amelioration of LPS-induced systemic inflammation (133, 136,

137). In order to disrupt the intestinal epithelial barrier and reach the underlying MPS cells, a

complex multicomponent self-assembling ON delivery system was designed (136). It included

oleyl-modified trimethyl chitosan and oleyl-PEG-cysteamine for permeation across intestinal

epithelium, oleyl-PEG-mannose for receptor-mediated uptake by mucosal macrophages, cationic


19

α-helical polypeptide (PVBLG-8) for endosomal escape, and sodium tripolyphosphate (TPP) as a

crosslinker (136). The intestinal absorption of these particles was compared in two in vitro models:

the non-follicle-associated epithelium (FAE) model based on polarized Caco-2 cell monolayers and

the FAE model obtained from co-culture of Caco-2 cell monolayers with Raji B lymphocytes

producing M cell phenotype (136). Increased permeability of chitosan/siRNA complex across the

non-FAE model was observed in comparison to naked siRNA as a result of the disturbance of

tight junctions (TJ), as indicated by the decreased transepithelial electrical resistance (TEER) and

the results of immunostaining of TJ proteins (136). Moreover, the permeation coefficient of the

chitosan-based delivery system increased 2.9-fold in the FAE model in comparison to that of the

non-FAE model, suggesting preferential uptake by M cells (136).

A similar delivery system relying on receptor-mediated uptake by M cells and consequently

by underlying mucosal macrophages involved direct functionalization of trimethyl chitosan with

mannose and cysteine groups (133). These mannose-modified carriers were shown to enter

macrophages via caveolae-mediated endocytosis and micropinocytosis in vitro (133, 136).

Alternatively, intracolonic instillation of galactose-modified chitosan complexed with AON against

TNF led to its uptake by activated intestinal macrophages overexpressing galactose receptor in

dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model (137). Zhang J. et al. reported

a similar polyplex system based on galactosylated trimethyl chitosan containing cysteine moieties

for the targeted delivery of Map4k4 siRNA to activated intestinal macrophages in a DSS-induced

colitis model (138). In a subsequent cancer therapy study by the same group, orally delivered

galactosylated cysteine-modified trimethyl chitosan-based polyplexes carrying shRNA and siRNA

(targeting survivin and VEGF, respectively) caused target genes silencing in a subcutaneously

implanted hepatoma tissue (135). Interestingly, siRNA and shRNA were detected not only in tumor

hepatocytes but also in MPS-cells-containing organs, such as liver, spleen, and lungs (135), despite

the low level of galactose receptor expression in quiescent macrophages (137, 138). Authors

attributed the systemic translocation of the nucleic acids and their hepatocyte uptake to the

permeation-enhancing properties of modified trimethyl chitosan (135). In a differentiated intestinal

epithelium in vitro model, this delivery system induced a dramatic reduction of TEER, which is an

indicator of disturbed TJ (135). These results suggest that the modified chitosan-based delivery

vehicle reached systemic circulation directly via paracellular transport through the absorptive

epithelium and unspecific uptake by underlying mucosal macrophages.

A newer approach has been reported to target liver hepatocytes taking advantage of the

intrinsic lipid transport system, using α-tocopherol-siRNA conjugate (Toc-siRNA) administered

rectally in mixed lipid nanoparticles (Scheme 1.5) (115). The main components of lipid


20

nanoparticles, PEG-60 hydrogenated castor oil (HCO-60) and linoleic acid, served as permeation

enhancers. Toc-siRNA conjugate was shown to bind to chylomicrons in the lymph duct and to be

transported to the systemic circulation. It was demonstrated that uptake by the liver hepatocytes

via remnant receptors caused target apolipoprotein B (ApoB) silencing and decrease in low-density

lipoprotein cholesterol and triglycerides serum levels (115). Notably, the size of the nanoparticles

and the site of their administration crucially affected the delivery of Toc-siRNA to the liver. While

the small uniform nanoparticles (15 nm in diameter) administered rectally were capable of siRNA

delivery to the liver, the presence of aggregates in the formulation and/or administration to the

small intestine abolished the delivery efficacy (115). Overall, the systemic translocation of nucleic

acid drugs after oral administration remains extremely challenging as it often requires complex

delivery vehicles and/or co-administration of potentially toxic absorbefacients (141–143). In

addition, many of the delivery systems rely on the uptake by M cells, the number of which varies

greatly between species (for example, the proportion of M cells in the FAE is 10% in human and

mice and 50% in rabbits), making the prediction of absorption in humans based on animal data

difficult (144).

Due to the accessibility of the digestive mucosa, local delivery of nucleic acids to the GI tract

targets has higher chances for clinical translation in comparison to targeting systemic disorders via

the oral route. Several GI ailments with unavailable or ineffective conventional treatments could

substantially benefit from nucleic acid therapy, such as GI infections (145), cancer (146), rare

genetic diseases (e.g., familial adenomatous polyposis (147)), and IBDs (Crohn's disease, ulcerative

colitis, pouchitis (148, 149)). In these cases, local administration would be the most desirable route

due to the minimal systemic exposure and potential adverse reactions (e.g., complement activation

or gene silencing in non-target organs) (148, 150).

To protect nucleic acids from the acidic gastric pH that leads to their depurination (151),

enteric formulations, enema, or complexation with polymers may be employed (150, 152). Chemical

modifications, as mentioned previously, can also be included to imbue nucleic acids with resistance

to the RNases and DNases found in the intestinal lumen (15). The strategy of delivering high doses

of chemically stabilized nucleic acids to the GI tract is currently being tested in clinical trials for the

treatment of IBDs (8), which is discussed in more detail in Chapter 2. Importantly, due to their

high negative charge density and large molecular weight, nucleic acids are poorly taken up by

intestinal cells. Therefore, strategies aimed at enhancing ON's cell uptake may improve their

efficacy.


21

Several particle-based delivery strategies have been developed to deliver nucleic acids

targeting cytokines (e.g., TNF), cytokine receptors (e.g., TNFR2), transcription factors (e.g., NFκB),

kinases (e.g., Map4k4), metalloproteinases (e.g., MMP-10), and oncogenes (e.g., β-catenin) to the GI

mucosa (Table 1.1). Most of the particle-based carriers for local GI therapy target mucosal immune

cells capable of disseminating to remote organs (115, 133, 135); therefore, these approaches could

potentially cause the same off-target effects as systemically delivered ONs. Generally, particle-

based carriers do not require chemical modification of nucleic acids because the vehicle can

effectively protect the ON from GI degradation. However, they need to overcome another

important barrier constituted by the thick, negatively charged mucus layer in the intestine with fast

renewal rates. This mucus layer acts as a sieve for large particulate carriers hampering their diffusion

to the epithelium, and additionally entraps nanoparticles via electrostatic and/or hydrophobic

interactions leading to their elimination through mucociliary clearance (153). Engineering mucus-

penetrating nanoparticles via conjugation of low-molecular-weight PEG chains has shown some

success in overcoming this barrier (153).

The first demonstration of local gene knockdown in the GI tract was achieved with lipoplex-

based ON delivery systems applied rectally or by intracolonic instillation (154, 155). Further

improvement in gene silencing by lipoplex-based vehicles has been attempted using chemically

modified siRNAs, given their increased nuclease resistance and reduced immunogenicity (69).

While simple and attractive as a proof-of-concept, cationic lipids or polymers have limitations due

to their reported toxicity (156, 157) and potentially negative impact on intestinal microbiota (143).

Approaches aimed at improving the safety of polycation-based delivery systems have thus been

developed, such as including a protective coating with poly(lactic acid) (PLA) and poly(vinyl

alcohol) (PVA) (158, 159), employing shorter polymer chains (159, 160), or incorporating

biodegradable disulfide bonds (161). Interestingly, a delivery system made of PVA-coated PLA

matrix containing siRNA/PEI polyplex was shown to be taken up and silence the target gene not

only in macrophages but also in intestinal epithelial cells (159); the mechanism of uptake by

polarized epithelium was however not suggested. In another study, some uptake by intestinal

epithelial cells was achieved via endocytosis of chitosan/PVA-coated PLGA nanospheres carrying

decoy ON (162). Notably, the particles adhered to and were taken up mainly by the inflamed

colonic tissue, while the uptake by the non-inflamed tissue was negligible (162). According to the

patent (WO 2013138930 A1) and company's web-site, enGene Inc. has developed a polyplex

system based on arginine/gluconic acid-modified chitosan capable of transfecting gut epithelium,

and plans to use this system for the treatment of IBDs and diabetes. Another possible approach to

enhance polyplex-mediated gene silencing is to modify the cationic polymer with lipophilic groups


22

(163, 164). Indeed, polyplex of alkyl-conjugated cyclodextrin and siRNA administered rectally to a

colitis mouse model induced target gene silencing in mucosal macrophages and decreased the

severity of the disease (165).

Some delivery vehicles use environmental triggers to release their cargo at defined locations

within the GI tract. For instance, the incorporation of a polyplex in a pH-sensitive

alginate/chitosan hydrogel was shown to release its cargo primarily in the colon at pH values of 5-

6 (159). Furthermore, the so-called nanoparticles-in-microsphere oral system (NiMOS) which relies

on the encapsulation of gelatin/siRNA nanoparticles in a microshell composed of lipase-labile

poly(ε-caprolactone) (PCL), was shown to protect the cargo during gastric transit and enable its

release in the small intestine (149, 166). This delivery system has been extensively studied for the

local delivery of siRNAs as well as pDNA to the small intestine and colon (166–169). A different

system employing thioketal nanoparticles has been employed to target ON specifically to the

inflamed intestinal tissue (140). This system is based on siRNA lipoplex coated with poly(1,4-

phenyleneacetone dimethylene thioketal), which is sensitive to reactive oxygen species (ROS) and

should allow the thioketal shells to degrade in the close proximity to the inflammation sites due to

the high levels of ROS produced by the mucosal macrophages (140). Despite the reported efficacy

in a colitis model, this delivery system employs potentially toxic cationic lipids, and in addition

carcinogenic solvent (i.e., benzene) is used for its preparation, making it less attractive for clinical

translation. Generally, the complexity of multicomponent systems often involving several

emulsification, complexation, and purification steps represents a major hurdle for their clinical

translation.

Another interesting GI delivery system, the transkingdom RNA interference technology

platform (tkRNAi), has been developed by Marina Biotech. Based on living attenuated Escherichia

Coli expressing shRNA for target gene silencing, this delivery system enters mucosal epithelial cells

via β1-integrin-mediated endocytosis, escapes the acidic phagosomes with the help of listeriolysin,

and releases its nucleic acid cargo in the cytosol (55, 56). One example of tkRNAi is the orally

administered CEQ508, aiming at silencing β-catenin to prevent the uncontrolled cell growth in

colons of familial adenomatous polyposis patients overexpressing this protein (147). The results

from animal models seem promising (55); however, clinical trials will be necessary to evaluate the

safety and efficacy of bacteria-based delivery systems.

23

Tab

le 1

.1. Str

ateg

ies

for

nucl

eic

acid

s del

iver

y to

th

e G

I tr

act.

Ref.

(154)

(155)

(140)

(162)

(115)

(158, 159)

(134)

(137)

(138)

(135)

(133)

(136)

(132)

(166, 169)

(167, 168)

(55)

(148, 152)

(139)

(108)

(170)

* ca

lcula

ted b

ased

on

co

mpo

nen

ts' w

eigh

t ra

tio

s duri

ng

pre

par

atio

n

‡ n

ot

avai

lab

le

Org

an

/cell

typ

e

tota

l co

lon

ic t

issu

e

tota

l re

ctal

tis

sue

IEC

, m

acro

ph

ages

colo

n

hep

ato

cyte

s, k

idn

ey, in

test

ine

mac

rop

hag

es, IE

C

MP

S o

rgan

s

colo

n, M

PS o

rgan

s

acti

vat

ed m

acro

ph

ages

hep

ato

ma,

liv

er, M

PS o

rgan

s

MP

S o

rgan

s

MP

S o

rgan

s

MP

S o

rgan

s

smal

l in

test

ine,

co

lon

smal

l in

test

ine,

co

lon

IEC

IEC

, m

acro

ph

ages

N.A

.

pro

lifer

atin

g H

T-2

9 c

ells

dif

fere

nti

ated

Cac

o-2

cel

ls

Carr

ier

do

se/

day

0.1

27 m

g*

0.0

004 m

g

N.A

.‡

37 m

g

164 m

g·kg-

1*

0.5

mg

0.3

25 m

g

N.A

.‡

275 m

g·kg-

1*

100 m

g·kg-

1*

7.7

mg·

kg-

1*

66.5

mg·

kg-

1*

4·1

09 G

eRP

s·kg-

1

1.2

mg·

kg-

1

58 m

g

5·1

010 c

.f.u

.

–

660 m

g

–

–

ON

do

se/

day

0.0

027 m

g

0.0

005 m

g

0.2

3 m

g·kg-

1

2 m

g

30 m

g·kg-

1

N.A

.‡

0.0

28 m

g

5 m

g·kg-

1

0.2

5 m

g·kg-

1

0.2

mg+

1 m

g ·k

g-1

0.0

5 m

g·kg-

1

0.2

mg·

kg-

1

0.0

2 m

g·kg-

1

N.A

.‡

0.1

mg

–

40-1

60 m

g

100 m

g

8-2

0 µ

M in

vitr

o

1-5

µM

in

vitr

o

Targ

et

CD

40

TN

F, C

CR

5

TN

F

NF

κB

Ap

oB

TN

F; C

D98

N.A

.‡

TN

F

Map

4k4

Surv

ivin

+V

EG

F

TN

F

TN

F

Map

4k4

TN

F; C

yD1

GF

P, βG

al, IL

-10

β-c

aten

in

ICA

M-1

; Sm

ad7

Ap

oB

; T

NF

luci

fera

se

Bcl

-2

Carg

o

AO

N

siR

NA

siR

NA

Dec

oy

ON

siR

NA

siR

NA

siR

NA

AO

N

siR

NA

siR

NA

+sh

RN

A

siR

NA

siR

NA

tRN

A +

siR

NA

siR

NA

pD

NA

shR

NA

AO

N

AO

N

AO

N

AO

N, si

RN

A

Deli

very

veh

icle

Lip

oso

me-

bas

ed lip

op

lex

LF

lip

op

lex

DO

TA

P lip

op

lex

wit

h R

OS-l

abile

th

ioket

al p

oly

mer

DO

TA

P lip

op

lex

in P

LG

A m

atri

x w

ith

chit

osa

n/

PV

A

To

c-si

RN

A c

on

juga

te, H

CO

-60, lin

ole

ic a

cid

PL

A-P

EI

po

lyp

lex

coat

ed w

ith

PV

A

Ch

ito

san

po

lyp

lex

Gal

acto

syla

ted

ch

ito

san

po

lyp

lex

Gal

acto

syla

ted

tri

met

hyl

ch

ito

san

-cys

tein

e, T

PP

Gal

acto

syla

ted

tri

met

hyl

ch

ito

san

-cys

tein

e

Man

no

se-m

od

ifie

d t

rim

eth

yl c

hit

osa

n-c

yste

ine,

TP

P

ole

yl-P

EG

-man

no

se, o

leyl

-mo

dif

ied

tri

met

hyl

chit

osa

n, o

leyl

-PE

G-c

yste

amin

e, P

VB

LG

-8, T

PP

β1,3

-D-g

luca

n-e

nca

psu

late

d P

EI

wit

h E

nd

op

ort

er

NiM

OS (

gela

tin

po

lyp

lex

in P

CL

mat

rix)

Liv

ing

E. C

oli

exp

ress

ing

invas

in

No

ne

(ch

emic

ally

sta

bili

zed

ON

)

So

diu

m c

apra

te (

per

mea

tio

n e

nh

ance

r)

Act

ivat

able

CP

P-P

NA

co

nju

gate

Am

ph

iph

ilic

do

cosa

no

yl-O

N c

on

juga

te

Particle Single molecule


24

Because of the previously discussed advantages of single-molecule-based conjugates over

particle-based delivery systems, our group has recently developed an activatable ON conjugate

targeting colonic epithelium (108). The conjugate combined neutrally charged PNA with cationic

CPP in a single molecule, where the positive charges of CPP were masked by PEG chains via

enzyme-labile linker (108). The PEGylated conjugate was designed to be inactive in the upper GI

tract and to release the active CPP-PNA only upon arrival to the colon, where bacterial

azoreductase cleaves the azobenzene-PEG chains. Despite being a good proof-of-principle

example, this system relies on an expensive and protease-labile CPP, which must be additionally

stabilized for in vivo applications by incorporating D-amino acids. Moreover, differing activity of

colonic bacteria-derived azoreductase between individuals (171) could potentially lead to higher

inter-patient variability of the concentration of released active de-PEGylated CPP-PNA conjugate.

Additionally, some diseases of the GI tract such as Crohn's disease are localized not only in the

colon but also in the small intestine, where the developed strategy would be inefficient due to the

absence of azoreductase-secreting bacterial species (172).

1.5 Scope of this thesis

Intestinal delivery of nucleic acid drugs is a promising therapeutic strategy against a wide

range of systemic and intestinal diseases (115, 145–149). However, the complex intestinal

environment imposes numerous challenges for the efficient delivery of nucleic acids. Chapter 2 of

this thesis discusses in detail the challenges associated with oral delivery of biomacromolecules,

such as nucleic acids, and reviews clinically tested formulations aimed at overcoming these hurdles.

In this thesis, we seek to identify a simple, potent, and robust nucleic acid delivery platform based

on amphiphilic ON conjugates for the target mRNA and protein silencing in intestinal epithelial

cells (Scheme 1.6). Such ON conjugates, diffusing freely through the mucus layer to the epithelial

cells, could be delivered to the desired location within the GI tract via enteric formulation,

endoscopic tube, or enema.

Previously, it was shown by our lab that the conjugate between a single-stranded AON and

the docosanoyl (DSA) group is capable of inducing the target mRNA silencing in proliferating

prostate cancer cells (112). DSA was significantly more potent than cholesteryl- or docosahexaenyl-

group in causing target silencing (112). In Chapter 3, we explore the versatility of DSA moiety as

a delivery vehicle for two of the most commonly used types of ONs –i.e. single-stranded AONs


25

and double-stranded siRNAs. We present the synthesis and screening of a series of amphiphilic

nucleic acid conjugates for the silencing of a model Bcl-2 mRNA. The silencing kinetics,

cytotoxicity, and contribution of sedimentation to the uptake of DSA-ON conjugates are shown.

In Chapter 4, we evaluate the developed single-molecule-based ON delivery system in conditions

mimicking the intestinal environment (in the presence of pancreatin or fat-containing media) and

using differentiated intestinal cell monolayers for transfection. Lastly, in Chapter 5 we present the

main findings of the thesis and discuss the directions for the future development of amphiphilic

nucleic acid conjugates as an intestinal delivery platform.

Scheme 1.6. Amphiphilic ON delivery strategy to the intestinal epithelium. Upon arrival to the

desired location within the GI tract via enteric formulation, enema, or endoscopic tube, the

amphiphilic ON conjugate will be taken up by the epithelial cells leading to the target gene silencing.

27

2 Oral delivery of biomacromolecules*

2.1 Introduction

The first attempt to deliver insulin orally in humans was undertaken as early as 1922, only

one year after the discovery of insulin by Drs. Frederick Banting and Charles Best (173), when

increasing doses of insulin were given orally to a single diabetes patient. The results were negative,

and already in this first study, the critical challenges of oral protein delivery became apparent: poor

and variable absorption, and low efficacy compared with subcutaneous injection. Although the

interest and efforts in the oral delivery of biomacromolecules have intensified over the past two

decades, safely and effectively delivering high-molecular-weight substrates via the oral route

remains highly challenging for formulation scientists (174, 175).

The gastrointestinal (GI) tract is a hostile environment for biomacromolecules because it is

evolutionarily optimized to break down nutrients and deactivate pathogens. The highly acidic pH

in the stomach results in the protonation of proteins and their unfolding, which exposes more

motifs that are recognized by protein-degrading enzymes (176). The enzymes in the stomach

(pepsin) and small intestine (e.g., chymotrypsin, amino- and carboxypeptidases, RNases and

DNases) cleave proteins and nucleic acids into smaller fragments and single units (176). In the

colon, enzymatic fermentation processes further degrade biomacromolecules (176). Because

therapeutically active biomacromolecules are equally affected by these processes, the fraction

surviving these degradation processes is generally low and variable, especially in the presence of

food (177). The macromolecular drug needs to overcome multiple barriers designed to prevent the

entry of dietary and bacterial antigens in order to reach the systemic compartment. To access the

epithelial cell layer, the biomacromolecule firstly needs to diffuse through the mucus layer covering

the intestinal epithelium (177). The latter is another important barrier, as the tight junctions which

* This chapter has been published: E. Moroz, S. Matoori, J.-C. Leroux (2016) Oral delivery of macromolecular drugs: where we are after almost 100 years of attempts. Advanced Drug Delivery Reviews, 101, 108–121. E.M. and S.M. contributed equally to this manuscript.

