Nucleic acid and cell based therapies

Gene Therapy

1980s and early 1990s Proteins of therapeutic use

nucleic acid based therapeutics center around

gene therapy and antisense Tehnology

Early 2007 only three nucleic acid based therapeutics gained worldwide approval.

antisense-based product (Vitravene)

aptamer (Macugen)

gene therapy product(Gendicine)

In cell based, fully differentiated cells or groups of cells used

organ or tissue transplantation

Principle of gene therapy

Stable Introduction of a gene into a genetic complement of cell in such a way that expression of gene achieves a therapeutic goal.

It is the replacement of defective gene with a new healthy gene

It is a curative approach to inborn error of metabolism.

Some diseases for which gene based clinical trials are in process

Basic approach to gene therapy

Vector systems used to deliver genes into mammalin cells

Practical approaches that may be pursued when undertaking gene therapy

In vitro

• In vitro gene therapy entails removal of target cells from the body followed by their incubation with nucleic acid-containing vector. After the vector delivers the nucleic acid into the human cells, they are placed back in the body.

In vivo

• In vivo gene therapy involves intravenous administration of the vector. The vector has been designed such that it will only recognize and bind the intended target cells. In this way, the nucleic acid is delivered exclusively to those cells

In situ

• In situ gene therapy entails direct injection of the vector immediately adjacent to the body target cells.

Some additional questions

choice of vector, target cell and protocol used

depends upon a number of

consideration.

gene therapy

treatment

protocol used

choice of target cells

Vectors used in gene therapy

Types of vectors

Vectors capable of introducing genes into recipient cells.

Viral based

Non –viral based

Retroviral vectors

Enveloped viruses.

Genome consists of ssRNA.

RNA transcribed and yields dDNA.

Integrates into host cell.

Structural genes in retroviral genome

• Code for viral protein.gag

• Codes for reverse transcriptase.pol

• Codes for viral envelope proteins.env

Other componentsLTR harbours promoter and enhancers.

promote integration

ψ packaging sequence.

viral RNA packaging

Retroviral genome

LTR Ψ gene of interest LTR

Retrovirus life cycle

Packing cells

Genetic material introduced into cell

Recombinant contain gag,pol,env and replication

deficient

One time,single hit gene transfer system e.g

MoMuLv

Properties of RetrovirusIntegrate their proviral DNA into replicating cells.

Efficiency of gene transfer in sensitive cells.

Long term,high level expression.

Integrates randomly.

Retro virus are promiscuous.

Complete copies are passed to daughter cells.

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Good,high level stocks of replication incompetent retroviral particles.

Studies carried out in animal species.

Advantages Disadvantages

Integrate into genome in a stable fashion Often damage during purification and concentration.

Effectively enter into various cell types. Ability to infect only dividing cells.

Transduction efficacy. Lack of selectivity.

Fairly high level expression. Not infect all dividing cells.

Easy to propagate. Integrate randomly into the chromosome of the recipient cells.

Drawback of retroviral gene therapy

In 2002, Patients with SCID-X1 received with retroviral therapy

developed leukemic like condition

Due to the proviral integration at the site near the LM02 proto-oncogene promoter leading to gene activation.

This resulted in initial ban on retro viral based gene therapy trials in some world regions.

Adenoviral and additional viral vectors

Some other vectors are used for the purpose of gene therapy in which includes

Adenoviruses

Adenoassociated viruses

Herpex virus

Sindbis virus

AdenovirusesRelatively large

Non enveloped structure

Double stranded DNA

Large genome and complex

Adenoviruses have some advantages or disadvantages

Advantages Disadvantges

Capable of gene transfer to non dividing cells

High immunogenic in man

Easy to propagate Duration of tranferredgene can vary

High gene expression Highly selective for cells

Adeno-associated virus

Small

Single stranded DNA

Need coinfectingadenoviruses replicate

Small genes can be introduced into adenoviral vector.

Facilitate long term gene expression

Herpes Simplex Virus Herpes simplex virus

Neurotropic vector

Can deliver genes to PNS and CNS.

Upon infection Remains latent

in non dividing

neurons

Genome is in unintegrated form

Difficult to generate

viable herpes simplex particle

Sindbis Virus

Member of alphavirus

familyssRNA

SimpleInfecting non dividing cells

Formation of engineered sindbis virus

Manufacture of viral vectors

Non-viral vectors

•Use naked plasmid dna or dnacomplexes as non-viral vector.

•Advantages:

• their low/non-immunogenicity;

•non-occurrence of integration of the therapeutic gene into the host chromosome

Methods of non-viral gene delivery:

physical (carrier-free)

chemical approaches (synthetic vector-

based gene delivery).

