thesis final finale bd (1)

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In silico vector engineering of recombinant

adeno-associated viral vectors for treatment of

Leber congenital amaurosis

Brett Davis Senior Thesis

Endicott College Spring 2014

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Acknowledgments

I would like to acknowledge Dr. Jessica Kaufman for her assistance in completing this

project, along with Dr. Jason Nichol as my thesis advisor. I would also like to thank the Biotech class of 2014 for their support.

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Table of Contents

Title Page Number

List of figures……………………………………………………………………………..4

Abstract with key words…………………………………………………………………..5

Introduction………………………………………………………………………………..6

Materials and Methods…………………………………………………………………...13

Results……………………………………………………………………………………19

Discussion………………………………………………………………………………..23

References………………………………………………………………………………..27

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List of Figures

Title Page Number

Figure 1: rAAV vector for LCA 2 (RPE65)……………………………………………..11

Figure 2: Genome Compiler main workshop area……………………………………….14

Figure 3: AAV inverted terminal repeat BLAST search results…………………………16

Figure 4: AAV vector template built in Genome Compiler……………………………..18

Figure 5: rAAV vector for LCA 1 (GUCY2D)………………………………………….20

Figure 6: rAAV vector for LCA 3 (SPATA7)…………………………………………...20

Figure 7: rAAV vector for LCA 4 (AIPL1)……………………………………………...20

Figure 8: rAAV vector for LCA 9 (NMNAT1)………………………………………….21

Figure 9: rAAV vector for LCA 11 (IMPDH1)………………………………………….21

Figure 10: rAAV vector for LCA 13 (RDH12)………………………………………….21

Figure 11: rAAV vector for LCA 15 (TULP1)…………………………………………..22

Figure 12: rAAV vector for LCA 16 (KCNJ13)…………………………………………22

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Abstract

The use of viruses as vehicles for the delivery of therapeutic DNA necessitates

engineering of recombinant vectors to be used in the treatment and testing of animal

models. It is necessary that the design process be done in silico to ensure efficiency and

plausibility of the gene therapy treatment. For the recombinant viral vector to be

engineered, it is not only necessary to incorporate therapeutic DNA, but also certain

features (e.g. promoter) needed for expression of the transgenic material. The goal of this

thesis project is to use Genome Compiler, a genetic design platform, to engineer adeno-

associated viral vectors for the treatment of different forms of LCA. The basis for the

design will be taken from Jacobson et al who successfully treated animal models with

knock-in LCA 2 (mutation in RPE65 gene) using recombinant AAV vectors. Genome

Compiler will allow for in silico vector engineering of many different types of LCA,

defined by mutations of different genes, using the published design of Jacobson et al as a

template.

Key Words: Adeno-associated virus (AAV), Leber congenital amaurosis (LCA), viral

vector design, in silico design, RPE65, Genome Compiler, synthetic design

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Introduction

Since its emergence on the biotech scene in the early 1990’s, the potential of gene

therapy has only been partially realized with roughly 1700 clinical trials since its

inception1. This period of time, while reflecting a certain level of progress and scientific

excitement, has been tumultuous and slow going1. The initial struggles in gene therapy

were primarily related to safety and biocompatibility concerns, which led to a significant

decrease in optimism that DNA based treatments would dominate the future of therapy1.

This adversity has more recently been replaced by success with positive results in treating

ADA deficiency, SCID-X1 and adrenoleukodystrophy, among others1. These cases have

built confidence and forward momentum for gene therapy studies in the current decade,

while diminishing the stigma towards such treatments from previous negative results. The

argument for utilizing gene therapy techniques has become more robust as the early

questions of safety and efficacy have been addressed.

The basic principle behind gene therapy, to use DNA as a therapeutic tool for

treating disease, is a simple one but complicated to implement and deliver 1, 5-7, 13, 14, 19.

The readily available and ever growing library of basic biological information (e.g.

sequence data) along with advances in genetic engineering processes has simplified the

design of DNA therapeutics 1,2. The difficulty arises in determining how a functional

copy of a gene can be delivered to a patient efficiently and safely. The primary form of

gene therapy involves packaging a functional gene into a vector to be delivered to the

treatment area. Vectors can be described as vehicles that carry foreign genetic material to

affected areas that can then be replicated and expressed for a desired, advantageous affect.

The type of vector determines the functionality; the therapeutic DNA copy is either

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incorporated into the host genome or delivered to the nucleus to be transcribed and

translated separately. These two results are most efficiently achieved by using viruses as

vehicles for delivering DNA to affected cells. The use of viruses in gene therapy has

resulted in the primary safety concern of biocompatibility in the process of delivering the

therapeutic DNA copy1, 3-7, 13-21. The aforementioned failure and subsequent successes in

gene therapy are directly related to an increased understanding of molecular medicine and

how viral vectors affect the body1.

In the recent history of gene therapy, retroviruses emerged as the vector candidate

that would improve the likelihood of success in clinical trials1, 5, 13-15. Retroviruses are

RNA based and utilize reverse transcriptase to become double stranded DNA from its

genome14. Inside of the nucleus, retroviruses incorporate into the host chromosome using

integrase, an enzyme produced by the virus that results in the provirus (incorporated in

host genome) state14. In the context of gene therapy, engineered genetic material (the

transgene) of another organism is incorporated into the RNA genome of the retrovirus.