CHAPTER 2: ORAL DELIVERY OF BIOMACROMOLECULES

28

seal the epithelial cells restrict the paracellular transport (i.e., the passage between cells) to small

molecules and ions (<600 Da) (178). The transcytotic pathway (i.e., the passage across the cell in

an endocytotic vesicle) is mediated by luminally expressed endocytotic receptors (e.g., vitamin B12

receptor, transferrin receptor), and therefore necessitates conjugation to the respective ligands in

order to be exploited in drug delivery (179). Another access point to the systemic compartment is

the phagocytotic M cells of Peyer's patches which sample luminal antigens and can take up

particular substrates in the low micrometer range (180). However, the proportion of M cells in the

gut epithelium is small and varies greatly between species, which complicates predictions of

absorption in humans based on animal data (144).

Not surprisingly, only six biomacromolecules have been approved by the Food and Drug

Administration (FDA) for oral delivery: two locally and two systemically delivered peptides, one

locally delivered non-peptidic macrocycle, and one locally delivered protein mixture. However,

several orally applied formulations of proteins, peptides, and nucleic acids are currently under

clinical evaluation. Often, these formulations contain at least one of the following excipients

(Figure 2.1): an enteric coating and/or protease inhibitors to prevent drug degradation and

permeation enhancers to enable paracellular transport of macromolecules (139, 181).

Mechanistically, absorption enhancement can be achieved by mechanically disrupting tight

junctions or the plasma membrane, lowering mucus viscosity, and modulating tight junction-

regulating signaling pathways (174). Additional strategies for the oral delivery of

biomacromolecules under clinical development include buccal delivery, utilizing carrier-mediated

transcytosis, and local delivery to GI targets.

The overwhelming majority of currently approved oral drugs and clinical candidates exhibit

a molecular weight of <1000 Da (182). Above this threshold, low bioavailability, inter- and

intraindividual variability, food effects, and long-term safety concerns of bioavailability-enhancing

excipients remain important challenges of oral delivery despite clear advances in knowledge after

nearly 90 years of trial and error. In this review, we address orally applied biomacromolecular

therapeutics (>1000 Da) already marketed or under clinical investigation for local or systemic

delivery with an emphasis on the drug formulations and the biopharmaceutical aspects. The oral

delivery of vaccines will not be covered in this manuscript, and the readers are referred to other

recent reviews for more information on this topic (183–186).


29

Figure 2.1. Strategies for oral delivery of macromolecular drugs that act on local or systemic

targets. In buccal or sublingual delivery, the drug targets the buccal or sublingual mucosa, which

avoids degradation pathways in the GI tract. Often, permeation enhancement is necessary to cross

the multi-layered buccal epithelium. Mucoadhesive bacteria that secrete the desired protein in situ

are a novel means of achieving sustained release in the oral cavity. To enhance stability against

degrading enzymes, macromolecular drugs can be chemically modified by polymer conjugation

(proteins), backbone and base modifications (nucleic acids), and cyclization as well as by

introducing D-amino acids (peptides). Sacrificial proteins, protease inhibitors, and enteric coatings

can also be included in the formulation to further improve GI resistance. To achieve meaningful

systemic exposure, absorption enhancement by tight junction-disrupting excipients is often needed.

A novel approach is the in situ production and secretion of therapeutic biomacromolecules by

genetically modified bacteria.

2.2 Local delivery

Macromolecular drugs that act on GI targets increasingly move into focus because local

delivery avoids the challenges of reaching the systemic compartments. Advantages of locally

delivering high-molecular-weight drugs include fewer restrictions regarding drug size and a

potentially more favorable safety profile due to minimal systemic exposure, reduced

immunogenicity, and the absence of permeation-enhancing excipients (187, 188). To conserve the

therapeutic activity in the GI tract, several formulation strategies have been employed, such as

enteric/colon-targeting capsules that protect against the harsh GI environment, supplementation

with sacrificial proteins that compete for degradation, and hindering enzymatic access using

polymer conjugation or protective antibodies which bind to known cleavage epitopes (176, 187,

189). The ailments that are targeted by locally acting macromolecules include inflammatory diseases


30

(Crohn's disease, ulcerative colitis), metabolic disorders (e.g., exocrine pancreas insufficiency),

constipation, and infections (Table 2.1) (176, 187).

Table 2.1. Macromolecular treatments for local GI therapy in clinical development (as of January

2016, source: Thomson Reuters Integrity®, ClinicalTrials.gov, and company press releases).

Type Generic

name, Mw† Modification/Formulation Indication Clinical phase

Pep

tide

Vancomycin (Vancocin®),

1449 Da

Cyclic structure, modified amino acid residues; gelatin capsule

Clostridium difficile-associated diarrhea

(CDAD) and staphylococcal enterocolitis

Marketed

Fidaxomycin (Dificid®), 1058 Da

Non-peptidic macrocycle; tablet CDAD Marketed

Surotomycin,

1681 Da Cyclic structure CDAD

Phase III (NCT01597505, NCT01598311)

Ramoplanin, 2554 Da

Cyclic structure CDAD Phase IIb (N.A.‡)

LFF-571, 1367 Da

Cyclic structure, thiazolyl peptide CDAD Phase II

(NCT01232595)

NVB302, 2115 Da

Cyclic structure, thioether bonds CDAD Phase I (N.A.)

Linaclotide (Linzess®), 1525 Da

Cyclic structure; gelatin capsule

Chronic idiopathic constipation (CIC) and irritable bowel

syndrome with constipation (IBS-C)

Marketed

Plecanatide, 1682 Da

Cyclic structure CIC and IBS-C

CIC phase III completed in 2015

(NCT01919697)

IBS-C phase III

(NCT02493452)

Dolcanatide (SP-333), 1682 Da

Cyclic structure, two D-amino acid substitutions

Opioid-induced constipation (OIC)

and ulcerative colitis (UC)

OIC phase II completed in 2014

(NCT01983306)

UC phase Ib (N.A.)

Anti

bo

dy

AVX-470 (Avaximab™-TNF), broad

Polyclonal antibody;

enteric capsule Pediatric UC

Phase II ongoing

(N.A.)

AG014, 47.8 kDa

Lyophilized Lactococcus lactis secreting certolizumab; enteric

capsule

Inflammatory bowel disease

Phase Ia completed in 2014

(N.A.)

† Mw: molecular weight ‡ N.A: unknown or non-existent NCT number


31

Table 2.1. (Continued) Macromolecular treatments for local GI therapy in clinical development

(as of January 2016, source: Thomson Reuters Integrity®, ClinicalTrials.gov, and company press

releases).

Type Generic

name, Mw§ Modification/Formulation Indication Clinical phase

En

zym

e Creon® (porcine

pancreatin) Enteric capsule

Exocrine pancreatic insufficiency

Marketed

Pro

tein

-sec

reti

ng

bac

teri

a

AG013, 6.5 kDa

Oral rinsing solution containing 2.0 × 1011 colony-forming units (CFU)/15 mL of Lactococcus lactis secreting human trefoil

factor 1

Chemotherapy- or radiation-induced

oral mucositis

Phase Ib completed in 2012

(NCT00938080)

AG011, 18 kDa

Lactococcus lactis secreting anti-inflammatory cytokine IL-10; enteric capsule in combination

with enema

UC Phase IIa completed in

2009, failed (NCT00729872)

Nucl

eic

acid

Mongersen (GED-0301),

6952 Da

Phosphorothioate AON; enteric coating

Moderate to severe Crohn's disease

Phase II completed

Phase I (NCT02367183)

Alicaforsen, 6368 Da

Phosphorothioate AON; nightly enema with hydroxypropyl

methylcellulose UC, pouchitis

Phase III (NCT02525523)

CEQ508

Suspension of genetically modified E. Coli expressing

invasin, listeriolysin, and short hairpin RNA targeting β-catenin

Familial adenomatous

polyposis

Phase I/II ongoing since 2011

(N.A.)

2.2.1 Peptides

Vancomycin

Isolated in 1953 from a soil sample in the jungle of the island Borneo, the antibiotic

vancomycin is produced by the bacterium Amycolatopsis orientalis (190). Vancomycin is a glycosylated

tricyclic heptapeptide (1449 Da) which contains modified amino acid residues (e.g., chlorinated

tyrosine) (190–192). Due to its highly hydrophilic (log P -3.1) and large cyclic structure, vancomycin

is only marginally absorbed and metabolized in the GI tract (193, 194). Intravenously applied

§ Mw: molecular weight


32

vancomycin was initially approved by the FDA to treat penicillin-resistant bacterial infections in

1958, and it is still indicated for severe infections caused by susceptible strains of methicillin-

resistant staphylococci, Clostridium difficile-associated diarrhea, and staphylococcal enterocolitis and

for patients who are allergic to beta-lactam antibiotics. Vancomycin is formulated as gelatin

capsules containing 125 or 250 mg of active compound (package leaflet). In 2011, a non-peptidic

macrocyclic macrolide antibiotic fidaxomicin (1058 Da, Dificid®, Cubist Pharmaceuticals) with

minimal systemic absorption and GI metabolism was approved for the treatment of C. difficile

infections. Fidaxomicin showed lower relapse rates compared with vancomycin. In view of its

much higher costs, fidaxomicin treatment may be most suitable for patients who are at highest risk

of relapse (195). Further cyclic peptidic compounds in clinical trials for the local treatment of C.

difficile infections include the lipopeptide surotomycin (1681 Da, phase III, Cubist

Pharmaceuticals/Merck), the lipoglycodepsipeptide ramoplanin (2554 Da, NTI-851, phase IIb,

Nanotherapeutics), the thiazolyl peptide LFF-571 (1367 Da, phase II but currently removed from

pipeline, Novartis), and the lantibiotic NVB302 (2115 Da, phase I, Novacta Biosystems Limited)

(196, 197).

Linaclotide

Linaclotide (1525 Da) is a truncated derivative of Escherichia coli heat-stable (ST) enterotoxin

that consists of 14 amino acid residues and three disulfide bonds (198). It acts as a guanylyl cyclase

C (GC C) agonist locally in the small intestine (Figure 2.2) (198–200). In 2012, it received FDA

approval (Linzess®, Forest Labs LLC) for the treatment of chronic idiopathic constipation and

irritable bowel syndrome with constipation. After oral administration, linaclotide is minimally

absorbed, and its plasma concentrations are below the limit of quantification (201). Because

linaclotide is stable in the stomach, it is formulated as hard gelatin capsules containing 0.145 or

0.290 mg of the active compound (package leaflet). However, it is cleaved in the small intestine to

a 13-amino acid active metabolite (201), which is eventually degraded in the intestine (202).


33

Figure 2.2. Schematic representation of the mechanisms of action, primary structure, and diarrhea

incidence of the GC C agonists linaclotide, plecanatide, and dolcanatide. The sequences of these

peptides are based on the physiological agonist uroguanylin and the structurally similar heat-stable

Escherichia coli enterotoxin (ST) but include sequence adaptations such as unnatural D-amino acids

(dN = D-asparagine, dL = D-leucine). Lines above and below the peptide sequences denote the

disulfide bonds. Upon binding of an activating ligand, GC C converts guanosine triphosphate

(GTP) to cyclic guanosine monophosphate (cGMP), increasing the intracellular cGMP

concentration and activating the cystic fibrosis trans-membrane conductance regulator (CFTR).

The resultant efflux of chloride ions out of the epithelial cell leads to a water influx into the

intestinal lumen, which normalizes bowel movements. Extracellular cGMP further inhibits

intestinal nociceptors. Thus, GC C agonists improve constipation and alleviate chronic abdominal

pain in patients suffering from inflammatory bowel syndrome with constipation. The incidence of

diarrhea, a common treatment-emergent adverse event associated with excessive treatment

response, is also indicated for the three peptides (dose and regimen in parentheses). The

information was collected from (199–204) and the website of Synergy Pharmaceuticals.

Plecanatide and derivatives

The GC C agonist plecanatide (1682 Da, Synergy Pharmaceuticals) is a synthetic analog of

uroguanylin, a naturally occurring GI regulator of GC C signaling, with one amino acid substitution

that results in stronger receptor binding (203). It is a bicyclic peptide consisting of 16 amino acid

residues with two disulfide bonds (203). Plecanatide completed the clinical phase III for the

treatment of chronic idiopathic constipation, and it is currently being evaluated in phase III for

irritable bowel syndrome with constipation. It showed a statistically significant increase in the

number of complete spontaneous bowel movements per week compared with placebo

(approximately 20% of durable responders at a dose of 3 mg per day vs. 12% in the placebo group).


34

In comparison to linaclotide, plecanatide showed reduced diarrhea incidence (10% vs. 20%) (204).

In addition to plecanatide, Synergy Pharmaceuticals is developing a more stable analog, dolcanatide

(SP-333), to treat opioid-induced constipation. Dolcanatide has two D-amino acid substitutions at

C- and N-terminus in order to improve stability in simulated gastric and intestinal fluids

(www.synergypharma.com). Dolcanatide has completed a randomized, double-blind, placebo-

controlled phase II clinical trial in patients with constipation who take opioid analgesics for chronic

pain (NCT01983306). Despite the increased stability in the intestinal environment, the dose of

dolcanatide needed to achieve a therapeutic effect was comparable with that for plecanatide.

Furthermore, the dolcanatide-mediated activation of GC C ameliorated inflammation in a colitis

mouse model, promoting its development in the treatment of ulcerative colitis (205).

2.2.2 Antibodies

Antibody delivery

The vast majority of clinically tested antibodies with GI luminal targets inactivate undesired

molecules, such as bacterial toxins, cytokines, viruses, and virulence factors (187). In the treatment

of IBDs, capturing TNF in the intestinal lumen is a promising alternative to systemic treatment

with anti-TNF antibodies (e.g., adalimumab, infliximab and certolizumab pegol) with regard to

systemic immunosuppression, development of neutralizing antibodies, and needle-free

administration (206–208). AVX-470 (Avaximab™-TNF, Avaxia Biologics) is an orally

administered polyclonal antibody which targets luminal TNF in the intestine (209). It is produced

by purifying total antibodies from the milk of dairy cows that are immunized with human TNF,

which results in approximately 0.3-0.9% (m/m) TNF-specific and mainly IgG1 subtype antibodies

(www.avaxiabiologics.com). Providing early immunity to neonates, high amounts of colostrum

antibodies seem to be partially resistant to GI digestion in adults with fully developed digestive

systems (210–212). AVX-470 is developed as an enteric-coated capsule for treating pediatric

ulcerative colitis and is currently in phase II of clinical trials. According to the company's website,

a phase Ib trial (NCT01759056) in 36 ulcerative colitis patients showed that the treatment resulted

in consistent encouraging trends across multiple disease parameters (colonic TNF levels,

endoscopic index of severity, serum C-reactive protein levels) after four weeks, and no allergic

reaction or human anti-bovine antibodies in serum were observed. Bovine immunoglobulin was


35

detected in stools and displayed TNF-binding activity, suggesting that the large excess of decoy

antibodies in the formulation (>99%) protected a fraction of the anti-TNF antibodies.

Genetically modified antibody-secreting bacteria

Intrexon's (formerly ActoGeniX) approach for luminal antibody therapy consists of

delivering attenuated bacteria to the GI tract, where the antibody is secreted in situ. Food-grade

Lactococcus lactis was genetically modified to secrete certolizumab, an anti-TNF Fab fragment (47.8

kDa). In an open-label clinical trial in healthy volunteers, lyophilized bacteria administered in

enteric capsules were recovered in both the small and large intestine by endoscopic sampling, and

the antibody was secreted by living bacteria in the colon lumen (www.dna.com). The treatment was

safe and well-tolerated. In addition, preclinical studies demonstrated the treatment's efficacy in

multiple colitis models and showed that Lactococcus lactis adhere especially to the inflamed mucosa,

which increases the concentration of the antibody in the close proximity of target cells (213).

However, questions remain regarding the concentration variability and the intestinal stability of the

secreted antibody.

2.2.3 Oral enzyme therapy

Enzyme replacement therapy via the oral route is an important therapeutic option in

metabolic disorders in which a certain enzymatic action in the GI tract is absent. Numerous oral

enzyme products are on the market for a variety of disorders such as exocrine pancreatic

insufficiency due to pancreatitis, cystic fibrosis and other conditions, and lactose, fructose, sucrose

and histamine intolerance (e.g., Creon®, Lacteeze®, Sucraid®, DAOSiN®) (176). Whereas the only

FDA-approved enzymatic drug is pancreatin (a mixture of bovine or porcine pancreatic amylase,

proteases, and lipases), the other oral enzyme products are generally under the legal framework of

dietary supplements, which usually do not require thorough efficacy studies.

Because these therapeutic enzymes are inactivated in the stomach by low pH and pepsin, one

or a combination of stabilization strategies are generally needed. Creon® (Abbvie), for instance,

consists of pancreatin formulated in enteric-coated capsules, whose dissolution is triggered by the

pH increase in the small intestine. An uncoated formulation of pancreatin (Viokace®, Aptalis


36

Pharma) which is used to treat exocrine pancreatic insufficiency is co-administered with proton

pump inhibitors to decrease gastric degradation. As an exception, sacrosidase and the acid lactase

(tilactase) produced by Aspergillus oryzae are intrinsically stable and active in acidic conditions (176),

and they do not require sophisticated delivery systems. However, average residence time of lactase

in the stomach might be insufficient for complete digestion of high amounts of lactose (214).

Therefore, a formulation of a lactase, which is active in neutral to basic pH of the small intestine,

was recently developed as a capsule containing enteric-coated pellets (Lactosolv®, Sciotec

Diagnostic Technologies) (214). The same approach has been successfully applied for other

enzyme deficiencies (Xylosolv® for fructose and DAOSiN® for histamine intolerance). As an

alternative, the administration of probiotics that secrete β-galactosidase, which is capable of lactose

hydrolysis, has been proposed; however, different clinical trials in patients with lactose intolerance

gave inconsistent efficacy results (215). Oral formulations of polymer-conjugated enzymes and

enzymes in enteric-coated capsules are under preclinical investigation for other pathologies such

as celiac disease and phenylketonuria (176, 216–220).

2.2.4 Cytoprotective/anti-inflammatory proteins

As a consequence of chemotherapy and radiation, cancer patients often develop a

complication called oral mucositis in which the mouth's mucosal lining is destroyed and ulcers

form. AG013 (Intrexon Corporation) is a bacteria-based system which delivers a mucosa-healing

protein for the treatment of oral mucositis. The protein, called human trefoil factor 1, is involved

in mucosal healing and tissue protection via multiple mechanisms, including inhibiting apoptosis

during cell migration and stabilizing the protective mucus layer (221). The formulation consists of

an oral rinsing suspension containing attenuated (i.e., unable to replicate due to thymidylate

synthase deficiency (221)), genetically engineered Lactococcus lactis which adhere to the buccal

mucosa and secrete the mucosa-healing factor locally for up to 24 h after administration (222). A

phase Ib study in head and neck cancer patients undergoing chemotherapy produced positive

results, showing a 35% reduction in the duration of oral ulcers (221).

A similar in situ system based on Lactococcus lactis which secretes the anti-inflammatory

cytokine interleukin-10 (AG011, Intrexon) was proposed for ulcerative colitis. However, a

combination of enema and enteric capsules containing interleukin-10-secreting Lactococcus lactis did

not result in mucosal healing in a phase IIa, double-blind, placebo-controlled study with 60


37

ulcerative colitis patients (223). Recently, another genetically modified strain of Lactococcus lactis was

engineered to deliver the endogenous serine protease inhibitor elafin. The expression of this anti-

inflammatory peptide is lowered in patients with active celiac disease, and elafin treatment was

effective in decreasing inflammation and intestinal permeability in a mouse model of celiac disease

(224).

2.2.5 Nucleic acids

AONs are short, single-stranded, synthetic sequences of nucleotides which can hybridize

with complementary mRNA and consequently decrease the expression of the encoded protein

(103). Several GI tract pathologies hold attractive targets for local nucleic acid therapy (e.g., IBD or

familial adenomatous polyposis) (149, 225). Because nucleic acids are readily degraded by the

RNases and DNases in the GI tract and depurinated in the acidic stomach environment, they

require stability-enhancing chemical modifications of the backbone or bases (151). Various

modifications of the ribose ring (e.g., 2′-O-(2-methoxyethyl) or 2′-F-ANA) not only exhibit

enhanced nuclease resistance but also show higher affinity to complementary mRNAs and thus

improved knockdown efficacy (25, 226).

Mongersen (GED-0301, Celgene) is a 21-mer phosphorothioate AON targeting the mRNA

of the Smad7 protein, which is overexpressed in IBD mucosal tissues and leads to suppression of

anti-inflammatory TGF-β signaling (152). Mongersen acts on epithelial and lamina propria cells,

and its bioavailability after oral administration is minimal (227). An enteric-coated oral formulation

of mongersen was evaluated in 166 patients with active Crohn's disease in a phase II trial (152).