Naked DNA:simplest method of non-viral transfection.

Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred .

low expression.

Lipoplexes: Plasmid DNA can be covered with lipids inan organized structure like a liposomecomplexed with DNA it is called a lipoplex

3 types of lipids: anionic (negativelycharged)

neutral

cationic (positively charged)

Polyplexes:Complexes of polymers with DNA are called polyplexes

consist of cationic polymers and their production is regulated by ionic interactions

polyplexes rapidly remove from circulation.

PEG attachment

cellular entry of (non-viral) gene delivery:

Target: appropriate cell surface

Therapeutic plasmid must enter the cell and reach the nucleus intact. Cellular entry is generally achieved via endocytosis.

Routes by which plasmid can reach nucleus:

direct nuclear entry transport through nuclear pores

manufacture of plasmiddna:

Source microorganism

fermentation

Cellular recovery and

lysisRemoval of cell

debris

Plasmid precipitation

Chromatographic purification

Concentration,if requiredFormulation

and packaging

Genetic diseases?

A genetic disease is any disease that is caused by an abnormality in an individual's genome.

Over 4000 diseases are characterized

Causes

Factors

Drug companies contribution.

Expression level

none of the first-generation gene-

delivering vectors

have proven

fully satisfactor

y.

Actual genes

responsible of these

diseases are

identified..HGP.

Some

shows

complexity

Example EX VIVO GT

1990 – 4 year old AshantiDaSilva had a geneticdisorder called severecombinedimmunodeficiency (SCID)

Defect in ADA gene results inan accumulation of dATP,which is toxic to certaintypes of T cells

Takes down the entireimmune system.

Example: IN-VIVO GT

mutation on 7th chromosome.

Defective cystic fibrosistransmembrane conductanceregulator (CFTR) gene.

Normally it serves as a pumpat the cell membrane to moveelectrically charged chlorideatoms out of the cells

If cells can’t move chloride out,they absorb water trying todilute the chloride in the cell

This leads to the production ofTHICK sticky mucus

Vectors used to deliver CF gene to airway epithelial cells:1993 vector used: Adenovirus

1995 liposome

have potential to avoid critical problems immune response, limited packaging capacity, and random integration .

Liposomes may be mildly effective, but their activity does not last.

Gene therapy

and cancer

1.4 million cases reported.

50% survival rate using surgery, chemo/radiotherapy

Low success rate due to:

1. requirement for improved, more target-specific vector systems.

2. A requirement for a better understanding of how cancer cells evade the normal immune response.

3. suffering from advanced and widespread terminal cancer (i.e. little/no hope of survival if treated using conventional therapies). Cancers at earlier stages of development will probably prove to be more responsive to gene therapy.

Boosting the immune response:

aim to boost the body's natural ability toattack cancer cells.

Our immune system has cells thatrecognize and kill harmful things that cancause disease, such as cancer cells.

Involved introduction of TNF…

Steps:

Anti-cancer strategy:

Pro-drug gene therapy:

GENE THERAPY AND AIDS

INTRODUCTIONUseful in treating medical condition inherited disease,cancer and infectious disease.

AIDS is a viral disease caused by intracellular pathogens.

Different approaches used to treat AIDS

•Introduction of a gene into pathogen susceptible cells is termed as intracellular immunization such as introduction of a gene into viral sensitive cell coding for an altered HIV protein such as gag tat env

•Mutant form of gag is capable of inhibiting viral replication

The transfer to sensitive cell of a gene coding for antibody fragments capable of binding to the HIV enveolpe proteins this interfere with viral assembly

Recombinant cell have also been generated which are capable of secreting soluble form of HIV cell surface receptor like CD4 antigen

These soluble viral receptor would bind with viriones

GENE BASED VACCINEoAdministration of a DNA vector housing the gene coding for a surface antigen protein from the target pathogen

oAny body cell could be targeted.The target cell export the resultant antigenic protein

oGene expression need only be transient to facilitate the induction of an immune response

Gene based vaccine have entered for clinical trials include malaria, hepatitis B and AIDS

Gene based vaccine have entered for clinical trials include malaria, hepatitis B and AIDS

Causes Of Disease Occurrence

the expression of oncogenes

overexpression of cytokines

overproduction of angiotensinogen Hypertension

Antisense Technology Definition

The specific sequence of nucleotide bind to mRNA or DNA when the sequence is known that will cause a

specific disease. This leads to gene turning off

Antisense technology Antisense Nucleotides

Short and single stranded stretches of Nucleic acid having a specific sequences of nucleotides.