Participants received 10, 40, or 160 mg of mongersen once daily for 2 weeks. In the mongersen

groups that received 40 and 160 mg, significantly more patients reached clinical remission, the

primary end point, after four weeks of treatment (55% and 65%, respectively), compared with 10%

in the placebo group. According to the company, follow-up data of this trial (e.g., on mucosal

healing by endoscopy) will be presented in the future.

Alicaforsen (Atlantic Healthcare) is a 20-mer phosphorothioate AON targeting the mRNA

of intercellular adhesion molecule 1 (ICAM-1), which recruits immune cells to the site of

inflammation and is overexpressed in the gut epithelium of IBD patients (148). Although a four-

week treatment with injectable alicaforsen fell short of inducing clinical remission in two phase II


38

trials (NCT00048113; NCT00048295), a six-week regimen of nightly 60 mL alicaforsen enemas

containing hydroxypropyl methylcellulose as a thickening agent was beneficial in treating moderate

to severe ulcerative colitis and pouchitis (i.e., an ileal pouch inflammation in colectomy patients)

(148, 150). According to the company's website (www.atlantichc.com), alicaforsen enema is

undergoing phase III clinical trials for the treatment of moderate to severe ulcerative colitis and

pouchitis.

Because the cellular absorption of naked ONs is limited, Marina Biotech is developing a

novel strategy to enhance ON cell uptake using a transkingdom RNA interference platform

(tkRNAi) based on genetically modified Escherichia coli. The most advanced tkRNAi candidate,

CEQ508, is designed to express and deliver short hairpin RNA silencing β-catenin, which is

overexpressed in gut epithelial cells of familial adenomatous polyposis patients and is responsible

for uncontrolled cell growth (147). The genome of CEQ508 has been altered to allow for whole

bacterium endocytosis by the epithelial cells and, ultimately, β-catenin silencing (56). In 2010,

CEQ508 received orphan drug status from the FDA, and a phase Ib clinical study was initiated;

however, due to financial problems, the development program was halted. Recently, the FDA

granted Fast Track designation to CEQ508, and clinical trials are expected to begin again

(www.marinabio.com). Other approaches for nucleic acid delivery to the mucosa are under

investigation, including nano/microparticles that target mucosal macrophages and colon-

activatable PNAs (108, 225).

2.3 Systemic delivery

Delivering macromolecules systemically after oral intake is highly challenging because, in

addition to the stability issues due to GI degradation, the limited permeability of the GI mucosa

generally leads to low and erratic absorption. Therefore, inter- and intraindividual differences in

pharmacokinetic parameters are generally high, especially in the presence of food. This chapter

provides an overview of oral macromolecular drugs that reached clinical trials or received market

approval (Table 2.2).


39

Table 2.2. Macromolecular treatments for oral administration in clinical development (as of

January 2016, source: Thomson Reuters Integrity®, ClinicalTrials.gov, and company press releases).

Type Generic name,

Mw** Formulation Indication Clinical phase

Pep

tid

e

Octeotride (Mycapssa™),

1019 Da

Oily suspension containing sodium

caprate Acromegaly

New Drug Application

(NDA) under review

Desmopressin (DDAVP®),

1128 Da Tablet, lyophilisate

Central diabetes insipidus, nocturnal

enuresis Approved

Cyclosporine (Neoral®), 1202

Da

Self-emulsifying system

Organ transplant rejection,

autoimmune diseases Approved

Acyline

(MER-104),

1533 Da

GIPET tablet (sodium caprate)

Prostate cancer, male oral contraception

Phase I/II completed in

2008 (NCT00603187)

Calcitonin (TBRIA™),

3432 Da

Citric acid-containing enteric coated tablet

Postmenopausal osteoporosis

NDA under review

Semaglutide (OG217SC),

4114 Da

Eligen®-based tablet (SNAC)

Diabetes Type II (T2D)

Phase III (N.A.††)

Pro

tein

Human insulin (Capsulin™),

5800 Da

Enteric coated tablet with hydrophilic aromatic alcohol

Diabetes Types I (T1D) and T2D

Phase IIb (N.A.)

Human insulin (ORMD-0801)

Enteric coated capsule containing several

protease inhibitors, permeation enhancers,

lipoidal carriers

T1D, T2D Phase II

(NCT02496000)

Long-acting insulin analogue

(OI338GT) GIPET tablet T1D, T2D

Phase I/II (NCT02470039)

Insulin-containing hepatocyte-

directed vesicles

Liposomes decorated with biotin

T1D, T2D Phase III

(NCT00814294)

Insulin-triethylene

glycol conjugate

(IN-105)

Tablet T1D, T2D Phase III (N.A.)

** Mw: molecular weight †† N.A: unknown or non-existent NCT number


40

Table 2.2. (Continued) Macromolecular treatments for oral administration in clinical development

(as of January 2016, source: Thomson Reuters Integrity®, ClinicalTrials.gov, and company press

releases).

Type Generic name,

Mw‡‡ Formulation Indication Clinical phase

Human insulin (Oral-lyn™)

Spray for buccal delivery

T1D, T2D Updating IND

Monarsen (EN 101, BL-

7040), 6139 Da

OMe modifications at the three terminal

bases at 3' end; aqueous solution

Myasthenia gravis (MG), ulcerative

colitis (UC)

MG discontinued

UC phase II completed in

2013 (NCT01506362)

Nucl

eic

acid

ISIS 104838 sodium salt,

7701 Da

MOE modifications at 5 terminal bases of both ends; enteric-

coated capsule containing sodium

caprate

Rheumatoid arthritis, Crohn's disease

Discontinued

Mipomersen sodium

(ISIS 301012), 7595 Da

MOE modifications at 5 terminal bases of both ends; enteric-

coated capsule containing sodium

caprate

Homozygous familial hypercholesterolemia

Discontinued

2.3.1 Peptides

Octreotide

Octreotide is a synthetic analogue of the endogenous hormone somatostatin, a regulator of

the neuroendocrine system (228). It was approved by the FDA in 1998 as a subcutaneous or

intramuscular treatment for acromegaly (usually a result of growth hormone-secreting tumors),

carcinoid tumors and vasoactive intestinal peptide-producing tumors. In comparison with its

mother compound, the cyclic octapeptide possesses a lower molecular weight (1019 vs. 1638 Da),

a higher logP (-1.69 vs. -8.50), and two D-amino acids. Additionally, the terminal carboxyl group is

reduced to an alcohol (229). The higher stability of octreotide over somatostatin in pepsin-

‡‡ Mw: molecular weight


41

containing simulated gastric fluid results in part from the inclusion of a D-amino acid in the pepsin-

vulnerable amide bond of octreotide, rendering octreotide non-recognizable by this enzyme (228).

Despite these structural improvements, orally administered aqueous octreotide showed an

unfavorable pharmacokinetic profile (low bioavailability, highly variable plasma concentrations) in

pigs and humans (230, 231). To improve the pharmacokinetics upon oral administration, octreotide

(Mycapssa™, formerly Octreolin™, Chiasma) was formulated as an oily suspension with the

permeation enhancer sodium caprylate, which transiently increases paracellular permeability for

approximately 1.5 h (232). The absorption of octreotide in the oily suspension was dose-dependent

and independent of the intestinal region (232). The passage through the stomach, however, led to

low bioavailability such that in the subsequent clinical studies, the suspension was formulated in an

enteric-coated capsule (232). As reported in a publication of four phase I and phase II studies, a

20-mg dose of Octreolin™ in fasting healthy volunteers led to a similar pharmacokinetic profile as

a subcutaneous injection of 0.1 mg (apart from a lag phase of 20 min) (233). A high-fat meal

decreased the bioavailability of oral octreotide by 90%, probably due to the premature dissolution

of the enteric-coated capsule caused by increased stomach pH (233). Furthermore, the presence of

food in the intestine may slow down diffusion to the intestinal mucosa, hindering octreotide

absorption (233). A baseline-controlled phase III clinical trial was conducted in acromegaly patients

who had previously received somatostatin receptor ligand injections and were switched to a twice-

daily regimen of oral octreotide. The intervention met its primary end point in that it resulted in

controlled levels of insulin-like growth factor-1 and growth hormone in 65% of the subjects after

seven months of the treatment, compared with 89% at baseline (234). In 6.5% of the subjects,

adverse GI events such as nausea, diarrhea and abdominal pain led to early termination of the study

(234). Hoffmann-La Roche bought the rights for development and commercialization of

Octreolin™ but terminated the development in 2014 and transitioned the drug back to Chiasma,

which has filed a New Drug Application (NDA) in 2015.

Desmopressin

The cyclic nonapeptide desmopressin (1-desamino-8-D-arginine vasopressin, 1128 Da) is a

synthetic derivative of the peptide hormone vasopressin (anti-diuretic hormone, ADH) (228, 235).

In comparison to the endogenous vasopressin, desmopressin exhibits desirable features such as a

significantly longer antidiuretic effect and strongly diminished vasopressor action (235).

Desmopressin was approved by the FDA in 1978 for the treatment of post-hypophysectomy

polyuria/polydipsia, central diabetes insipidus, and nocturnal enuresis. The oral formulation


42

(DDAVP®, Ferring Pharmaceuticals) was approved in 1992 by the FDA for the latter two

indications. A variety of formulations of desmopressin are currently marketed for parenteral, nasal,

and oral applications. Desmopressin exhibits exceptionally high stability in human gastric fluid,

presumably due to its rigid cyclic structure and the absence of pepsin cleavage motifs (228). In

contrast, the stability of desmopressin in intestinal fluids is limited but enhanced compared with

that of vasopressin (228). This finding may be due to the replacement of the L-arginine at position

8 with its D-enantiomer in desmopressin, which seems to hinder the recognition by trypsin (228).

Because of its hydrophilicity (logP -1.95), the permeability of desmopressin across a Caco-2

monolayer is low and concentration-dependent, which suggests passive permeation (236). The oral

bioavailability of a standard-dose desmopressin acetate tablet (0.2 mg) is only approximately 0.1%

(237). In 2005, a lyophilisate tablet for sublingual administration of desmopressin was approved

(DDAVP® Melt, Ferring Pharmaceuticals). Despite its circumventing the harsh conditions of the

GI tract, the bioavailability of this formulation is only approximately 0.25% (238). With regard to

the broad therapeutic window of desmopressin, the antidiuretic action does not seem to be

influenced by the low and variable oral bioavailability. Indeed, food intake did not change the

efficacy (urine production and osmolarity) or safety (plasma sodium concentrations) of

desmopressin tablets despite significantly altering the pharmacokinetic parameters (239).

Furthermore, the incidence of water intoxication-induced hyponatremia, a serious adverse drug

reaction related to desmopressin overdose, was lower in oral than in nasal application, suggesting

a low risk of this adverse drug reaction due to erratic absorption (240).

Cyclosporine

Originally isolated in 1969 from the fungus Tolypocladium inflatum in a Norwegian soil sample,

cyclosporine (Cyclosporine A) has become a widely used immune-suppressive peptide approved

by the FDA for the prophylaxis of organ transplant rejection and the second-line treatment of the

autoimmune diseases rheumathoid arthritis and psoriasis. Cyclosporine is a cyclic peptide

composed of 11 amino acid residues (1202 Da). The exceptionally high stability in gastric and

intestinal fluids is most probably related to cyclosporine's high structural rigidity and low potential

for H-bonding-mediated interaction with peptidases (228). The high hydrophobicity (logP 3.6) of

cyclosporine enables its transcellular passage by passive diffusion, but it also makes it susceptible

to p-glycoprotein-mediated expulsion on the apical side of enterocytes (241). Interestingly,

structural motifs such as the β-turns of cyclosporine seem to be important features in cell

permeability (242). The low solubility in aqueous solutions due to the high hydrophobicity led to


43

the development of a self-emulsifying formulation in soft gelatin capsules or as an oral solution,

approved by the FDA in 1995 (Neoral®, Novartis) (243). Compared with the originally developed

oil-in-water emulsion (SandImmun®, Novartis) this formulation is characterized by lower intra-

and interindividual variability, a more linear dose-exposure relationship, and a markedly decreased

influence of food fat content (243–245). Nonetheless, the narrow therapeutic window generally

necessitates monitoring cyclosporine plasma concentrations to determine the optimal dose.

Acyline

The linear decapeptide acyline (MER 104, Merrion Pharmaceuticals, 1533 Da) competitively

inhibits the receptor binding of gonadotropin-releasing hormone, thereby inhibiting the secretion

of luteinizing and follicle-stimulating hormone and interfering with the production of downstream

sex hormones (e.g., testosterone, estrogen). It is a candidate for treating hormone-dependent

conditions (breast and prostate cancer, endometriosis) and male oral contraception. Acyline

contains a variety of modifications (e.g., five D-amino acids, two acylated 4-aminophenylalanines)

enhancing its stability and receptor-binding properties (246). It is formulated with the

Gastrointestinal Permeation Enhancement Technology (GIPET, Merrion) as an oral dosage form.

GIPET is composed of an enteric-coated matrix tablet that contains the permeation enhancer

sodium caprate (181, 247). In a phase I clinical trial, acyline tablets (10, 20, and 40 mg) were

administered in eight healthy men after overnight fasting and under continued fasting for 4 h (248).

High variability was observed among the subjects, and no dose effect on pharmacokinetic

parameters (maximum concentration and time of maximum concentration, half-life, and area under

the curve, AUC) was observed (248). After 12 h, all three doses suppressed luteinizing and follicle-

stimulating hormone serum concentrations by approximately 70% and 30%, respectively (248).

Suppression of the downstream hormone testosterone was also observed, and the effects of the

lowest and highest doses could be discriminated (248). One study participant reported a GI-related

adverse event (nausea and vomiting) (248). While acyline is still listed as being under active

development by the company's website, no trials have been published since 2009.

Calcitonin

Calcitonin is a natural peptide hormone regulator of calcium homeostasis (249). It was

approved by the FDA in 1978 and is indicated for the treatment of hypercalcemia and symptomatic

Paget's disease and as a second-line treatment of postmenopausal osteoporosis. Marketed

calcitonin products are administered via the parenteral and nasal routes and contain salmon


44

calcitonin as the active ingredient because this peptide exhibits forty times higher potency

compared with the human version (249). Salmon calcitonin is a 32 amino acid peptide (3432 Da)

with a highly negative logP (-28.49) (228). Due to its size and peptidase-recognizable motifs, it is

rapidly degraded in gastric and small intestinal fluids in vitro, and it only marginally diffuses across

Caco-2 monolayers (228, 250). The bioavailability of salmon calcitonin in a rat model ranged from

0.0011% after intrajejunal to 1% after colonic administration (250). SMC021 (Novartis) is an oral

salmon calcitonin formulation which includes the permeation enhancer N-(5-chlorosalicyloyl)-8-

aminocaprylic acid (5-CNAC, Eligen® Technology, Emisphere Technologies). In two phase I trials,

oral salmon calcitonin tablets were tested in healthy postmenopausal women and patients with

osteoarthritis, and it reached the systemic compartment and suppressed bone resorption markers

(251, 252). However, the amount of water administered with the tablet and the time of

administration with respect to food intake were identified as influencing factors (Figure 2.3) (251,

252). These studies presumably contributed to the decision to terminate SMC021 development.

Another oral calcitonin formulation (TBRIA™, formerly Ostora, Tarsa Therapeutics) that is

currently in clinical development consists of an enteric-coated tablet, which releases its content at

a pH of 5.5 in the duodenum (253). The tablet contains citric acid to decrease the small intestinal

pH and thus diminishes the activity of small intestinal peptidases whose pH optimums are in the

neutral to basic range (253). Despite reports that citrate's calcium chelating properties may increase

paracellular absorption by disrupting tight junction complexes by depleting intracellular calcium,

permeation enhancement was low in Caco-2 monolayers (254). In a phase III trial, oral or nasal

calcitonin or placebo were administered to 565 postmenopausal women who were also receiving

vitamin D and calcium supplements (253). The oral salmon calcitonin formulation given at a dose

6 times higher than the nasal one met the primary endpoint of this clinical trial in that it was superior

to nasal calcitonin and placebo in increasing lumbar spine bone mineral density after 48 weeks of

treatment (253). Adverse events were mainly limited to the GI tract (253). Nausea and dyspepsia

were significantly more common in the group receiving the calcitonin tablet compared to the nasal

formulation which could have been associated with the administration route and the higher dose

in the oral treatment group (253). In a recently reported phase II trial, no food effect on therapeutic

efficacy was observed (255). Following the phase III clinical trial, Tarsa has filed an NDA with the

FDA (under review). The FDA's benefit-risk assessment of this formulation will likely be

influenced by their warning about a higher incidence of cancer in patients who received calcitonin

compared with placebo, even though a causal relationship could not be established (256).


45

Figure 2.3. Influence of water and food on the plasma profile of SMC021 (salmon calcitonin

formulated with the permeation enhancer 5-CNAC). (A) The higher amount of water co-

administered with the tablet resulted in lower calcitonin plasma concentrations (60 min before a

meal). (B) The time span between tablet administration and meal intake further influenced systemic

exposure to calcitonin. (C) In a twice-daily dosing scheme, the morning dose resulted in higher

calcitonin plasma concentrations than the evening dose, potentially due to the fasting before and

after the morning dose in contrast to meals ingested 1.5 h before and 1 h after tablet administration.

Figures adapted from (251, 252) with permission.


46

Semaglutide

Semaglutide (Novo Nordisk, 4114 Da) is a glucagon-like peptide 1 (GLP-1) analogue

consisting of 31 amino acid residues. GLP-1 derivatives are used to treat type 2 diabetes because

they improve glucose control by three mechanisms: stimulating insulin release in a glucose-

dependent manner, suppressing glucagon activity under hyperglycemia, and delaying gastric

emptying leading to slower glucose absorption (257). Semaglutide has more favorable

pharmacokinetics than its rapidly inactivated mother compound owing to several structural

modifications. An amino acid substitution at position 8 (alanine to α-aminoisobutyric acid) impairs

dipeptidyl peptidase-4-mediated degradation (257). Furthermore, the conjugation of C18 fatty

diacid via a spacer to the lysine in position 26 prolongs plasma half-life by increasing binding to

albumin and reducing renal clearance, making semaglutide suitable for once weekly subcutaneous

dosing (257). Oral semaglutide (OG217SC, formerly NN9924, Novo Nordisk) was formulated as

a tablet containing the permeation enhancer N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC,

Eligen® Technology, Emisphere Technologies) (181). In a phase II trial involving 600 type II

diabetes patients, OG217SC tablets were administered once daily (2.5 to 40 mg) for 26 weeks

(www.novonordisk.com). HbA1c improvements were dose-dependent from 0.7 to 1.9%, making

the 40 mg oral dose comparable with the 1 mg subcutaneous dose. Oral semaglutide was well

tolerated and adverse events were largely dose-dependent, transient, and confined to the GI tract.

According to the company's website, Novo Nordisk will initiate phase IIIa development of

OG217SC.

2.3.2 Proteins

Oral insulin

Human insulin consists of 51 amino acids (5800 Da) organized in two chains (A chain 21 aa,

B chain 30 aa) linked with two disulfide bridges (228). Although insulin formulations have only

been approved for subcutaneous and pulmonary administration, their application via the oral route

remains an attractive and vital field of research. In addition to the greater convenience of orally

applying insulin, absorbing insulin from the gut into the portacaval vein and the liver mimics the

pancreatic insulin secretion better than the subcutaneous route, and promises to decrease


47

peripheral hyperinsulinemia (258). Multiple insulin formulations for oral administration have

entered clinical trials, ranging from enteric-coated capsules and formulations with permeability

enhancers, liposomes, and polymer conjugates to buccal sprays (259). Referring the readers to three

comprehensive reviews (258–260) on oral insulin delivery, we will present here the most clinically

advanced insulin-containing drug delivery systems for oral or buccal administration with published

results from clinical trials.

Several oral insulin delivery systems are based on enteric-coated capsules with bioavailability-

improving adjuvants. Unfortunately, the exact compositions of the formulations were generally not

communicated (258). Capsulin™ (Diabetology) is formulated as an enteric-coated capsule that

contains insulin, a non-disclosed “dissolution aid”, and an aromatic alcohol (e.g., phenoxyethanol,

benzyl alcohol and phenyl alcohol, according to the patent (261)) for absorption enhancement

(262). In a randomized controlled crossover phase IIa study, Capsulin™ was administered 60 min

before breakfast and the evening meal in sixteen patients with type II diabetes (262). Although

significant hypoglycemic action of Capsulin™ was shown, postprandial blood glucose remained

above baseline within 2 h after administration. Moreover, a dose-dependent increase in plasma

insulin levels upon Capsulin™ administration was not observed in the reported clinical trials, and

the effect of food remained unclear (259). Capsulin™ is supposedly starting phase IIb clinical trials

after Diabetology entered a licensing agreement with the Indian pharmaceutical company USV

Limited in 2012 (www.diabetology.co.uk) (260).

ORMD-0801 (Oramed) is another insulin-containing enteric capsule (263). According to the

filed patent, the undisclosed formulation likely contains one or several protease inhibitors (e.g., soya

bean tryspin inhibitor, ethylenediaminetetraacetate, EDTA), permeation enhancers (e.g., EDTA),

and lipoidal carriers (e.g., vegetable, fish or synthetic omega-3 fatty acid) (261). In a pilot study

involving eight type I diabetes patients, ORMD-0801 capsules were administered 45 min before

meals as an add-on to the standard insulin treatment and dietary regimen (264). The add-on

significantly decreased the frequency of high blood glucose readings and glucose AUC, but the

response varied among the patients, as reported in other early-phase clinical trials of ORMD-0801

(259, 264). The ORMD-0801 is currently in phase II clinical trials for type I and type II diabetes.

An insulin formulation that used the permeation enhancer SNAC was tested in sixteen

healthy fasting volunteers (265). Plasma glucose decreased 20 – 40 min after insulin administration

in a dose-dependent manner (265). Despite the high amount of administered SNAC (2.1 g), no

adverse events were reported (265). In the meantime, the development of this formulation was

terminated by Emisphere Technology (260). Furthermore, an oral insulin formulation OI338GT


48

(formerly NN1953, Novo Nordisk), that is based on GIPET is currently in phase I and II clinical

development for diabetes type I and II, respectively, but published results were not available at the

time of submission of this review.

IN-105 (Biocon) is an insulin derivative based on conjugating a methoxy-triethylene glycol

propionyl moiety to a lysine on the B chain of human insulin which aims at improving stability in

the GI tract (266, 267). In a cross-over study, 20 diabetes type II patients received IN-105 as tablets

(10 to 30 mg) 20 min before ingesting a standardized high-carbohydrate meal (268). The

postprandial blood glucose levels were significantly decreased for all IN-105 doses in a dose-

dependent manner, but insulin concentration levels varied, and hypoglycemic events were reported

for all doses except the lowest (268). In an unpublished six-month phase III clinical trial, IN-105

failed to meet the primary efficacy endpoint of achieving an HbA1c (glycated hemoglobin) reduction

of 0.7% after placebo adjustment (259). In 2012, Biocon and Bristol-Myers Squibb signed a license

option agreement for worldwide development and marketing of IN-105 (except India), but the

exact state of the current development is unclear.

Insulin was further encapsulated in “hepatocyte-directed vesicles” (Diasome

Pharmaceuticals). The unilamellar liposomes (25 to 125 nm) are composed of the structural

phospholipid distearoyl lecithin, negatively charged dicetyl phosphate, cholesterol, and a liver-

targeting agent (biotin-phosphatidylethanolamine), which aims at decreasing peripheral

hyperinsulinemia (269). However, scientific publications that demonstrate the uptake and liver-

targeting effect are not available. The formulation is dried using a proprietary procedure and

formulated as an oral gelatin capsule (270). In a placebo-controlled clinical trial, liposomal insulin

was given 30 min before every meal as an add-on therapy to six type II diabetes patients (270). It

was significantly more effective in lowering mean postprandial plasma glucose concentrations and

AUC than the placebo but no dose-response relationship was observed (270).

Buccal insulin

Oral-lyn™ (Generex Biotechnology Corporation) is an oral formulation for buccal delivery

that contains recombinant human insulin and non-disclosed absorption enhancers (258).

According to the patent, these GRAS status excipients are likely to be sodium alkyl sulfate, EDTA,

sodium salicylate, and a lecithin-based or bile salt-derived agent, claimed by the company to form

insulin-containing mixed micelles and enhance absorption (261, 271). The insulin liquid

formulation is propelled into the mouth as a fine-particle aerosol by the RapidMist™ device (271,

272). In a study with 31 subjects who suffered from impaired glucose tolerance, six puffs of Oral-


49

lyn™ right before and six puffs 30 min after a standard oral glucose tolerance test decreased plasma

glucose levels approximately 30% after 2 and 3 h compared with no treatment, and hypoglycemia

was not observed in this time frame (272). However, an increase in plasma insulin levels was only

observed 30 min after the administration of the first six puffs (272). According to an announcement

by Generex, the company has improved the formulation to increase insulin bioavailability and the

insulin amount per puff, and is planning to update its IND on file at the FDA.

In conclusion, the goal of reproducibly delivering insulin via the oral route has not yet been

achieved. Inter- and intraindividual variability often undermines reliable dosing, especially in the

presence of food. Presumably, non-buccal insulin formulations will have to be taken at specific

times before food is ingested, and the meals cannot be fully or partially skipped to avoid

hypoglycemia. These restrictions in eating habits and the limited reliability counterbalance the

advantages of needle-free insulin delivery. The most appropriate role of the currently tested oral

insulin formulation may be as an add-on to improve blood glucose control, provided that the risk

of hypoglycemic episodes is small.

2.3.3 Nucleic acids

Despite the polyanionic nature and high molecular weight (>5000 Da) of AONs (131),

several attempts at systemic AON delivery via the oral route have been undertaken. Mipomersen

(formerly ISIS 301012, Genzyme/Isis Pharmaceuticals) is a 20-mer phosphorothioate AON

stabilized with 2′-O-(2-methoxyethyl) modifications at the five terminal bases at both ends. It is

used to treat homozygous familial hypercholesterolemia because it targets the liver apoB mRNA

which encodes apolipoprotein B, an essential component of several fat carriers (e.g., chylomicrons,

LDL, VLDL). The AON was formulated with the permeability enhancer sodium caprate, and upon

its administration once daily for 90 days, the average plasma bioavailability was 6% (273). A

maximum reduction of apoB of 12-15% was observed on day 55 of the treatment. The formulation

for subcutaneous administration of mipomersen was further developed and finally approved by

the FDA in 2013 (Kynamro®, Genzyme). In 1999, Isis Pharmaceuticals and Elan formed Orasense

to develop an oral formulation of ISIS 104838, a 20-mer phosphorothioate AON that targeted the

TNF mRNA. It contains 2′-O-(2-methoxyethyl) modifications at the five terminal bases at both

ends (139). Several enteric-coated capsules with varying durations of sodium caprate release (total

660 mg of sodium caprate and 100-140 mg of AON per capsule) were evaluated in healthy humans


50

for the oral delivery of ISIS 104838 (139). Each subject received five capsules per treatment, and

all formulations were well-tolerated. However, the inter-individual variability in AON oral

bioavailability was high, ranging from 0.7% to 27.5% in ten fasting subjects and with 12% being

the average bioavailability of the best formulation (274). In 2005, Isis Pharmaceuticals terminated

the development of ISIS 104838.

Monarsen (EN 101, Amarin Corporation) is an AON under investigation for myasthenia

gravis (MG), a neuromuscular autoimmune disorder. It downregulates a pathologic mRNA splicing

variant of acetylcholinesterase, which constitutes an established target in MG (275). Monarsen is a

20-mer phosphodiester ON with 2′-O-methyl modifications at the three terminal bases of the 3'

end (275). It was given orally for four days as an aqueous solution to 16 patients who suffered from

MG (276). Thirteen patients showed improved Quantitative MG scores; however, the study was

not placebo-controlled. In the following phase IIa study, oral monarsen given for 7 days at a

maximum daily dose of 40 mg decreased Quantitative MG scores by 20.3% compared to baseline

(277). Further development of monarsen for MG was discontinued. Recently, monarsen was

reported to exert an off-target anti-inflammatory effect through activation of Toll-like receptor 9,

which additionally decreased AChE activity. Therefore, the observed therapeutic effect might be

attributable to the indirect action of AON rather than the target knockdown (278). This finding

led to further development of monarsen for the oral treatment of ulcerative colitis by BioLineRx

under the name BL-7040. This drug has completed phase II clinical trial with positive results,

showing reduced neutrophil levels and histological improvement (www.biolinerx.com).

2.4 Safety of bioavailability-increasing excipients

To address the challenges posed by the low stability and intestinal absorption of orally

applied macromolecular drugs, excipients such as permeation enhancers and/or protease inhibitors

are often added to oral drug delivery systems. Given that the body is exposed to the excipients as

much as it is exposed to the drug, their safety profiles are essential features of the final product,

especially in case of long-term use. This section reviews protease inhibitors and permeation

enhancers with a focus on excipient toxicity. Enteric coatings will not be covered due to their

widespread use and established safety profiles.


51

2.4.1 Protease inhibitors

A major obstacle to the local and systemic delivery of peptides and proteins is their

susceptibility to enzymatic degradation in the GI tract (279). Protease inhibitors reversibly or

irreversibly inactivate luminal and/or brush border membrane-bound enzymes in the GI tract

(279). A conceptual drawback of protease inhibitors is the impaired digestion of dietary proteins

(279). In rodents, protease inhibitors led to the activation of a feedback loop that resulted in

enhanced secretion of proteases, and pancreas hypertrophy and hyperplasia (280–283). Inhibition

of protease activity should therefore be restricted to the area of the liberation and absorption of

the macromolecular drug.

Protease inhibitors are grouped into amino acid-based, (poly)peptidic, and non-amino acid-

based inhibitors (279). Those based on amino acids, (poly)peptides, and their derivatives

competitively bind to the enzyme active site (279). However, their efficacy is often limited due to

generally low inhibitory activity and rapid dilution, digestion, and absorption in the GI tract. For

instance, in a small short-term clinical study, the peptidic serine protease inhibitor aprotinin was

co-administered with human calcitonin in the colon, and although no adverse events related to

aprotinin were reported (284, 285), the excipient failed to increase the bioavailability of human

calcitonin. In rats, aprotinin and several other protease inhibitors (soybean trypsin inhibitor,

sodium glycocholate, camostat mesilate, and bacitracin) fell short in enhancing insulin absorption

upon small-intestinal administration (285). In view of the low potency and presumably high doses

needed to generate an effect, concerns regarding the safety profiles of the excipients and their

potential systemic availability have been raised. The systemic exposure of peptidic aminopeptidase-

inhibitor bacitracin, for instance, was associated with nephrotoxicity (286). Non-amino acid-based

chelating agents inactivate proteases by scavenging enzyme cofactors (calcium, zinc, cobalt,

manganese, magnesium). The chelating agent EDTA is widely used in the pharmaceutical and food

industries, but its binding capacity for calcium is not sufficiently high to inhibit the activity of the

calcium-dependent luminal endoproteases trypsin and chymotrypsin (279). Due to its considerably

higher affinity to zinc, EDTA may be more useful in targeting zinc-dependent exonucleases,

provided that it is not excessively diluted. However, the binding to essential trace elements may

lead to nutritional deficiencies, especially upon chronic administration. Furthermore, small-

molecule protease inhibitors can be associated with local and systemic adverse reactions. The

synthetic trypsin inhibitor camostat mesylate, for instance, increased insulin bioavailability upon

co-administration in the colon but was associated with pancreas hypertrophy (281, 285). This serine

protease inhibitor further showed cross-reactivity with other proteases, leading to off-target effects


52

(e.g., enhanced pulmonary mucociliary clearance due to inhibition of airway channel-activating

proteases) (287, 288).

2.4.2 Permeation enhancers

A variety of compounds with different mechanisms of action are grouped under the term

permeation enhancer (289). They enhance membrane permeability by disrupting the mucus layer

on the intestinal epithelium, transiently opening tight junctions, disrupting the membrane bilayer

packing, and directly binding to the substrate to form an absorbable complex (289). The most

important and clinically advanced permeation enhancers are salts of medium-chain fatty acids (290).

Sodium caprate, the salt of decanoic acid, opens tight junctions, and thus enhances permeability via

the paracellular pathway (291). Even though cytotoxic effects of sodium caprate were observed in

Caco-2 monolayers, repeated oral administrations of sodium caprate did not result in

morphological changes in the gastrointestinal mucosa of several animal species (274, 292–294).

Orally administered sodium caprate were investigated in several clinical studies and, even upon

long-term administration, generally well-tolerated apart from being associated with GI adverse

events in some patients (290). Rectal administration of sodium caprate-containing suppositories,

however, led to transient mucosal damage in healthy subjects (295). Furthermore, the safety profile

of sodium caprate remains insufficiently investigated in certain high-risk populations, notably in

patients with increased GI permeability at baseline (e.g., irritable bowel diseases) or subjects exposed

to GI-irritant drugs such as nonsteroidal anti-inflammatory drugs and ethanol (290).

Another strategy for enhancing membrane permeation is the formation of a membrane-

permeable complex between the substrate and a complexing agent. SNAC and 5-CNAC are

claimed to form a non-covalent complex with the substrate, protecting it from intestinal

degradation and increasing its hydrophobicity in order to enable passive permeation of the

epithelium, after which the complex disassembles (296). However, this mechanism of action has

been the subject of controversial discussions (181, 289). Interestingly, SNAC damaged Caco-2 cells

in various cytotoxicity assays but the toxicity profile in rats was favorable apart from the appearance

of histopathological changes in the stomach which the authors partly related to the application

method (gavage) (296–298). It was generally well-tolerated in clinical studies, with occasional

reports of adverse GI events (nausea, vomiting) (265, 299–301).

Conceptually, enhancing intestinal permeation entails the risk of increasing the exposure to

dietary antigens, which potentially increases the risk of triggering chronic autoimmune diseases


53

(141). However, currently published clinical trials have not confirmed this hypothesis.

Furthermore, surfactant-type permeation enhancers impair the protective properties of the mucus

layer, facilitating the diffusion of luminal bacteria to the intestinal epithelium and ultimately

disturbing the host-microbiota relationship. For instance, it was recently found that the ubiquitous

pharmaceutical excipients and food additives polysorbate-80 and carboxymethyl cellulose induced

a microbiome composition shift, inducing low-grade inflammation and obesity in wild-type mice

and promoting colitis in a predisposed mouse model (142). While the clinical relevance of these

intriguing in vivo findings have yet to be established, alterations in the composition of the gut

microbiota were also reported in healthy humans upon long-term administration of the permeation

enhancer chitosan (143). These studies show that even widely used excipients with GRAS status

may lead to local and systemic pathophysiologic effects, underlining the need for more thorough

long-term preclinical and clinical testing.

2.5 Conclusion

In spite of the considerable progress made in pharmaceutical technology since the first oral

insulin application in 1922, the administration of biomacromolecules via the oral route remains one

of the greatest challenges for formulation scientists. In the systemic delivery of orally administered

biomacromolecules, the harsh environment of the GI tract and the intestinal epithelial barrier often

result in considerable luminal degradation, low and erratic absorption and bioavailability, and highly

variable plasma concentrations. Food effects and drug interactions (e.g., proton pump inhibitors)

may further influence the pharmacokinetic profile. Therefore, the oral administration is currently

restricted to potent low-molecular-weight peptides with large therapeutic windows or with plasma

concentration monitoring. Although novel approaches such as buccal delivery are under clinical

investigation, it remains to be seen if the systemic delivery of orally applied macromolecules above

5000 Da, notably insulin, can be achieved sufficiently reproducibly to reach a favorable efficacy

and safety profile. In the current trials, permeation enhancers in combination with protease

inhibitors are often used to overcome the absorption and enzymatic barriers. However, serious

risks might be associated with the long-term application of these excipients, including increased

exposure to dietary antigens and pathogens due to the sustained increased permeability of intestinal

mucosa and potentially pathogenic changes caused by alterations in the intestinal microbiome.

Therefore, thorough long-term toxicity studies of these excipients are needed.


54

In contrast, the local delivery to GI targets avoids the challenges of crossing the intestinal

epithelium. GI resistance is often conferred by chemical stabilization strategies, which are especially

straightforward for peptides and short nucleic acids. Furthermore, novel approaches such as

genetically modified probiotics that produce and secrete the desired proteins in situ are on the

horizon. However, luminal degradation, potential immunogenicity, and the risk of inadvertent

systemic exposure (e.g., in patients with increased intestinal permeability) remain challenging.

Moreover, the therapeutic areas of local delivery are mainly restricted to diseases of the GI tract

(GI infections, IBD, and certain metabolic diseases).

In view of these challenges, we believe that a better understanding of the molecular

interactions between targets and their biomacromolecular substrates and ligands will eventually

yield low-molecular-weight drugs, which are more suitable for oral delivery than their

macromolecular parent compounds.

55

3 Amphiphilic nucleic acid conjugates§§

3.1 Introduction

Nucleic acids are highly attractive class of therapeutics due to their potential to regulate any

selected gene of interest. Given their capacity to modulate conventionally undruggable targets,

ONs have been extensively investigated as potential therapeutics to treat cancer, viral infections,

genetic diseases, and immunological disorders (302, 303). Despite the clear therapeutic potential of

ONs, their poor permeability across cellular membranes (due to their intrinsic polyanionic nature

and high molecular weight) and susceptibility to degradation by ubiquitous nucleases hamper their

clinical translation (302). Recent advances in the development of various delivery vehicles (e.g.,

polymer-, lipid-, peptide-, nano/microparticles-, or viral-based) have helped overcoming some of

the ON delivery problems; however, issues such as systemic toxicity, low concentration at target

sites and pharmaceutical complexity of the delivery systems still represent obstacles to the clinical

translation of ON therapeutics (127, 304). The conception of stable ONs with enhanced affinity

via various chemical modifications is one of the most remarkable achievements in this field. For

example, the combination of PS backbone modification with OMe and MOE moieties in the sugar

units or bicyclic ribonucleosides are the most widely used chemical strategies under clinical

investigation (15, 305, 306). Another approach is modification with FANA, which upon binding

to the target mRNA induces its RNase H-mediated degradation (30, 307). Several chemically

stabilized ONs are already marketed (e.g., mipomersen for homozygous familial

hypercholesterolemia (306)) or in late-phase clinical trials (15).

Nucleic acid therapy could be especially beneficial for several disorders of the gastrointestinal

(GI) system that currently lack appropriate treatments such as IBDs, colon cancer, and familial

adenomatous polyposis (8). The delivery of nucleic acids directly to the GI mucosa could achieve

§§ Part of this chapter has been published: E. Moroz, S.H. Lee, K. Yamada, F. Halloy, S. Martínez-Montero, H. Jahns, J. Hall, M.J. Damha, B. Castagner, J.-C. Leroux (2016) Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Molecular Therapy – Nucleic Acids, 5, e364.

CHAPTER 3: AMPHIPHILIC NUCLEIC ACID CONJUGATES

56

high local concentrations while minimizing systemic exposure (308) and subsequent side-effects

(41). Indeed, a group of carrier-free ONs targeting GI mucosa for IBD therapy is progressing

through clinical trials, although high doses of ONs are required to obtain the positive therapeutic

effects (8, 227, 308). Therefore, a safe and efficient delivery approach to the GI mucosa would be

desirable to facilitate the cellular uptake of ON and to decrease dosing. Two major strategies are

currently investigated to improve the ON delivery efficacy to the intestinal tissue: particle-based

systems (e.g., polyplex- or lipoplex-based (155, 303)) and single-molecule-based conjugates (e.g., free

ON or CPP-ON conjugates (108, 227)). ON incorporated in particles can be taken up primarily by

M cells in the Peyer's patches of the intestine and eventually by mucosal macrophages via

phagocytosis (132). Therefore particle-based systems would be suitable for the targeting of disease-

related genes expressed mainly in the immune cells, such as TNF. In contrast, single-molecule-

based system, such as free ONs, can be taken up not only by lamina propria immune cells but also

by epithelial cells (227). Considering that in some intestinal diseases the dysregulated expression of

genes is localized in the gut epithelium (147, 309, 310), single-molecule-based systems could open

promising therapeutic avenues for previously inaccessible epithelium-specific targets. In addition,

these systems possess the advantage of reduced carrier toxicity and immunogenicity (83, 91, 311),

as well as simpler characterization processes, compared to particle-based carriers (91, 311).

In a recent investigation from our group, the conjugation of a lipid moiety to chemically

modified ONs enabled significant target gene knockdown in a prostate cancer cell line in the

absence of transfection reagents (112). ON conjugates with the long-chain DSA group had higher

silencing efficacy in comparison to cholesterol or docosahexaenoic acid derivatives. However, only

one type of ONs (i.e., single-stranded AONs) with only one type of chemical modifications (i.e.,

FANA) was investigated. Therefore, the present work aimed at exploring the versatility of DSA as

a delivery vehicle for single-stranded AONs and double-stranded siRNAs targeting a model Bcl-2

mRNA. The cytotoxicity and the silencing efficacy of the resulting conjugates were assessed in vitro

in an intestinal cell line. The most potent conjugate was further evaluated for its silencing kinetics

including the important transfection controls in two intestinal cell lines, and contribution of

sedimentation to the uptake of DSA-ON conjugate was investigated.