Oligonucleotide binds to DNA or mRNA.

Binding prevents/block the Transcription and Translaion process

Antisense oligonucleotides and their mode of action

Identify the suitable sequences of mRNA

Blind or shot gun used to designed the oligonucleotides

Computer programs For mRNA sequencing and its accessible region for oligonucleotides

Antisense oligonucleotides and their mode of action

Advantages of OligosIt acts as therapeutic agent in

Cancer

Viral diseases such as HIV,hepatitis B Herpes and Papilloma Infection

Example Cancer

BCL2 gene

These gene increase the neoplastic progression

Inhibit Cell apoptosis

Survivin gene

Present in cancer patient.

Cell division and apotosis inhibiton.

Lung ,colon,breast and prostate cancer.

Clusterin genes

Codes for cytoprotectivechaperon protein.

Upregulation results in various human cancer.

Advantages

Low Toxicity

Required in minute

quantity as mRNA is

present in nanomolar

concentration

Easily Formed by automatic synthesizer

Disadvantages

sensitivity to nucleases

very lowserum half lives

poor rate of cellular uptake

orally inactive.

Oligonucleotide pharmacokinetics and delivery

Intravenous administration

Sub cutaneous administration

Intra dermal administration

Charged Oligo's enter in cell by receptor mediatedendocytosis

UnCharge Oligos enter in the cell through Passivediffusion and endocytosis

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Liposomes mediated and polymeric carriers are used for the delivery of oligos

Oligos appear to be ultimately metabolized within the cell by the action of nucleases, particularly 3´-exonucleases. Breakdown metabolic products are then mainly excreted via the urinary route.

Phosphodiester linkage

Manufacture of oligos

Oligonucleotides

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.

Commonly made in the laboratory by solid-phase chemical synthesis.

Oligonucleotides

“Oligo” – Prefix meaning few (~ 2-10).

Nucleosides can be obtained from either natural sources (i.e. salmon sperm) or chemically synthesized.

We use phosphoramidite method for manufacturing oligos.

Phosphoramidite Method

Developed in 1980.

The principle of this method was developed by McBride and Caruthers in 1983.

Currently considered as the standard synthesis.

Used in most automated synthesizers today.

Cycle

The cycle consists of four steps:

1. De-protection

2. Coupling

3. Oxidation

4. Capping

De-protection

Trityl group attached to the 5’ carbon of the pentose sugar of the recipient nucleotide is removed by trichloroacetic acid (TCA) leaving a reactive hydroxyl group.

Coupling

The phosphoramidite monomer is added in the presence of an activator such as a tetrazole

This structure then reacts with the hydroxyl group of the recipient and the 5’ to 3’ linkage is formed.

Tetrazole = Class of synthetic organic heterocyclic compound, consisting of a 5-member ring of four nitrogen atoms and one carbon atom. The simplest is tetrazole itself, CH₂N₄. They are unknown in nature.

Oxidation

The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide.

The traditional method of achieving this is by treatment with iodine in water.

Capping

Any remaining free 5’-hydroxyl groups are blocked at the capping step in an irreversible process.

After having synthesized the full length sequence, the oligonucleotide is then released from the solid support using a base, such as aqueous ammonia or a mixture of ammonia and methylamine. This will also remove protection groups from the nucleobases.

The oligonucleotide is now ready for purification.

oligonucleotide is purified with RP-HPLC where the retention time is to a large.

RNA interference and ribozymesRNAi represents a sequence-specific post-translational inhibition mechanism of gene expression,

Induced ultimately by dsRNA

Entry of dsRNA triggers its cleavage into short (21–23 nucleotide long) sequences called short interfering RNAs

This cleavage is catalyzed by a Enzymeknown as Dicer

RNA inducedsilencing complex (RISC)antisense’ siRNA strand then facilitates RISCbinding to a specifi c mRNA via Watson–Crickbase complementarity, which is thendegraded by RISC nuclease activity.

Ribozyme RNA sequences can function as catalysts. This is known as Ribozymes

Many ribozymes will cleave

Target mRNA where there exists a particular triplet nucleotide sequence G–U–C

Ribozyme

AptamersAptamers are single-stranded DNA or RNA-based sequences allowing them to bind a specific target molecule.

Identification of specific aptamers binding the target molecule

SELEX (systematic evolution of ligands by exponential enrichment)

affinity-based purifi cation,

target validation

drug discovery, diagnostics and therapeutics.

low immunogenicity

In order to prevent renal removal, aptamers are usually conjugated to PEG.

Their half-lives can most effectively be extended via chemical modification