57

3.2 Materials and Methods

3.2.1 Materials

DSA, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP),

N,N-diisopropylethylamine (DIPEA), N,N′-diisopropylcarbodiimide (DIC), trichloroacetic acid

(TCA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile (ACN), N,N-

dimethylformamide (DMF), ethanol (EtOH), pyridine, triethylammonium acetate (TEAA) buffer 1

M, concentrated aqueous ammonia, and methylamine (40% solution in water) were obtained from

Sigma-Aldrich (Buchs, Switzerland). Methanol (MeOH), 4-dimethylaminopyridine (DMAP), and N-

(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl), were purchased from

Acros Organics (Geel, Belgium). DNA and RNA phosphoramidites were obtained from Thermo

Fisher Scientific (Waltham, MA). Monomethoxytrityl (MMT)-protected 6-amino-hexanol

phosphoramidite and 3% dichloroacetic acid (DCA) in DCM were obtained from Glen Research

(Sterling, VA). 5-Ethylthiotetrazole was purchased from ChemGenes (Wilmington, MA). 5-

(Benzylthio)-1H-tetrazole was obtained from Carbosynth (Compton, UK). Lithium perchlorate

(LiClO4) was obtained from Alfa Aesar (Haverhill, MA). Parafilm M® was purchased from Bemis

Company (Neenah, WI). Hexafluoroisopropanol (HFIP) was obtained from Fluorochem

(Hadfield, UK). Duplex annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5)

was purchased from Integrated DNA Technologies (IDT, Coralville, IA). HCT-116 cell line was

kindly provided by Prof. Azzalin's group at ETH Zurich, and Caco-2 cells were purchased from

ATCC (Manassas, VA). DMEM with GlutaMAX™ medium, Opti-MEM medium, fetal bovine

serum (FBS), non-essential amino acids, penicillin-streptomycin solution, trypsin, Lipofectamine®

2000 (LF), phosphate-buffered saline (PBS; 1 mM KH2PO4, 3 mM Na2HPO4, 155 mM NaCl, pH

7.4), and RNase-free distilled water were obtained from Invitrogen (Carlsbad, CA). MycoAlert™

PLUS Mycoplasma Detection Kit was purchased from Lonza (Basel, Switzerland). Cell Counting

Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD).

Thermanox™ coverslips were purchased from Thermo Fisher Scientific (Waltham, MA). Low-

binding microcentrifugation tubes (DNA Lobind®) were purchased from Eppendorf-Vaudaux

(Schönenbuch, Switzerland). RNeasy Mini kit and specific primers for α-splicing variant of human

Bcl-2 mRNA (Hs_BCL2_1_SG; QT00025011) and human β-actin (Hs_ACTB_2_SG;

QT01680476) were obtained from Qiagen (Valencia, CA). High-capacity cDNA reverse

transcription kit and Power SYBR Green PCR Master Mix were purchased from Applied

Biosystems (Foster City, CA).


58

3.2.2 Synthesis of ONs and their derivatives

Unmodified Bcl-2 targeting siRNA (112) and mismatch siRNA (63) were synthesized by

Bioneer Co. (Daejeon, South Korea). Unmodified antisense strand of siRNA and L-OMe conjugate

were obtained from Microsynth (Balgach, Switzerland). All other ONs were synthesized according

to the standard protocol for automated phosphoramidite solid-phase synthesis. DSA (L) was

conjugated via an aminohexanol-linker to the 5′ end of ONs in line with previously described

method (112). All L-ON conjugates were purified by reverse-phase HPLC, analyzed by liquid

chromatography – mass spectrometry (LC-MS), and quantified via UV spectrophotometry

(NanoPhotometer P 330, Implen, Germany). Details of synthetic procedure and analytical data are

presented in the appendix. The ONs' molar extinction coefficients at 260 nm were calculated using

the software of IDT website (OligoAnalyzer tool, www.idtdna.com/calc/analyzer). FANA and

MOE extinction coefficients were calculated using DNA and OMe values, respectively. These

modifications were assumed to have negligible effect on the extinction coefficients, as previously

described (35, 63). The complementary single strands of siRNAs were combined in the duplex

annealing buffer at a concentration of 50 µM each, heated up to 95 °C for 1 min and cooled slowly

to 4 °C overnight to ensure proper annealing. Various ONs and L-ON conjugates were dissolved

in RNase-free deionized water at a concentration of 100 µM and stored at -20 °C.

3.2.3 Cell culture

HCT-116 cells were maintained in DMEM medium supplemented with GlutaMAX™

containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5%-CO2

humidified atmosphere. The cells were seeded in a 24-multiwell plate at a density of 4 × 104

cells/well. For the inverted transfection, the cells were seeded on Thermanox™ coverslips in a 24-

multiwell plate at a density of 1 × 105 cells/well. The cells were incubated for 1 day before

experiments. Caco-2 cells were maintained in DMEM medium supplemented with GlutaMAX™

containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1% of non-essential

amino acids at 37 °C in a 5%-CO2 humidified atmosphere. Cells with passage number between 58

and 77 were seeded in a 12-multiwell plate at a density of 7 × 104 cells/well.

All experiments were performed on mycoplasma-free cell lines (regularly checked by

MycoAlert™ PLUS Mycoplasma Detection Kit), and only cells in the exponential phase of growth

were used for seeding.


59

3.2.4 Cytotoxicity assay

To compare the cytotoxicities of different L-ON conjugates, HCT-116 cells were seeded in

a 96-multiwell plate at a density of 7 × 103 cells/well the day before the experiment. The cells were

treated with various concentrations of L-ON conjugates or with free PS siRNA at 1 μM in 50 μL

of serum-deficient Opti-MEM medium for 15 h. Alternatively, the cells were treated with 50 nM

of various siRNAs complexed with LF according to the manufacturer's instructions in 50 μL of

serum deficient Opti-MEM medium for 5 or 15 h. The Opti-MEM medium containing no L-ON

or siRNAs was used as a control. The medium was exchanged for 100 μL of DMEM supplemented

with 10% FBS, and the cells were further incubated for 1 or 2.5 days, after which the cell viability

was assessed using tetrazolium-based CCK-8 reagent following the manufacturer's instructions.

3.2.5 Screening of Bcl-2 silencing efficiencies of various L-ONs

To assess the silencing efficiency of various L-ON conjugates, the conjugates at various

concentrations (0.25-1 µM) were incubated with HCT-116 and Caco-2 cells. Given the absence of

intact serum in the intestinal environment, all transfection experiments were performed in serum

deficient Opti-MEM medium. The Opti-MEM medium containing no ONs was used as a control.

Following overnight incubation (15 h), the transfection medium was exchanged with fresh DMEM

supplemented with 10% FBS. After 1, 2.5, or 3.5 days of further incubation cells were washed with

PBS, and total RNA was isolated using RNeasy Mini kit (Abs260/ Abs230 >1.8) according to the

previously optimized method (112). The expression levels of Bcl-2 mRNA relative to the internal

control β-actin mRNA were quantified by two-step quantitative real-time PCR as previously

described (112). Briefly, cDNA was synthesized from 1.2 µg of total mRNA using high-capacity

cDNA reverse transcription kit according to the manufacturer's instructions. Quantitative real-time

PCR was performed using Power SYBR Green PCR Master Mix and specific primers for human

Bcl-2 and β-actin on a 7900HT Fast Real Time PCR instrument (Applied Biosystems) according

to the manufacturer's instructions. Briefly, the reaction mixtures were incubated at 50 °C for 2 min

and 95 °C for 10 min followed by 40 cycles of denaturation (95 °C for 15 s) and

extension/detection (60 °C for 1 min). Relative gene expression levels were calculated using the

delta delta Ct (2-ΔΔCt) method (the fluorescence threshold was set to 0.4). Results are expressed as

the Bcl-2 mRNA level change between ON-treated and ON-free medium treated cells. L-ON

potency (effective concentration causing 50% of target gene silencing, EC50) was calculated using

a four-parameter logistic function to fit the dose-response data via SigmaPlot software.


60

3.2.6 Target Bcl-2 mRNA silencing study with different incubation times

HCT-116 and Caco-2 cells were seeded in a 24-multiwell plate at a density of 4 × 104

cells/well and in a 12-multiwell plate at a density of 7 × 104 cells/well, respectively. After one day,

the cells were transfected with L-FANA conjugate or siRNA/LF complexes in Opti-MEM

medium in the absence of serum for 5 or 15 h. Bcl-2 mRNA expression was evaluated by real time

PCR 72 h after starting the transfection as described above.

3.2.7 Comparison of transfection efficiencies in upright and inverted

transfection setups

The transfection medium consisted of 250 μL of Opti-MEM containing either 1 μM of

carrier-free Bcl-2 targeting L-FANA conjugate or 50 nM of Bcl-2 specific siRNA complexed with

LF according to the manufacturer's instructions as a particle-mediated delivery control. HCT-116

cells grown on the coverslips were washed with Opti-MEM, and the coverslips were transferred

using tweezers into a new 24-multiwell plate for the transfection. For the upright transfection, the

cells were placed on the bottom of the multiwell plate followed by the addition of the transfection

medium. For the inverted transfection setup, the transfection medium was added to the empty

well, and the cells grown on coverslips were carefully deposited upside down onto the surface of

the medium. The coverslips floated on the surface of the medium due to the surface tension of the

medium. After overnight incubation (15 h), the cells were transferred to a new plate and further

cultured for 2.5 days in 500 μL of DMEM supplemented with 10% FBS. Subsequently, total RNA

was isolated and expression levels of Bcl-2 mRNA were assessed as described above.

3.2.8 Statistical analysis

All treatment groups were compared pairwise using the one-way ANOVA test combined

with Tukey's (Holm-Sidak) post-hoc test assuming normal data distribution. The statistical analysis

was performed using SigmaPlot software. The differences between treatment groups were

considered statistically significant at p-values below 0.05.


61

3.3 Results

3.3.1 L-ON library screening for Bcl-2 mRNA knockdown efficacy

In a previously published study by our group, it was reported that lipophilic DSA conjugated

to ONs modified with 2′-F-arabinonucleosides were more potent in transfecting prostate cancer

cells than nucleic acids derivatized with cholesterol or docosahexaenoic acid (112). In a search for

novel and safe amphiphilic ONs capable of modulating gene expression in intestinal cells, we

synthesized and characterized a set of conjugates by linking lipophilic DSA (L) to different single-

stranded antisense ONs and double-stranded siRNAs targeting the mRNA of the oncoprotein

Bcl-2 (Table 3.1) as previously described (112).

Table 3.1. The sequences and modification strategies of ONs.

Designation Sequenceb Backbonec Reference

DNA *5′-TCTCCCAGCGTGCGCCAT-3′ PS (27)

OMe *5′-TCTC CCAGCGTGCGCCAT-3′ PS (305)

MOE *5′-TC mTC mCCAGCGTGCGC mC mAT-3′ PS (306)

FANA *5′-TCTcccAGCgtgCGCcat-3′ PS (307)

FANAnca *5′-CGCagaTTAgaaACCttt-3′ PS (35)

siRNA *5′-GCAUGCGGCCUCUGUUUGAUU-3′ 3′-UUCGUACGCCGGAGACAAACU-5′

PO (112)

PS siRNA *5′-GCAUGCGGCCUCUGUUUGAUU-3′ 3′-UUCGUACGCCGGAGACAAACU-5′

PS (32)

siRNAnc *5′-GUACGACAACCGGGAGAUAUU-3′ 3′-UUCAUGCUGUUGGCCCUCUAU-5′

PO (63)

anc – negative control, bATGC are DNA, AUGC are RNA, ATC are OMe- or MOE-RNA, atgc

are 2′-F-ANA, Cm – 5-methylcytosine, upper sequence of siRNA – sense strand, lower sequence –

antisense strand, * – site of DSA conjugation, cPS – all phosphorothioate linkages, PO – all

phosphodiester linkages.


62

The antisense ONs were based on a single parent DNA sequence (oblimersen (27, 112)) and

were modified by incorporating OMe-, MOE-ribonucleosides, or FANA connected via PS linkages

(L-DNA, L-OMe, L-MOE, and L-FANA, respectively). These chemical modifications of

nucleotides are commonly employed to enhance the binding affinity of ONs for target mRNAs as

well as to increase their stability against nuclease digestion (312, 313). Typically, antisense ONs'

mode of action involves recruitment of RNase H, which recognizes the thus formed DNA:RNA

heteroduplex leading to a processive cleavage of the mRNA strand in a catalytic fashion (27). Since

oligoribonucleotides and their 2′-O-modified analogues generally do not support RNase H binding,

a “gapmer” design, in which a central DNA stretch is flanked by modified ribonucleotides (OMe

and MOE) at both termini, is preferred for preserving an RNase H binding site, while providing

the ON with higher nuclease resistance (28, 29). In contrast to 2′-O-modified ribonucleotides, the

FANA modification can be incorporated throughout an ON sequence, since upon binding to

complementary mRNA, the FANA nucleotide adopts a conformation similar to that of DNA and

therefore supports RNase H recognition (30). Since an “altimer” design showed higher potency

compared to “gapmer” design in our previous work (112), it was chosen for the FANA-modified

ON in the present study. In order to test whether DSA is capable of facilitating the intracellular

delivery of double-stranded siRNAs, it was conjugated to the unmodified phosphodiester siRNA

(L-siRNA) and its fully phosphorothioated counterpart (L-PS siRNA) (32, 112).

L-MOE, L-PS siRNA, and L-OMe caused a small but significant decrease in cell viability

already 1 day after transfection (Fig. 3.1). This result indicates that the effect of the L-ON conjugate

on cell viability depends on the type of ON used and on its chemical modification. Longer

incubation times could potentially lead to increased cell apoptosis due to the Bcl-2 silencing (27). All

L-ON conjugates exhibited significant cytotoxicity at 2.5 days post-transfection in comparison to

medium treated cells, while the difference between the conjugates was not statistically significant.

Interestingly, not only the conjugates targeting the Bcl-2 mRNA caused appreciable cell death, but

also the conjugate with unrelated sequence (L-FANAnc) indicating the mild cytotoxicity (ca. 20%)

of chemically-modified ONs, which could result from their unspecific protein binding (314, 315)


63

Figure 3.1. Viability of HCT-116 cells after transfection with various ON derivatives at a dose of

1 µM in Opti-MEM medium overnight. Cell viability was assessed 1 or 2.5 days after medium

exchange. Results are expressed as mean + SD (n=3). (***p < 0.001, **p < 0.01, or *p < 0.05 vs. Opti-

MEM medium treated cells).

As shown in Fig. 3.2, both single- and double-stranded L-ONs decreased target gene

expression levels in colon carcinoma cells in the absence of transfecting reagent. L-ON conjugates

showed the following order of efficacy (based on statistically significant differences at various

concentrations): L-FANA > L-OMe > L-siRNA >> L-DNA, where L-DNA did not exhibit a

statistically significant reduction. There was no significant difference between the efficacies of L-

FANA and L-MOE or L-PS siRNA and L-OMe at any of the concentrations tested. L-ON

conjugates downregulated their target gene expression in a dose-dependent manner, and the EC50

values of L-PS siRNA, L-OMe, L-MOE, and L-FANA were 0.86, 0.69, 0.47, and 0.28 µM,

respectively (Fig. 3.2B). These results indicate that also the silencing potency of the L-ON

conjugate depends on the type of ON used and on its chemical modification. Unfortunately, the

potency of AONs could not be evaluated in the lipoplex-based assay due to their severe cytotoxicity

on proliferating cells.


64

Figure 3.2. The silencing efficacy and cytotoxicity of L-ON conjugates. (A) Bcl-2 mRNA silencing

in HCT-116 cells after transfection with various L-ON conjugates at a concentration of 1 µM. Results

are expressed as mean + SD (n=3-5). *** p < 0.001, ** p < 0.01, or * p < 0.05 vs. medium treated

cells. (B) Dose-dependent silencing of Bcl-2 mRNA in HCT-116 cells by transfection with L-PS

siRNA, L-OMe, L-MOE, and L-FANA conjugates. Results are expressed as mean + SD (n =3-4).

DSA coupled to the unmodified siRNA was less efficient in terms of Bcl-2 mRNA silencing

than its conjugate with PS siRNA in the absence of transfection reagent. Since L-PS siRNA

possessed slightly lower silencing potency compared to L-siRNA upon the LF-mediated

transfection (Fig. 3.3), the superior silencing properties of carrier-free L-PS siRNA could have been

due to its higher resistance to extra- and intracellular nucleases (32) and/or higher uptake of PS-

modified ONs (26). The conjugation of lipid to the 5′ end of the sense strands of siRNAs did not

reduce their potency when transfected with the help of LF. The PS modification of L-PS siRNA


65

resulted in a decrease of silencing efficacy in comparison to L-siRNA when transfected with LF

probably due to the impaired recognition by the RNA-induced silencing complex (RISC)

machinery (32). Free PS siRNA did not decrease the bcl-2 mRNA level indicating the importance of

lipid moiety for the efficient cellular delivery. All siRNA/LF lipoplexes exhibited significant

cytotoxicity. Interestingly, L-PS siRNA caused significantly lower cytotoxicity in comparison to

unconjugated PS siRNA when delivered as a LF-lipoplex (p < 0.01) (Figure 3.3, closed dot, right y-

axis), which could result from their differing intracellular trafficking.

We selected the L-FANA conjugate as a promising candidate for further characterization

based on a combination of lowest EC50 with a favourable cytotoxicity profile.

Figure 3.3. Bcl-2 mRNA silencing (bar, left y-axis) and viability (closed dot, right y-axis) of HCT-

116 cells after transfection with various siRNA derivatives complexed with LF in Opti-MEM

medium at a dose of 0.05 µM for 5 h and free PS siRNA at 1 µM overnight. Results are expressed

as mean ± SD (n=3-4). *** p < 0.001, ** p < 0.01, or * p < 0.05 vs. Opti-MEM medium treated

cells, * p < 0.05 between two treatment groups.


66

3.3.2 Bcl-2 mRNA silencing by L-FANA conjugate in two colon carcinoma

cell lines

We further investigated the target gene silencing effect of L-FANA by transfecting two

human colorectal carcinoma cell lines, HCT-116 and Caco-2, with L-FANA and its controls. At a

concentration of 0.7 µM, L-FANA downregulated Bcl-2 mRNA in both cell lines by 87 and 83%,

respectively (Fig. 3.4). Transfection of free FANA and negative control conjugate with an irrelevant

nucleic acid sequence (L-FANAnc) did not change the Bcl-2 mRNA expression levels, indicating

that target gene inhibition is caused by a sequence-specific antisense mechanism and that lipid

conjugation contributes to the improved intracellular delivery of FANA.

Figure 3.4. Dose-dependent silencing of Bcl-2 mRNA in HCT-116 and Caco-2 cells by L-FANA

conjugate compared to the silencing efficacy of the FANA without lipid and to L-FANAnc with

non-targeting sequence (nc). Results are expressed as mean + SD (n =3-4). *** p < 0.001 or ** p <

0.01 vs. medium treated cells.

3.3.3 Bcl-2 mRNA silencing in upright and inverted transfection setups

In order to assess whether sedimentation influenced the transfection efficiency, experiments

with cells in inverted position were performed (Fig. 3.5). Recent studies with nanoparticles

demonstrated that cellular uptake can be dramatically reduced in the inverted configuration (cells

on top), as opposed to the conventional in vitro setup where cells are on the bottom of the culture


67

plate and sedimentation can artificially promote internalization (63, 108, 316). Indeed, we observed

that transfection efficiency of siRNA/LF lipoplex was significantly reduced using the inverted

setup, whereas the silencing efficiency of L-FANA conjugate was the same in both cell setups.

These results indicated that L-FANA was predominantly taken up by cells via sedimentation-

independent uptake routes, which was in line with the outcome of our previous work on peptide-

nucleic acid conjugates (108).

Figure 3.5. Comparison of transfection efficiency of siRNA/LF lipoplex and L-FANA conjugate at

concentrations of 0.05 µM and 1 µM, respectively, in HCT-116 cells with upright and inverted setup.

Results are expressed as mean + SD (n =3-4). *** p < 0.001 vs. siRNA/LF in inverted setup.

3.3.4 Bcl-2 mRNA silencing with different incubation times

It could be envisaged that sedimenting particulate carriers could be rapidly taken up by cells

and achieve target silencing with relatively short transfection times. Indeed, in the case of the

siRNA/LF lipoplex, the taget mRNA silencing efficacy did not depend on the duration of the

transfection, and even a relatively short incubation time with the lipoplex (5 h) was enough to

achieve potent mRNA silencing. It is likely that this is a consequence of the fast uptake of

sedimented particles (Fig. 3.6) (317). This is in contrast to the effect of L-FANA, where increasing

the exposure time to the ON conjugate from 5 h to 15 h resulted in significantly higher knockdown

efficacy (38 vs. 65%, respectively, at 0.5 µM; Fig. 3.6), further supporting a presumed sedimentation-

independent uptake.


68

Figure 3.6. The influence of incubation time on knockdown efficacy in HCT-116 cells transfected

with 0.5 µM of L-FANA or 0.05 µM of siRNA/LF in Opti-MEM medium. The cells were

transfected for 5 or 15 h, followed by incubation with complete medium for 2.5 days. Upon

extending the incubation time from 5 h to 15 h, target gene knockdown efficacy by L-FANA

significantly increased in contrast to siRNA/LF complex. Results are expressed as mean + SD

(n=3-4). * p < 0.05.

3.3.5 Kinetics of Bcl-2 mRNA silencing by L-FANA or siRNA/LF lipoplex

Previously, it was reported that amphiphilic siRNAs could be sequestered in the

endolysosomal compartment and require prolonged post-transfection times (>3 days) in order to

elicit target gene silencing (117). In contrast, L-FANA conjugate exhibited rapid mRNA silencing

and did not require multiple cell divisions in order to reach nucleus (Fig. 3.7). We observed strong

Bcl-2 mRNA silencing already after 1 day post-transfection, which remained unaltered for at least

three days. Of note, the silencing by siRNA/LF lipoplex was also achieved as early as 1 day post-

transfection, and lasted for at least 3.5 days.


69

Figure 3.7. The Bcl-2 mRNA silencing kinetics in HCT-116 cells transfected with 1 µM of L-FANA

or 0.05 µM of siRNA/LF in Opti-MEM medium overnight. Knockdown efficacy by L-FANA

conjugate and siRNA/LF complex remained stable when extending the post-transfection time

from 1 to 3.5 days (no statistically significant difference). Results are expressed as mean +

SD (n=3-4).

3.4 Discussion

Local delivery of nucleic acid drugs is a promising therapeutic strategy against intestinal

diseases but faces numerous challenges associated with the complexity of the GI environment. In

the present work, we sought to identify a potent and robust nucleic acid delivery platform based

on amphiphilic ON conjugates. A set of L-ON conjugates targeting a model Bcl-2 mRNA was

prepared using chemically modified AONs and siRNAs. The selected sequence of the AONs was

based on oblimersen, a drug previously tested in numerous clinical trials, which has a thoroughly

optimized sequence site. The sequence of siRNAs was tested in several previous studies from our

laboratory showing efficient Bcl-2 mRNA and protein silencing when delivered by various

particulate nanocarriers (63, 74, 112). Although all L-ON conjugates carried the same DSA lipid

moiety, they induced different degrees of cytotoxicity. This could be attributed to the modification-

dependent protein binding of ONs (314, 315) or Bcl-2-level-dependent cell death (27). Superior

silencing efficacy of L-FANA and L-MOE conjugates compared to those of L-DNA and L-OMe

may be attributed to a combination of factors including higher nuclease resistance and higher


70

affinity for the complementary mRNA (313, 318, 319). By introducing a few chemically modified

sugar rings in the structure of AON, we could drastically increase its potency. It is worth

mentioning that the order of efficacy of different ON modifications could be different for other

sequences/targets. Additional efforts to increase ON's affinity towards the target mRNA could

enhance L-ON silencing efficacy even further.

A fully phosphorothioated L-PS siRNA conjugate showed significant Bcl-2 silencing upon

carrier-free transfection, while PS siRNA without DSA did not, suggesting that the uptake of L-PS

siRNA was mediated by the lipid moiety. A phenomenon called gymnosis was recently reported in

which target genes are sequence-specifically suppressed in cells by chemically modified ONs, such

as locked nucleic acids and FANA, in the absence of transfection reagents or delivery moiety

conjugation. For an efficient and non-toxic gymnotic transfection, prolonged exposure of

proliferating cells (6-10 days) to high (µM) concentrations of ONs is required (35, 320). Considering

that the average transit times in the small and large intestines of healthy humans are 3 h (321) and

27 h (322) respectively, gymnotic ON delivery is likely to be too slow for intestinal tissues, at least

in the absence of frequent dosing. Moreover, the renewal cycle of intestinal epithelial cells lasts

barely 3-5 days (323), therefore the intestinal ON therapy needs an efficient delivery strategy to

allow the rapid knockdown of the target mRNA and protein. In the present study, DSA

conjugation to ONs promoted the delivery of single-stranded antisense ONs and even double-

stranded siRNAs during limited exposure times. In contrast to previous findings on amphiphilic

siRNAs showing their prolonged endosomal entrapment (> 3 days) (117), DSA-FANA conjugate

exhibited rapid target mRNA knockdown which lasted for more than 3 days post-transfection. The

knockdown was dose-dependent in two intestinal cells lines, and longer incubation with L-FANA

conjugate resulted in stronger silencing. Overall, these results present L-ON conjugates as a

promising nucleic acid delivery tool capable of gene silencing in intestinal cells.

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4 Effect of intestinal conditions on

transfection***

4.1 Introduction

Amphiphilic lipid-ON conjugates can bind to the proteins that function as lipid carriers, such

as low-density lipoproteins (113), chylomicrons (115), or albumin (112, 116). Notably, a previous

study from our group has demonstrated that their binding to albumin could be reversed via addition

of excess of short-chain fatty acid in the medium, successfully releasing the amphiphilic ON

conjugate (112). Due to their binding to serum proteins, these amphiphilic conjugates might

constitute an effective delivery approach primarily for liver targeting or topical applications with

reduced serum content (105, 112, 121–126). However, their applicability to the intestinal mucosa

remains questionable since the harsh GI environment containing digestive enzymes coupled with

the known difficulties transfecting differentiated epithelium in vitro suggests that ON delivery will

be limited (Scheme 4.1) (160, 324–331). Moreover, the presence of food-derived fat in the intestine

could potentially act as sink for lipid-ON conjugates, preventing their cell uptake (Scheme 4.1).

In the present work, we investigated the integrity and transfection efficiency of amphiphilic

conjugates under conditions mimicking the intestinal environment (inclusion of food-derived fats

(soybean oil emulsion) and pancreatin-derived digestive enzymes). To investigate the silencing of

L-FANA in polarized intestinal epithelium, the non-FAE model based on differentiated Caco-2

cells was chosen. It is widely used in pharmaceutical laboratories both in academia and in industry,

as it has proven to be an adequate and simple model to assess drug metabolism, toxicity, and

intestinal absorption/permeation.

*** Part of this chapter has been published: E. Moroz, S.H. Lee, K. Yamada, F. Halloy, S. Martínez-Montero, H. Jahns, J. Hall, M.J. Damha, B. Castagner, J.-C. Leroux (2016) Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Molecular Therapy – Nucleic Acids, 5, e364.

CHAPTER 4: EFFECT OF INTESTINAL CONDITIONS ON TRANSFECTION

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Scheme 4.1. Potential barriers for cellular delivery of amphiphilic ON conjugates in the intestine.

TJ – tight junctions.

4.2 Materials and Methods

4.2.1 Materials

HCT-116 cell line was kindly provided by Prof. Azzalin's group at ETH Zurich and Caco-2

cells were initially purchased from ATCC (Manassas, VA). DMEM with GlutaMAX™ medium,

Opti-MEM medium, fetal bovine serum (FBS), non-essential amino acids, penicillin-streptomycin

solution, trypsin, LF, phosphate-buffered saline (PBS; 1 mM KH2PO4, 3 mM Na2HPO4, 155 mM

NaCl, pH 7.4), and RNase-free distilled water were obtained from Invitrogen (Carlsbad, CA).

Porcine pancreatin (4×USP), ammonium persulfate, TEAA buffer 1 M, Intralipid® (20% (w/v) of

soybean oil, 1.2% of egg yolk phospholipids, 2.25% of glycerol, pH 6-8.9; mimic of high-fat meal),

chloroform (CHCl3) Triton X-100, NaF, Na3VO4, Tris, and skim milk powder were obtained from

Sigma-Aldrich (Buchs, Switzerland). Sodium decanoate was purchased from TCI (Tokyo, Japan).

Duplex annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5) was purchased from

Integrated DNA Technologies (IDT, Coralville, IA). Potassium dihydrogen phosphate was

obtained from Merck (Kenilworth, NJ). MeOH, N,N,N′,N′-tetramethylethylenediamine

(TEMED), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Acros Organics

(Geel, Belgium). HFIP was obtained from Fluorochem (Hadfield, UK). EDTA and polysorbate 20


73

were purchased from AppliChem (Darmstadt, Germany). Gel Red nucleic acid gel stain was

obtained from Biotium (Hayward, CA). MycoAlert™ PLUS Mycoplasma Detection Kit was

purchased from Lonza (Basel, Switzerland). Cell Counting Kit-8 (CCK-8) was obtained from

Dojindo Molecular Technologies (Rockville, MD). Thermanox™ coverslips, DNA loading dye,

NaCl, and micro BCA™ protein assay kit were purchased from Thermo Fisher Scientific

(Waltham, MA). Transwell® inserts were obtained from Corning Inc. (Corning, NY). Low-binding

microcentrifugation tubes (DNA Lobind®) were purchased from Eppendorf-Vaudaux

(Schönenbuch, Switzerland). RNeasy Mini kit and specific primers for α-splicing variant of human

Bcl-2 mRNA (Hs_BCL2_1_SG; QT00025011) and human β-actin (Hs_ACTB_2_SG;

QT01680476) were obtained from Qiagen (Valencia, CA). High-capacity cDNA reverse

transcription kit and Power SYBR Green PCR Master Mix were purchased from Applied

Biosystems (Foster City, CA). Complete® EDTA-free protease inhibitors' cocktail was purchased

from Roche Diagnostics (Mannheim, Germany). Poly(vinylidene difluoride) membranes (PVDF)

were obtained from Bio-Rad Laboratories (Hercules, CA). Mouse anti-human Bcl-2 monoclonal

antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody

were purchased from Dako (Glostrup, Denmark). Rabbit anti-β-actin polyclonal antibody and

HRP-conjugated goat anti-rabbit IgG polyclonal antibody were obtained from Abcam (Cambridge,

UK). ImmunoCruz® Western blotting luminol reagent was purchased from Santa Cruz

Biotechnology (Dallas, TX). Super RX X-Ray films were obtained from Fujifilm (Tokyo, Japan).

4.2.2 Cell culture

HCT-116 cells were maintained in DMEM medium supplemented with GlutaMAX™

containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5%-CO2

humidified atmosphere. The cells were seeded in a 24-multiwell plate at a density of 4 × 104

cells/well. Caco-2 cells were maintained in DMEM medium supplemented with GlutaMAX™

containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1% of non-essential

amino acids at 37 °C in a 5%-CO2 humidified atmosphere. For the differentiated Caco-2 cell

monolayer transfection, the cells with passage number between 58 and 77 were seeded in

Transwell® inserts with polyester membrane with pore size of 0.4 µm in a 12-multiwell plate at a

density of 1.12 × 105 cells/well as previously described (332). The medium was exchanged every

other day, and cells between 13 and 17 days of differentiation were used for the experiments. The

differentiation of monolayers was monitored by measuring TEER using an EVOM epithelial

voltmeter with STX2 electrode (World Precision Instruments, Sarasota, FL).


74

All the monolayers achieved TEER higher than 1000 Ωcm2 after the two weeks of culturing,

indicating the completion of differentiation process. TEER values of individual wells measured

just before the transfection with ONs were set to 100% and their change was monitored for the

three following days.

All experiments were performed on mycoplasma-free cell lines (regularly checked by

MycoAlert™ PLUS Mycoplasma Detection Kit), and only cells in the exponential phase of growth

were used for seeding.

4.2.3 Cytotoxicity assay

For the L-ON cytotoxicity experiments, Caco-2 cells were seeded in a 96-multiwell plate, and

the culture medium was exchanged every other day for two weeks to obtain differentiated

monolayers. The cells were treated with various concentrations of L-ON conjugates in 50 μL of

serum-deficient Opti-MEM medium for 15 h. The Opti-MEM medium containing no L-ON was

used as a control. The medium was exchanged for 100 μL of DMEM supplemented with 10%

FBS, and the cells were further incubated for 24 h, after which the cell viability was assessed using

tetrazolium-based CCK-8 reagent following the manufacturer's instructions.

4.2.4 Interaction of FANA and L-FANA with oil

For polyacrylamide gel electrophoresis (PAGE) analysis of ONs interaction with an oil-in-

water emulsion, 1 μL of FANA or L-FANA (100 μM) was mixed with 9 μL of emulsion

(Intralipid®) diluted with Opti-MEM to 0-2% of soybean oil, incubated for 30 min at room

temperature, and mixed with 1 µL of DNA loading dye. Samples were then loaded onto 20% (w/v)

polyacrylamide gel prepared in a Tris-acetate-EDTA buffer (TAE: 40 mM Tris-acetate, 1 mM

EDTA, pH 8.0) by free-radical polymerization with ammonium persulfate/TEMED as an initiator.

Gel was then immersed in TAE buffer and electrophoresed at constant voltage of 150 V for 60

min. ONs were revealed following manufacture's protocol for Gel Red nucleic acid gel stain and

fluorescence was recorded on a ChemiDoc XRS.

4.2.5 L-FANA conjugates stability in simulated intestinal fluid (SIF) and

soybean oil emulsion

To investigate the influence of digestive enzymes and food-derived fats present in intestine

on the biological function of L-FANA, its stability and efficacy were tested in simulated intestinal

environment containing pancreatic enzymes and lipids. For PAGE analysis 1 μL of L-FANA (100


75

μM) was mixed with 9 μL of USP SIF (2.5 g/L porcine pancreatin (4×USP), 50 mM KH2PO4, pH

6.8) or water and incubated for 2 or 15 h at 37 °C. Samples were kept at 95 °C for 15 min to heat-

inactivate the enzymes and mixed with 1 µL of DNA loading dye. Alternatively, 1 μL of L-FANA

was pre-mixed with 1 μL of 5% soybean oil emulsion in Opti-MEM (prepared by diluting 20%

Intralipid® with Opti-MEM) for 30 min followed by incubation at 37 °C for 2 h with 8 μL of

active/heat-inactivated SIF or water. Samples were then loaded onto 20% (w/v) polyacrylamide gel

prepared in a TAE buffer by free-radical polymerization with ammonium persulfate/TEMED as

an initiator. The gel was then immersed in TAE buffer and electrophoresed at constant voltage of

150 V for 60 min. L-FANA was revealed following manufacture's protocol for Gel Red nucleic

acid gel stain, and fluorescence was recorded on a ChemiDoc XRS (Bio-Rad Laboratories,

Hercules, CA).

For the LC-MS analysis, 2 μL of L-FANA (100 μM) were mixed with 18 μL of SIF or water

and incubated for 2 h at 37 °C, then at 95 °C for 15 min, and centrifuged at 14000 × g at room

temperature for 10 min. The supernatants were mixed with 50 μL of CHCl3, shaken to extract

possible hydrophobic contaminants from SIF. After incubating the mixtures at room temperature

for a few minutes, water layers were collected and stored at -20 °C until analysis. Analytical LC-MS

was carried out on Agilent 1200/6130 system (Agilent Technologies, Santa Clara, CA) using a

reverse phase column (Waters Acquity OST C18, 2.1 x 50 mm, 1.7 µm; Waters Corporation,

Milford, MA) with solvent A being 400 mM HFIP and 15 mM TEAA in water, and solvent B being

MeOH. The flow was set at 0.3 mL/min and a gradient was run at 65 °C from 5% to 90% B in 14

min.

4.2.6 Transfection of L-FANA pre-incubated with SIF and soybean oil

emulsion

The influence of pre-incubation of L-FANA conjugates with SIF on the transfection efficacy

was investigated. HCT-116 cells were seeded one day prior to the experiment in a 24-multiwell

plate at a density of 4 × 104 cells/well in DMEM containing 10% FBS. Five microliters of L-FANA

were pre-incubated with 45 µL of SIF for 2 h at 37 °C followed by heat-inactivation of enzymes

for 15 min at 95 °C. The mixtures were diluted ten times with Opti-MEM to a final L-FANA

concentration of 1 μM, and incubated with cells overnight (15 h). ON-free Opti-MEM medium

was used as a control. Subsequently, the transfection medium was changed to DMEM

supplemented with 10% FBS. After 2.5 days of further incubation, total RNA was isolated and

expression levels of Bcl-2 mRNA were assessed as described above. To study the silencing activity

of L-FANA released from SIF-digested emulsion, 2.5 μL of L-FANA (100 μM) were pre-incubated


76

with 6.25 µL of 20% Intralipid® for 30 min at room temperature followed by incubation with 25

µL of SIF for 2 h at 37 °C. The released L-FANA was separated from cytotoxic oil digestion

products by preparative PAGE before transfection experiments. Gels were prepared and

electrophoresed as described above. After the electrophoresis, the L-FANA band was excised,

weighed, and immersed in 250 μL of Opti-MEM. For L-FANA extraction, samples containing gel

pieces were flash-frozen in liquid nitrogen, heated at 90 °C for 15 min, and agitated at room

temperature for 3 h. The concentration of extracted L-FANA in the supernatant was 0.6 µM. The

supernatants were incubated with cells overnight (15 h). The expression levels of Bcl-2 mRNA

were assessed as described above.

4.2.7 Transfection of differentiated Caco-2 cell monolayers

The differentiated Caco-2 cell monolayers grown in Transwell® inserts for two weeks were

washed with Opti-MEM medium from apical and basal sides. The 500 µL of Opti-MEM medium

containing either naked ON derivative (0.625-5 µM) or siRNA (0.2-0.4 µM) complexed with LF

according to the manufacturer's instructions was added to the apical compartment. The basal

chamber was filled with 1.5 mL of Opti-MEM medium. The Opti-MEM medium in both

compartments was used as a control. Following the overnight incubation (15 h), the transfection

medium was exchanged with fresh DMEM supplemented with 10% FBS in both compartments.

For Bcl-2 mRNA expression analysis by qRT-PCR, cells were lysed 2.5 days after transfection as

described above. For Bcl-2 protein expression analysis by Western blot, cell monolayers were lysed

4 days after transfection in 25 µL of lysis buffer (20 mM Tris–HCl pH 7.7, 150 mM NaCl, 5 mM

EDTA, 1% v/v Triton X-100, 25 mM NaF, 1 mM PMSF, 1 mM Na3VO4 supplemented with

Complete® protease inhibitors) in line with previously described method (63). Briefly, cell lysates

were scraped from the Transwells®, centrifuged at 10,000 × g for 15 min at 4 °C to remove cell

debris, and protein concentration in the supernatants was determined by the micro BCA assay

according to the manufacturer's instructions. Fifty micrograms of total protein per sample were

resolved on 12% SDS-PAGE under reducing conditions and transferred to a PVDF membrane.

The membrane was washed once with Tris-buffered saline buffer supplemented with polysorbate

20 (Tween-20) (TBS-T) (20 mM Tris–HCl pH 7.7, 150 mM NaCl, 0.1% v/v polysorbate 20) and

blocked with TBS-T containing 5% w/v skim milk (blocking buffer) for 1 h. The membrane was

cut in two at 35 kDa; the lower part containing Bcl-2 (26 kDa) was incubated with anti-Bcl-2

antibody diluted to 1:100 in blocking buffer, and the upper part containing β-actin (42 kDa, loading

control) was incubated with anti-β-actin antibody diluted to 1:4000 overnight at 4 °C. Membranes

were washed 3 times for 5 min with PBS-T followed by 1.5-h incubation with the corresponding

HRP-conjugated secondary antibodies diluted to 1:4000 in blocking buffer. Membranes were


77

washed 3 times with TBS-T, and protein bands were detected with ImmunoCruz® luminol reagent

and revealed on Super RX X-Ray films using an AGFA Curix 60 film processor (AGFA, Mortsel,

Belgium). The relative intensities of the bands were analyzed using Image J software (National

Institutes of Health, Bethesda, MD).

4.2.8 Statistical analysis

All treatment groups were compared pairwise using the one-way ANOVA test combined

with Tukey's (Holm-Sidak) post-hoc test assuming normal data distribution. The statistical analysis

was performed using SigmaPlot software. The differences between treatment groups were

considered statistically significant at p-values below 0.05.

4.3 Results

4.3.1 Stability and silencing properties of L-FANA in SIF

The local intestinal gene silencing poses specific hurdles for nucleic acid delivery systems.

Upon arrival to the desired GI site of action via enteric formulations, endoscopic tube, or enema,

the L-ON conjugate will be exposed to the intestinal digestive (and/or bacterial) enzymes.

Therefore, the delivery system and its nucleic acid cargo need to be stable against the attack of

intestinal enzymes. To this end, we tested whether the pre-incubation of L-FANA conjugate in SIF

containing pancreatic digestive enzymes would impair its transfection efficacy (Fig. 4.1).

Importantly, L-FANA conjugate was stable even after 15 h of incubation with SIF, as assessed by

the PAGE analysis (Fig. 4.1A), and the lipid moiety was not cleaved off upon incubation with

pancreatic enzymes containing lipases (Fig. 4.1B). For the silencing experiments, L-FANA and its

negative control (L-FANAnc) conjugates were incubated with pancreatic enzymes at pH of 6.8 for

2 h at 37 °C, and after the heat-inactivation of the digestive enzymes and dilution with cell culture

medium the mixture was directly added to the HCT-116 cells. The difference between relative

expression levels of target Bcl-2 mRNA after the treatment with L-FANA with or without

pancreatin pre-incubation was not statistically significant (31% and 19%, respectively; Fig. 4.1C).

This result shows that the pre-incubation of L-FANA conjugate with pancreatin did not strongly

affect its silencing efficacy due to the enzymatic degradation or unspecific protein binding.

Treatments with pancreatin alone or pancreatin with L-FANAnc did not change the target mRNA


78

expression, indicating that the silencing effect was caused by L-FANA in a sequence-specific

manner.

Figure 4.1. The stability and silencing properties of L-FANA in SIF (pancreatin 1X USP). (A) PAGE

images of L-FANA conjugate after incubation with SIF for 2 h and 15 h at 37 °C. Band at the top of

the gel could correspond to the unspecific staining of insoluble material from pancreatin. (B) Results

of LC-MS analysis of L-FANAnc conjugate in water and after 2 h incubation with SIF (i and ii,

respectively). L-FANAnc conjugate: retention time 12 min; mass 6417.9 and 6419.5 Da in water

and SIF, respectively (theoretical 6419.3 Da). (C) Target Bcl-2 mRNA knockdown efficiency after

the transfection of HCT-116 cells with L-FANA pre-incubated with SIF for 2 h; nc – L-FANAnc

conjugate with non-specific sequence. Results are expressed as mean + SD (n=3-4). *** p < 0.001

vs. Opti-MEM medium treated cells.


79

4.3.2 The effect of oil on L-FANA silencing properties

It was previously demonstrated that amphiphilic ON conjugates can bind to lipid-containing

particles (e.g., low-density lipoprotein or chylomicrons (113, 115)), implying the possibility of

interaction of L-ON conjugates with food-derived fats in the intestine. Using a gel-based binding

assay, we observed that L-FANA indeed bound to soybean oil in a concentration-dependent

manner, while the migration of unconjugated FANA was not retarded in the presence of oil

(Fig. 4.2).

Figure 4.2. PAGE images of L-FANA conjugate and unconjugated FANA incubated with oil-in-

water emulsion containing 0-2% of soybean oil in Opti-MEM for 30 min. The interaction is mediated

by the docosanoyl moiety of L-FANA conjugate, as FANA without lipid group did not bind to the

emulsion.

The decrease of silencing efficacy observed for L-FANA in the presence of the emulsion

could be attributed to the interaction between the oil and the lipid moiety of the conjugate

interfering with the cellular uptake (Fig. 4.3A, B). However, the interaction of L-FANA with the

emulsion could be disrupted after the digestion of the oil droplets by the SIF (Fig. 4.3C).

Importantly, the released L-FANA from SIF-digested oil fully preserved its silencing capacity (Fig.

4.3B). This finding suggests that the interaction of amphiphilic ONs with food-derived fat would

probably not hamper their cellular uptake and subsequent silencing activity.


80

Figure 4.3. The silencing properties of L-FANA in 0.5% soybean oil emulsion (Oil). (A) Schematic

representation of L-FANA interaction with oil droplets and its release after digestion in SIF. (B)

Target Bcl-2 mRNA knockdown efficiency after the transfection of HCT-116 cells with L-FANA

pre-incubated with soybean oil emulsion in Opti-MEM for 30 min. Oil + L-FANA (digested) –

released L-FANA after pre-incubation with oil emulsion and SIF-mediated digestion. Results are

expressed as mean ± SD (n=3-4). *** p < 0.001 vs. Opti-MEM medium treated cells. (C) PAGE

images of L-FANA conjugate after incubation with a mixture of soybean oil and SIF at 37 °C for

2 h. +inactive – SIF inactivated at 95 °C for 15 min before incubation with L-FANA and emulsion.

Bands at the top of the gels could correspond to the unspecific staining of insoluble material from

pancreatin.

4.3.3 L-FANA silencing properties in differentiated intestinal monolayer

Intestinal epithelial cells possess characteristics that make their in vivo transfection by non-

viral and non-bacterial carriers rather challenging: they are polarized cells that express tight


81

junctions and microvilli, and have reduced proliferative and endocytic capacity compared to

undifferentiated cancer cells (323, 333–336). Caco-2 carcinoma cells are able to differentiate into

such monolayer system, and have been used as a model to assess gene delivery efficacy to polarized

intestinal epithelium (330). To compare the delivery of modified ON into proliferating cells and

difficult-to-transfect epithelial cell monolayers, the L-FANA conjugate was tested on differentiated

Caco-2 cells. To illustrate the resistance to transfection of fully differentiated Caco-2 cell

monolayers, experiments were performed with the Bcl-2-targeting siRNA complexed with LF (Fig.

4.4A). Transfection of siRNA/LF at a concentration of 0.2 µM elicited a small decrease of Bcl-2

mRNA expression (21%, Fig. 4.4B), which was not accompanied by its downregulation at the

protein level (Fig. 4.4C). We could not achieve dose-dependent Bcl-2 mRNA knockdown even

with siRNA concentrations as high as 0.4 µM, at which the control siRNAnc possessing a target-

unrelated sequence caused appreciable suppression of Bcl-2 mRNA, probably due to the toxicity

caused by high concentrations of LF (327). In contrast, Bcl-2 silencing was successfully achieved

in proliferating Caco-2 cells at a dose of 0.05 µM of siRNA (Fig. 4.5).

Strikingly, the single-molecule-based L-FANA conjugate was able to knockdown Bcl-2

mRNA in differentiated Caco-2 cell monolayers in a sequence-specific and dose-dependent manner

(Fig. 4.4B). At L-FANA concentrations of 1.25, 2.5, and 5 µM the target Bcl-2 mRNA expression

was reduced to 56%, 32%, and 26% of the initial level, respectively. At 5 µM the negative control

L-FANAnc was inactive. Conjugation of DSA was found to be essential for successful delivery, as

unconjugated FANA did not have any effect on Bcl-2 mRNA expression even at 5 µM. Consistent

with mRNA silencing, western blot analysis demonstrated that L-FANA reduced efficiently the

target Bcl-2 protein level (by approximately 53% at 2.5 µM; Fig. 4.4C).


82

Figure 4.4. In vitro evaluation of L-FANA silencing properties for differentiated intestinal monolayer

delivery. (A) Schematic representation of L-FANA and siRNA/LF transfection to differentiated

intestinal epithelium. (B) Target Bcl-2 mRNA knockdown efficiency by transfection of differentiated

Caco-2 cell monolayers with L-FANA and siRNA/LF lipoplex in Opti-MEM medium, nc –

corresponding negative controls L-FANAnc and siRNAnc with non-specific sequences. Results are

expressed as mean + SD (n=3-5). *** p < 0.001, ** p ≤ 0.01 vs. Opti-MEM medium treated cells, **

p < 0.01 between two treatment groups. (C) Western blot assay of Bcl-2 and β-actin protein

expression after transfection of differentiated Caco-2 cell monolayers with L-FANA (2.5 µM),

siRNA/LF (0.2 µM), and their control groups in Opti-MEM medium.


83

Figure 4.5. Transfection of proliferating Caco-2 cells with siRNA/LF and negative control siRNA

(siRNAnc)/LF lipoplexes in Opti-MEM medium at a dose of 0.05 µM for 15 h. This result shows

that the Bcl-2 mRNA silencing by siRNA is sequence-specific. Results are expressed as mean

+ S.D. (n=3-4). ** p < 0.01 vs. Opti-MEM medium treated cells.

Importantly, the treatment with relatively high dose of L-FANA neither caused cytotoxicity

(Fig. 4.6A) nor decreased the TEER of Caco-2 monolayers (Fig. 4.6B), which is a measurable

indicator of the monolayer integrity (337). In comparison, the treatment with sodium decanoate, a

widely used absorbefacient disrupting the tight junctions (8), caused a pronounced drop in TEER.

Based on this result, it is likely that L-FANA conjugates will be taken up primarily by epithelial cells

in vivo with minimal systemic translocation via paracellular route.

Figure 4.6. In vitro evaluation of L-FANA toxicity on differentiated intestinal epithelium. (A)

Viability of differentiated Caco-2 monolayer cells after treatment with 1-5 µM of L-FANAnc in

Opti-MEM medium. Results are expressed as mean ± SD (n=3). (B) TEER change upon overnight

treatment (15 h) with FANA derivatives (5 µM) or permeation enhancer sodium decanoate (DecNa;

4 mM) in Opti-MEM medium. Results are expressed as mean ± SD (n=3-4). *** p < 0.001, ** p ≤

0.01 vs. Opti-MEM medium treated cells.

Rela

tive B

cl-

2 m

RN

A level

(tre

ate

d v

s. m

ock

)

0.0

0.2

0.4

0.6

0.8

1.0

**

siRNA siRNAnc


84

4.4 Discussion

For a successful in vivo delivery of ONs to intestinal epithelium, L-ON conjugates must

remain functional in the intestinal environment, and they should be able to silence target genes in

difficult-to-transfect epithelial cell monolayers. One concern regarding their biological activity in

the intestine was that amphiphilic ON conjugates might bind to intestinal lipophilic content,

potentially leading to reduced activity. Here, in an in vitro model system, we demonstrated that free

L-FANA is released from oil phase after digestion by pancreatic enzymes and retains its silencing

ability. The combination of the extreme stability of FANA-modified ONs towards nucleases (313)

and hydrolysis at low pH (338), together with the high stability of DSA under similar conditions

found in the GI tract (339) makes DSA-FANA conjugates particularly suitable for GI applications.

Differentiated intestinal epithelial cells featuring microvilli and expressing tight junctions

represent a difficult target for nucleic acid delivery (160, 324–330). Their reduced proliferation and

endocytosis rate in comparison to undifferentiated cells are largely responsible for poor

transfection efficacy (335, 336). Regarding particle-based delivery systems, the resistance to

transfection observed with differentiated cells may be partially attributed to the structure of their

apical membrane featuring microvilli, which limits access to the absorptive membrane (330, 331).

Several studies have described a variety of attempts to overcome this barrier by, for example, pre-

treatment with membrane-disturbing agents (330), transfection of proliferating Caco-2 in the

suspension state followed by accelerated differentiation (325), formulation with small lipid

nanoparticles (331), use of β1-integrin-mediated endocytosis (55) or by electroporation (324). As

previously reported for siRNA/LF, we were unable to efficiently downregulate Bcl-2 mRNA in

polarized Caco-2 cell monolayers with these lipoplexes. In contrast, the single-molecule-based L-

FANA conjugate was able to effectively silence the Bcl-2 gene expression at both mRNA and

protein levels in differentiated monolayers. Importantly, despite lower potency of carrier-free

amphiphilic conjugates in comparison to siRNA/LF lipoplexes in classical in vitro assays based on

rapidly proliferating cancer cells, they by far outperform particulate delivery vehicles under more

stringent conditions, such as the inverted setup or using differentiated cells. The involvement of

cell surface receptors, such as fatty acid receptors, better access of L-FANA to the adsorptive cell

membrane due to the smaller size, or other uptake routes different from LF-mediated endocytosis

may explain the efficient target silencing by L-FANA conjugate in a non-dividing polarized

epithelium. As mentioned previously, several studies have extrapolated the possible internalization

routes of amphiphilic ON conjugates in proliferating cells, including adsorptive endocytosis, SID-


85

1 or β2-integrin-mediated endocytosis, depending on the cell line and/or the lipophilic moiety

tested (112, 113, 117–121). Moreover, it is possible that the uptake mechanism and intracellular

trafficking can vary among different states of cell differentiation (328); therefore, further studies

are necessary to elucidate the uptake mechanism of L-FANA by the differentiated epithelium.

87

5 Conclusions and Outlook

In order to fully exploit the therapeutic potential of nucleic acid drugs and facilitate their

clinical translation, non-toxic delivery strategies that efficiently overcome extracellular and

intracellular barriers must be developed. Such delivery systems should be tailored according to the

administration route and target tissue. Some success has already been achieved for the treatment

of liver disorders with chemically stabilized nucleic acids or particle- or -based delivery systems,

that are administered i.v. or subcutaneously. Notwithstanding, the development of oral

formulations would be the most desirable way to bring these therapies forward, especially for

patients with chronic disorders requiring regular dosing. However, the oral administration of

nucleic acids requires their systemic translocation from the GI tract and current attempts to enable

this are still quite far from reaching the clinic as they often involve complex delivery systems with

questionable safety profiles.

On the other hand, the local delivery of ON therapeutics stands a better chance for clinical

translation in a nearer future. The availability of the GI mucosa makes it particularly attractive for

the local delivery of nucleic acid therapies. Indeed, high doses of ONs can be delivered directly to

the disease site in the GI tract without the risk of complement activation or systemic target silencing.

However, important barriers have to be considered, such as the thick mucus layer hampering the

diffusion of delivery systems to the epithelium, the high enzymatic activity leading to nucleic acid

degradation, and the tight junctions and microvilli of intestinal cells further limiting ON's uptake.

Several particle-based vehicles have been investigated for the local delivery of nucleic acids to the

intestinal mucosa, the majority of which relies on their uptake by the intestinal M cells and

subsequently by the underlying macrophages to disseminate them to remote organs. This strategy

could be applied for the treatment of inflammatory diseases, although not without the risk of

systemic immunosuppression. However, other GI disorders such as familial adenomatous

polyposis, cannot be treated using this approach as the therapeutic targets are located in the

epithelial cells. Recently, a bacteria-mediated strategy for gene silencing in the intestinal epithelium

has been proposed (55, 56), but potential issues associated with immunogenicity of the invasive

CHAPTER 5: CONCLUSIONS AND OUTLOOK

88

bacteria and reproducibility of dosage have to be considered. In principle, carrier-free nucleic acid

drugs should have higher mucosal permeability in comparison to particulate carriers, as well as

more predictable dose-effect relationships and more controllable synthesis and characterization

processes. Additionally, their distribution within the intestinal lumen should be more homogeneous

and unaffected by precipitation, leading to steady and evenly distributed gene silencing.

In this work, amphiphilic conjugates were tested for the first time to achieve target gene

silencing in an intestinal epithelium model. The main findings have been summarized in Fig. 5.1.

A series of amphiphilic conjugates consisting of DSA and nucleic acids with different architectures

and mechanisms of action (single-stranded AONs and double-stranded siRNAs) was synthesized

to explore the capacity of DSA as a delivery moiety. To compare their efficacy, the ONs were

designed to target a model mRNA of the oncoprotein Bcl-2. While the different conjugates

exhibited various degrees of cytotoxicity and target silencing, the L-FANA conjugate was selected

as the most potent candidate with a favourable safety profile in an in vitro model. Presumably,

although the FANA-modified conjugate was slightly better in terms of Bcl-2 mRNA silencing in

comparison to the OMe- and MOE-modified counterparts, the order of efficacy could be different

for other sequences/targets.

Further investigation of the silencing properties of the L-FANA conjugate in two colon

carcinoma cell lines demonstrated that the delivery of amphiphilic ON conjugates was indeed

mediated by the DSA group, since the unconjugated control failed to significantly downregulate

the target mRNA. The rapid mRNA silencing induced by L-FANA was sequence-specific and

dose-dependent, and lasted for more than 3 days. This well-defined single-molecule-based

approach was compared to conventionally employed lipoplexes in an inverted transfection setup.

It was found that the silencing efficacy of L-FANA was not altered by inverting the orientation of

cells, whereas the knockdown induced by lipoplex particles was completely abolished, probably

due to sedimentation. This suggests sedimentation-independent uptake mechanism for L-FANA

conjugate, which could potentially lead to more homogeneous gene silencing within the intestinal

lumen in vivo.

The oral administration of ON delivery systems implies that they retain the silencing ability

in intestinal conditions. Accordingly, the L-FANA conjugate was stable in presence of digestive

enzymes and its silencing activity was preserved. Indeed, the conjugate's interaction with an oil-in-

water emulsion used to mimic the content of the intestinal tract was successfully disrupted upon

treatment with pancreatin, which released the intact L-FANA conjugate and restored its silencing

efficacy. The most encouraging finding was however that the L-FANA conjugate was able to


89

silence the Bcl-2 mRNA in the difficult-to-transfect fully differentiated Caco-2 cell monolayers. In

this aspect our single-molecule-based approach was remarkably superior to conventionally

employed lipoplexes, which were unable to transfect the intestinal epithelium. To the best of our

knowledge, this is the first study showing the efficient gene silencing in polarized epithelial cells by

an ON conjugate in the absence of complexation, delivery device, or membrane-disturbing agents.

The TEER measurements of the differentiated intestinal monolayer revealed that L-FANA

conjugates did not disturb tight junctions, which is necessary for high molecular weight drugs to

achieve systemic bioavailability via paracellular transport. Therefore, there is low probability of

systemic translocation during L-FANA treatment in vivo, thus limiting potential systemic off-target

events and allowing these conjugates to be taken up primarily by cells lining up the intestinal

mucosa. Importantly, although the L-FANA conjugate was selected as the most potent one based

on mRNA silencing efficacy, the general conclusions drawn from this study regarding binding to

fats and monolayer transfection are likely valid for other stabilized L-ON conjugates.

Overall, we have demonstrated successful intestinal epithelium transfection with single-

molecule-based conjugates without the help of transfection or membrane-disturbing agents. This

is a valuable achievement not only in view of its future pharmaceutical development but also of its

application in molecular biology research, where it provides a carrier-free tool to study gene

functions in differentiated intestinal epithelium. In the current work, the in vitro experiments were

thus designed from a pharmaceutical technology perspective (e.g., interactions with food-derived

fats, degradation by intestinal enzymes), and the characterization data gathered should serve as a

basis for future in vivo experiments in suitable GI disease models.

Future work should focus on better understanding the trafficking of the L-ON in proliferating

and differentiated cells, as well as elucidating its mechanism of uptake. This could be achieved by

tracking the fluorescently/radioactively labelled L-ON conjugates or by using the endocytosis

inhibitors for instance. Additionally, even though the bacterial load in the small intestine is relatively

low, the colon hosts billions of bacterial cells and their effect on the L-ON interaction with the

intestinal epithelium should thus be studied. Finally, while we have presented a proof-of-concept

study in an in vitro model targeting the Bcl-2 mRNA, the L-ON silencing properties should be

tested in vivo using other clinically relevant targets, such as ICAM-1 or Smad7 for the treatment of

IBDs. The head-to-head comparison of efficacy of L-ON conjugates and unconjugated ONs will

also be necessary to validate the efficacy of this approach. Further development of this system

could include the attachment of a targeting ligand such as FANA aptamer, to enhance the cell

uptake and add cell-type specificity to the system. Based on the main findings of this thesis, L-ON

conjugates like L-FANA represent a suitable approach to further improve the efficacy of ON


90

enteral treatments currently under investigation, a road which we hope to have paved with the

promising results presented herein.

Figure 5.1. Summary of the main findings of the present thesis. (A) DSA is a versatile delivery carrier

for AONs and siRNAs, while the L-FANA was the most promising conjugate. (B) L-FANA

preserved its silencing efficacy in the inverted transfection setup in contrast to a conventional

particulate lipoplex-based carrier. (C) Given the digestion-resistant properties of L-FANA,

pancreatic enzymes could disrupt its interactions with food-derived fats and restore its silencing

efficacy. (D) L-FANA could efficiently silence the target Bcl-2 gene in the differentiated epithelium

in contrast to the siRNA lipoplex.

91

6 Appendix

6.1 Synthesis of L-MOE and L-siRNA conjugates

Instruments, reagents, and conditions for standard phosphoramidite solid-phase synthesis

were used for the synthesis of the L-MOE, L-PO sense strand of the siRNA, and L-PS siRNA (L-

PS sense strand of the siRNA, PS antisense strand of the siRNA) as previously described (32).

RNA, MOE, and DNA phosphoramidites were prepared as 0.08, 0.08, and 0.16 M solutions in dry

ACN, respectively. 5-(Benzylthio)-1H-tetrazole (0.24 M in ACN) was used to activate the

phosphoramidites for coupling. Phosphoramidite coupling times were 2 × 120 s for DNA and 2

× 180 s for MOE. In case of PO and PS RNAs, the coupling times were 2 × 120 s and 2 × 90 s,

respectively. For DSA conjugation, first a MMT-protected 6-amino-hexanol phosphoramidite

linker was attached to ON′s 5′ end using a regular coupling cycle and coupling time of 2 × 180 s,

followed by three consecutive detritylation steps for MMT removal (3% DCA in DCM, 3 × 60 s).

Prior to DSA coupling to MOE or PO sense strand of the siRNA, the solid support was washed

three times with ACN, transferred to a screw tube and resuspended in 300 μL of anhydrous THF

and DMF mixture (1:1 v/v) containing 17 mM of DSA, 34 mM of EDC HCl, and 160 mM of

DMAP. The tubes were sealed with Parafilm M® and shaken overnight at 45 °C. Following the

extensive washing with THF/DMF mixture, deprotection and cleavage of ONs from the solid

support was accomplished with 200 µL of 25% aqueous ammonia for 16 h at 50 °C. Alternatively,

in case of DSA coupling to PS sense strand of the siRNA, the solid support was successively

washed with DCM (1 × 5 min), 5 % DIPEA in DCM (5 × 1 min), DCM (5 × 1 min) and ACN (5

× 1 min). Subsequently, the solid support was transferred to an Eppendorf® tube and resuspended

in 200 μL of anhydrous THF and DMF mixture (9:2 v/v) containing 0.25 M DSA, 0.25 M PyBOP,

and 0.75 M DIPEA. The reaction mixture was shaken for 3 h at 45 °C, and washed with DMF and

ACN. The deprotection and cleavage of L-PS sense strand of the siRNA from the solid support

was accomplished with 500 μL of AMA (a 1:1 mixture of concentrated ammonium hydroxide and

aqueous methylamine) at 65 °C for 20 min. The cleavage solution was recovered, the CPG was

washed two times with 200 µL of aqueous EtOH (1:1 v/v), and all the liquid fractions were

APPENDIX

92

combined and evaporated to dryness. After suspending the crude products in 200 µL of aqueous

MeOH (10% v/v), the supernatants were purified by reverse phase HPLC. The purification of

ONs was carried out on an Agilent HPLC using a reverse phase column (Waters XBridge OST

C18, 10 × 50 mm, 2.5 µm) with solvent A being 100 mM TEAA in water, and solvent B being

MeOH. HPLC flow was set at 5 mL/min and a gradient was run at 65 °C from 12.5 to 98% B in

11 min.

Analytical LC-MS was carried out on Agilent LC-MS using a reverse phase column (Waters

Acquity OST C18, 2.1 × 50 mm, 1.7 µm) with solvent A being 400 mM HFIP and 15 mM TEAA

in water, and solvent B being MeOH. The flow was set at 0.3 mL/min and a gradient was run at

65 °C from 8 to 95% B in 14 min for all the ONs except for the L-PS sense strand of the siRNA

(7-80% B in 14 min).

6.2 Synthesis of L-DNA, L-FANA, negative control L-FANA

conjugates, and FANA

Standard phosphoramidite solid-phase synthesis instruments, reagents, and conditions were

used for the synthesis of the L-DNA, FANA, L-FANA, and negative control L-FANA (L-FANA

n.c. (35)) as previously described (112). FANA and DNA phosphoramidites were prepared as 0.15

and 0.1 M solutions in dry acetonitrile (ACN), respectively. 5-ethylthiotetrazole (0.25 M in ACN)

was used to activate the phosphoramidites for coupling. FANA phosphoramidite coupling times

were 600 s, with the exception of guanosine, which was allowed to couple for 900 s, and DNA

coupling times were 200 s, and 300 s for guanosine. For L-ON conjugates preparation, MMT-

protected 6-amino-hexanol phosphoramidite was attached to ON 5′ end using a coupling cycle

(without capping) with a coupling time of 600 s, followed by oxidation and detritylation (3% TCA

in DCM, 240 s). Prior to the coupling with the DSA moiety, the solid supports were successively

washed with 5% DIPEA in DCM and DCM. The solid supports were transferred to Eppendorf®

tubes and resuspended in a solution of DCM containing 200 mM of docosanoic acid, 400 mM

DIPEA, and 200 mM DIC. The Eppendorf® tubes were sealed with Parafilm M®, crowned with a

cap lock, and shaken overnight at 40 °C. Following the excessive washing with DCM and DMF,

deprotection and cleavage from the solid support was accomplished with 1 mL of concentrated

aqueous ammonia and EtOH mixture (3:1 v/v) for 48 h. Cleavage solutions was removed under

reduced pressure, the pellets were resuspended in 1 mL of autoclaved water, and the supernatants

was purified by reverse phase HPLC. Purification of L-ON conjugates was carried out on an

Agilent HPLC using a reverse phase C18 column (250 × 10 mm) with solvent A being 100 mM

TEAA in water supplemented with 5% ACN (pH 7.0), and solvent B being ACN. HPLC flow was

APPENDIX

93

set at 4 mL/min and a gradient was run at 50 °C from 0 to 100% B in 30 min. After collecting the

target materials, volatile TEAA was removed by repeated triple co-evaporation with water and

ethanol. Purification of FANA was performed using a Protein Pak DEAE 5PW analytical anion

exchange HPLC column with a solvent A being water and solvent B being 1 M LiClO4 in water. A

gradient was run from 0 to 50% B in 46 min. Following purification, LiClO4 salt was removed

using illustra NAP-25 size exclusion columns (GE Healthcare, Little Chalfont, UK) according to

the manufacturer's protocol.

Analytical LC-MS analysis of FANA and L-ON conjugates was carried out on Agilent LC-

MS using a reverse phase column (Waters Acquity OST C18, 2.1 × 50 mm, 1.7 µm) with solvent

A being 400 mM HFIP and 15 mM TEAA in water, and solvent B being MeOH. The flow was set

at 0.3 mL/min and a gradient was run at 65 °C from 8 to 95% B for all the ONs except for the L-

FANA n.c. conjugate (10-90% B in 14 min). The data were processed and deconvoluted using the

Bruker DataAnalysis software version 4.1.

6.3 Characterization of ONs

General remarks: Solvent A: H2O with 400 mM HFIP and 15 mM TEAA; solvent B: MeOH.

PO sense strand of the siRNA:

3.6 min (C18, gradient from 8 to 95% B in 14 min at 65 °C), 6641.3 Da (theoretical 6643.0 Da).

APPENDIX

94

L-PO sense strand of the siRNA:


PS sense strand of the siRNA:


APPENDIX

95

PS antisense strand of the siRNA:


L-PS sense strand of the siRNA:

12.6 min (C18, gradient from 7 to 80% B in 14 min at 65 °C); 7465.0 Da (theoretical 7466.7 Da).

APPENDIX

96

L-MOE:


L-DNA:

11.2 min (C18, gradient from 8 to 95% B in 14 min at 65 °C), 6186.0 (theoretical 6186.4).

APPENDIX

97

L-FANAnc:


L-FANA:


APPENDIX

98

FANA:


99

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List of Abbreviations

ACN acetonitrile

AMD age-related macular degeneration

AON antisense oligonucleotide

ApoB apolipoprotein B

ASGR asialoglycoprotein receptor

Bcl-2 human B-cell lymphoma 2 gene

CD40 cluster of differentiation 40

cDNA complementary DNA

CPP cell-penetrating peptide

cRGD cyclic pentapeptide Arg-Gly-Asp-d-Phe-Lys

DCA dichloroacetic acid

DCM dichloromethane

DecNa sodium decanoate

DIC N,N′-diisopropylcarbodiimide

DIPEA N,N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMEM Dulbecco's Modified Eagle Medium

DMF N,N-dimethylformamide

DNA deoxyribonucleic acid

DOTAP N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride

DSA; L docosanoic acid

DSS dextran sulfate sodium

EC50 effective concentration causing 50% of target gene silencing

LIST OF ABBREVIATIONS

126

E. Coli Escherichia Coli

EDC HCl N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

EDTA ethylenediaminetetraacetic acid

EtOH ethanol

FAE follicle-associated epithelia

FANA 2′-deoxy-2′-fluoro-arabinonucleic acid

FBS fetal bovine serum

GalNAc N-acetylgalactosamine

GFP green fluorescent protein

GI gastrointestinal

HCO-60 PEG-60 hydrogenated castor oil

HFIP hexafluoroisopropanol

HPLC high performance liquid chromatography

IBD inflammatory bowel disease

IEC intestinal epithelial cells

LC-MS liquid chromatography–mass spectrometry

LF Lipofectamine® 2000

LNA locked nucleic acid

lncRNA long non-coding RNA

L-ON lipid-oligonucleotide conjugate

LPS bacterial lipopolysaccharide

Map4k4 mitogen-activated protein kinase kinase kinase kinase 4

MeOH methanol

miRNA microRNA

MMP-10 metalloproteinase-10

MOE 2′-O-(2-methoxyethyl)-RNA

MPS mononuclear phagocyte system

mRNA messenger RNA

nc negative control

NFκB nuclear factor kappa B


127

NiMOS nanoparticles-in-microsphere oral system

Oil soybean oil-in-water emulsion

OMe 2′-O-methyl-RNA

ON oligonucleotide

PAMAM poly(amidoamine)

Papp apparent permeability coefficient

PBS phosphate-buffered saline

PCL poly(ε-caprolactone)

pDNA plasmid DNA

PEG poly(ethylene glycol)

PEI poly(ethyleneimine)

PLA poly(lactic acid)

PLGA poly(D,L-lactide-co-glycolide)

PMO morpholino ON

PNA peptide nucleic acids

PO phosphodiester linkages

PS phosphorothioate linkages

PSMA prostate-specific membrane antigen

PVA poly(vinyl alcohol)

PVBLG-8 poly(γ-(4-((2-(piperidin-1-yl)ethyl)-aminomethyl)benzyl-L-glutamate)

PVDF poly(vinylidene difluoride)

PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

qRT-PCR quantitative real-time polymerase chain reaction

RISC RNA-induced silencing complex

RNA ribonucleic acid

RNAi RNA interference

SELEX systematic evolution of ligands by exponential enrichment

SID-1 systemic RNA interference defective protein 1

SIF simulated intestinal fluid

siRNA small interfering RNA


128

shRNA short hairpin RNA

TAE tris-acetate-EDTA buffer

TCA trichloroacetic acid

TEAA triethylammonium acetate

TEER transepithelial electrical resistance

TEMED N,N,N′,N′-tetramethylethylenediamine

THF tetrahydrofuran

TLR Toll-like receptor

TNF tumor necrosis factor

TNFR2 TNF receptor 2

Toc-siRNA α-tochopherol-siRNA conjugate

TPP sodium tripolyphosphate

tRNA transfer RNA

UV ultraviolet

VEGF vascular endothelial growth factor

129

Curriculum Vitae

Personal Details

Name: Elena Moroz

Born: 26.01.1990 in Krasnoyarsk, USSR

Nationality: Russian

Address: HCI J 394 ETH Zurich

Vladimir-Prelog-Weg 1-5/10

8093 Zurich, Switzerland

Phone: +41 44 633 06 65

Email: [email protected]

Education

05/2012 – present Doctoral studies, research group of Prof. Dr J.-C. Leroux, ETH Zurich, Switzerland

09/2006 – 07/2011 Bachelor and Master in Chemistry, Lomonosov Moscow State University, Russia. Graduated with honors (summa cum laude)

09/2004 – 07/2006 Advanced Chemistry and Mathematics Section, School #144, Krasnoyarsk, Russia. Graduated with honors

Work experience

09/2010 – 04/2012 Researcher, R&D projects on carbon nanotubes- and polyaniline-based composite materials, MSU – LG Chem Joint Laboratory, Moscow, Russia

03/2011 Research internship on interaction of DNA with oppositely charged carbon nanotubes, Graduate School of Environmental Studies, Nagoya University, Japan

07/2010 – 08/2010 Research internship on arrays of polyelectrolyte multilayer chambers for site-specific release of drugs, Patterning Fabrication, Institute of Materials Research and Engineering, Singapore

CURRICULUM VITAE

130

Teaching experience

09/2013 – 12/2015 Teaching assistant at the general chemistry lab course

02/2013 – 05/2013 Teaching assistant at the galenik lab course

Supervisor of master students Aikaterini Papagiannopoulou, Carmen Egger, and Vivien Nachtsheim

Awards

2016 First Prize Poster Award, Swiss Pharma Science Day, Bern, Switzerland

2012 Winner of LG Chem Scholarship, Moscow, Russia

2011 Prize winner in MSU Chemistry Department Project Contest, Moscow, Russia

2010 Prize winner, 6th Saint-Petersburg Young Scientists Conference “Modern Problems of Polymer Science”, Saint-Petersburg, Russia

2008 Prize winner, “XVIII Mendeleev Contest of Students”, Belgorod, Russia

2008 Prize winner, Undergraduate and Postgraduate Students International Conference on Fundamental Sciences “Lomonosov-2008”, Moscow, Russia

2006 Winner of “Russian Federation talented young people support” award, Russia

2006 Prize winner of All-Russian chemistry Olympiad final stage, Ufa, Russia

Language proficiency

Russian native

English fluent

German advanced

Japanese beginner

131

Scientific Contributions

Publications

Moroz E., Lee S.H., Yamada K., Halloy F., Martinez-Montero S., Jahns H., Hall J., Damha M. J., Castagner B., and Leroux J.-C. Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Mol. Ther. Nucleic Acids, 2016, 5, e364

Moroz E.1, Matoori S.1, and Leroux J.-C. Oral delivery of macromolecular drugs: where we are after almost 100 years of attempts. Adv. Drug Deliv. Rev., 2016, 101, p. 108–121 (1equal contribution)

Lee S.H., Moroz E., Castagner B., and Leroux J.-C. Activatable cell penetrating peptide–peptide nucleic acid conjugate via reduction of azobenzene PEG chains. J. Am. Chem. Soc., 2014, 136 (37), p. 12868–12871

Buerkli C.2, Lee S.H.2, Moroz E., Stuparu M.C., Leroux J.-C., and Khan A. Amphipathic homopolymers for siRNA delivery: probing impact of bifunctional polymer composition on transfection. Biomacromolecules, 2014, 5 (5), p. 1707–1715 (2equal contribution)

Milakin K.A., Korovin A.N., Moroz E.V., Levon K., Guiseppi-Elie A., and Sergeyev V.G. Polyaniline-based sensor material for potentiometric determination of ascorbic acid. Electroanalysis, 2013, 25 (5), p. 1323–1330

Moroz E.V., Zakharova J.A., Sergeyev V.G., and Zezin A.B. Ternary complexes polyelectrolyte – oppositely charged surfactant – carbon nanotubes. Formation and properties. Doklady Physical Chemistry, 2011, 441 (1), p. 212–214

Zakharova J.A., Moroz E.V., and Otdel'nova M. V. Poly-(N-ethyl-4-vinylpyridinium) charge density crucially affects the phase state of its complexes with oppositely charged surfactant in aqueous solution. Doklady Physical Chemistry, 2011, 438 (1), p. 94–97

Oral Presentations

Moroz E.V., Grishagin I.V., and Pergushov D.V. Interpolyelectrolyte complexation in chloroform. “XVIII Mendeleev Contest of Students”, Belgorod, Russia, 22 – 26 April 2008.

Moroz E.V. and Zakharova Yu.A. The influence of polyelectrolyte charge density on thermosensitivity of its complexes with surfactant. Sixth Saint-Petersburg Young Scientists Conference “Modern Problems of Polymer Science”, Saint-Petersburg, Russia 18 – 21 October 2010.

Moroz E.V., Lee S.H., Castagner B., and Leroux J.-C. Amphiphilic nucleic acid drugs delivery to colonic tissues. Swiss Galenik Meeting, Geneva, Switzerland, 1 – 2 July 2013.

Moroz E.V., Lee S.H., Jahns H.I., Hall J., Yamada K., Damha M.J., Castagner B., and Leroux J.-C. Amphiphilic nucleic acid conjugates for the treatment of colonic diseases. Doktorandentag ETH Zürich, Zurich, Switzerland, 10 September 2014.

Moroz E., Lee S.H., Jahns H.I., Hall J., Yamada K., Damha M.J., Castagner B., and Leroux J.-C. Amphiphilic nucleic acid conjugates for local treatment of intestinal diseases. 1st European Conference on Pharmaceutics – Drug Delivery, Reims, France, 13 – 14 April 2015.

SCIENTIFIC CONTRIBUTIONS

132

Poster Presentations

Grishagin I.V. and Moroz E.V. Preparation of interpolyelectrolyte complexes of star-shaped polyacrilic acid in chloroform. Undergraduate and Postgraduate Students International Conference on Fundamental Sciences “Lomonosov-2007”, Moscow, Russia, 11 – 14 April 2007.

Moroz E.V. and Grishagin I.V. Preparation and properties of interpolyelectrolyte complexes of star-shaped polyacrilic acid in low-polar organic media. Undergraduate and Postgraduate Students International Conference on Fundamental Sciences “Lomonosov-2008”, Moscow, Russia, 8 – 11 April 2008.

Moroz E.V. and Zakharova Yu.A. Polyelectrolyte charge density determines phase behaviour of its complexes with surfactant. Fifth Kargin Polymer Conference “Polymers-2010”, Moscow, Russia, 21 – 25 June 2010.

Moroz E.V., Lee S.H., Castagner B., and Leroux J.-C. Nucleic acid drugs conjugates for efficient uptake in colon tissues. Scholarship Fund of the Swiss Chemical Industry, 5th Symposium, Zurich, Switzerland, 19 November 2012.

Moroz E.V., Lee S.H., Castagner B., and Leroux J.-C. Colon-specific delivery of nucleic acid drugs via oral administration. 1st Rare Diseases Summer School, Wädenswil, Switzerland, 4 – 6 July 2013.

Moroz E., Yamada K.; Deleavey G., Damha M. J., Lee S. H., Castagner B., and Leroux J.-C. Amphiphilic antisense oligonucleotides for local delivery to the colon. Pharma Posterday, Zürich, Switzerland, 20 August 2013.

Moroz E., Yamada K., Deleavey G., Damha M. J., Lee S. H., Castagner B., and Leroux J.-C. Chemically modified nucleic acid conjugates for local delivery to the colon. Swiss Pharma Science Day, Bern, Switzerland, 28 August 2013.

Moroz E.V., Lee S.H., Yamada K., Damha M.J., Castagner B., and Leroux J.-C. Amphiphilic nucleic acid conjugates for local treatment of colonic diseases. 10th International Conference and Workshop on Biological Barriers, Saarbrücken, Germany, 16 – 21 February 2014.

Lee S.H., Moroz E., Castagner B., and Leroux J.-C. Cell penetrating peptide–peptide nucleic acid (CPP-PNA) conjugates for inflammatory bowel disease therapy. 2nd International Congress on Research of Rare and Orphan Diseases, Novartis Campus, Basel, Switzerland, 5 – 8 March 2014.

Moroz E., Lee S.H., Yamada K., Jahns H.I., Halloy F., Hall J., Damha M.J., Castagner B., and Leroux J.-C. Nucleic acid conjugates for colonic disease oral therapy. AAPS Annual Meeting and Exposition, Orlando, USA, 25 – 29 October, 2015.

Moroz E., Lee S.H., Yamada K., Halloy F., Martinez-Montero S., Jahns H., Hall J., Damha M. J., Castagner B., and Leroux J.-C. Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Swiss Pharma Science Day, Bern, Switzerland, 31 August 2016.

133

Acknowledgements

I gratefully acknowledge the financial support from the Gebert Rüf Foundation (GRS-

041/11), the Swiss National Science Foundation (310030_135732), the Canadian Foundation for

Health Research, and the JSPS Strategic Young Researcher Overseas Visits Program for

Accelerating Brain Circulation, which enabled the research presented in this thesis. I also thank

Prof. Dr Jean-Christophe Leroux, Dr Jong Ah Kim, Athanasia Dasargyri, and Anna Polomska for

their critical reading and thoughtful comments on this thesis. Thank you Peter Tiefenböck and

Prof. Dr Bruno Gander for the German translation of the abstract for these thesis.

I would like to express my sincerest gratitude to Prof. Dr Jean-Christophe Leroux for

accepting me in his research group, giving me this extremely interesting project, providing the best

environment to carry our my research, and for his patience, motivation, and immense knowledge.

I am most grateful to Dr Soo Hyeon Lee for teaching me all the magic of nucleic acids, for being

an amazing supervisor and even better friend. I am grateful to my collaborators Drs Ken Yamada

and Saúl Martínez-Montero from the group of Prof. Dr Masad J. Damha in McGill University, and

François Halloy and Dr Hartmut Jahns from the group of Prof. Dr Jonathan Hall at ETH Zurich

for the synthesis of lipid-ON conjugates. Without their precious work it would not be possible to

conduct this research. Many thanks in particular to Prof. Dr Jonathan Hall for agreeing to take the

time to be my co-examiner. Thanks also to Prof. Dr Michael Detmar and his group members for

giving me access to their instruments.

I thank all the present and former labmates not only for the stimulating discussions inside

and outside of the lab, but also for all the fun we have had in the last four years. In particular,

thanks to Jong Ah Kim, Davide Brambilla, Diana Andina, Xiangang Huang, Jessica Schulz, Bastien

Castagner, Paola Luciani, Lorine Brülisauer, Arnaud Felber, Vincent Forster, Mattias Ivarsson,

Ander Estella, Virginie Schmid, and Monica Langfritz for their help and friendship. I truly thank

Ania Polomska and Nancy Dasargyri who have walked through the doctoral studies with me right

from the start and became my best friends here. Thank you Mauri Roveri for being the best

officemate of all time. Thank you my Russian-speaking friends from ETH Zurich and outside,

particularly Dima Mazunin, Ira Bolotova, and Assem Zhakupova, for making me feeling that I have

never left home.

I am grateful to my school chemistry teacher Elena Molchanova and my master’s thesis

supervisor Dr Julia Zakharova for enlightening me the first glance of science. I would like to thank

my parents and brother for always supporting me throughout my life. Finally, I am grateful to my

boyfriend, Hayato, for giving me the reason to leave the lab and come home.

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