The recombinant viral-human vector is delivered to affected cells that incorporate the

retroviral and transgenic DNA into the host genome. The desired result is the

transcription and translation of the therapeutic gene copy to make functional protein

capable of treating a certain disorder. The primary advantage of retroviral gene therapy is

that it allows for a possible permanent outcome and treatment. This is achievable due to

retroviruses being proviruses that will continue to be expressed after cell division,

resulting in more copies of functional transgenic protein being made 14, 15. The primary

concern for using retroviral vectors is, however, directly related to this genome

incorporating quality. This characteristic is inherently concerning in that it raises a

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distinct possibility of cancer causing oncogenesis and mutagenesis5, 13-15. Insertion of the

recombinant retroviral vector into host chromosomes is relatively random, and there can

be disruption of functioning genes or more critical genes controlling cell division5, 13-15.

In a recent study, the successful disease treatment of X-SCID using a retroviral gene

therapy approach had the adverse result of leukemia developing in several patients 5, 13-15.

This has caused gene therapy researchers to either develop solutions to address

oncogenesis of retroviral treatment or turn to other vector options 1, 5, 13-15, 24.

The primary response to retroviral vectors causing cancer was increased interest

in using adenoviral vectors 1, 24. Adenoviruses are comprised of a double stranded DNA

genome that enter cells but are transient; they do not incorporate into host cell

chromosomes. Contrary to retroviruses, adenoviruses have a desired affect as gene

therapy vectors by carrying transgenic DNA that is transcribed separately from the host

genomic DNA. Among the advantages of adenoviruses are the large size (37kb) for

incorporation of therapeutic DNA and the wide range of dividing and non-diving cell

types they can affect24. Additionally, the strains used to construct recombinant vectors are

well characterized and therefore have a predictable functionality24. The drawback in

using adenoviruses for gene therapy is the limited amount of time cells are able to

produce protein from the transgene. This is a result of the transient nature of

adenoviruses; failure to incorporate into the host genome addresses the issue of causing

cancer but expression fails to carry through cell divis ion. Adenoviral treatments often

times require large amounts of product to have an impact on treating a certain disorder.

On the issue of safety, the high profile case in 1999 involving the death of a research

participant treated with an adenoviral vector was a clear message that adenoviruses can

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be particularly harmful to humans 25. This case limited the growth of adenoviral studies

greatly and in turn put gene therapy into a state of regress thereafter 1, 25.

The status of gene therapy as a treatment option was dependent on using viral

vectors that would be efficient, but would place the primary importance on safety. The

emergence of adeno-associated viral (AAV) treatments in the last seven years has

allowed for a reemergence and revival of gene therapy by addressing the principal issue

of safety 1, 3, 4, 16-21. The genome of the virus is 4.7 kb in length and is single stranded

DNA26. There are two open reading frames of the virus, described as rep (replication) and

cap (capsid). Rep is responsible for replication of the virus and cap encodes for the

protein envelope surrounding the DNA26. A key difference between AAV from

adenoviruses is the incorporation of the viral DNA into the host genome. Unlike

retroviruses, AAV have a designated site for integration on chromosome 9 in a 2 kb

region of the long arm26. The primary concern of gene therapy treatments being safety,

AAV has a distinct advantage in having no known pathogenicity 3, 6, 7, 19, 20. This non-

disease causing virus’ safety profile is supported by its limited immunogenicity or ability

to cause an immune response 6, 19, 20. Additionally, recent studies primarily associated

with retinal gene therapy have found that AAV has the ability to transduce post-mitotic

cells efficiently while maintaining high levels of expression 20. The primary drawback in

using AAV as a vector for gene therapy is its relatively small genome. AAV can only

package a certain amount of exogenous material; this includes an enhancer, a promoter, a

polyadenylation signal, and a human gene of interest. The amount of exogenous material

cannot surpass 4.7 kilobases, which limits the use of AAV in gene therapy for disorders

caused by mutations in larger genes.

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In 2006, the University of Florida did a proof of concept study using AAV vectors

to treat Leber congenital amaurosis 2 (LCA 2) in different animal models17. The positive

results of this study, improved vision and lack of immune response in at least 70% of

animals (for each experiment), paved the way for the 2009 study at University of

Pennsylvania in human subjects 4,17. This subsequent study, along with the safety and

efficacy update published in 2010, confirm successful treatment of LCA 2 using AAV

vectors 4, 16, 17.

Congenital disorders are genetic defects present from birth and in certain cases the

disorder’s symptoms can worsen throughout ones lifetime 8, 22. Leber congenital

amaurosis (LCA) is one such disorder, causing partial blindness at birth and worsening

sight later in life 4, 8, 22. More specifically, LCA describes a set of retinal dystrophies that

arise in early childhood with the primary symptoms of vision loss, nystagmus

(involuntary eye movement), and severe retinal dysfunction 8, 22. These symptoms occur

along with many others, such as photophobia (abnormal intolerance to light) and high

hyperopia (farsightedness), which contribute to an overall diagnosis of blindness 22. This

disorder affects an estimated 1 in 30,000 to 1 in 80,000 live births 22. LCA is primarily

identified through behavior of those afflicted in the first years of life 4, 22. In most cases

vision worsens and is lost entirely in a patient’s 30’s or 40’s 4. LCA has historically been

viewed as a managed disease rather than a treatable one, with correction of refractive

error and use of low vision aids being the primary sources of managing the disorder while

patients still retain some vision22. There are currently sixteen types of LCA defined by

OMIM, with mutations in specific genes accounting for each variation of the disorder 8.

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This thesis project involved the use of bioinformatics tools to design novel adeno-

associated viral vectors (AAV) for treating different forms of Leber congenital amaurosis

(LCA). This was be accomplished by modeling the vector designs after those used in the

initial proof of concept studies (2006) that used adeno-associated viral vectors to treat

LCA 2. The image below describes the features of the two rAAV used in the POC study

(Figure 1)17. The human cytomegalovirus (CMV) immediate early (ie) enhancer is

present to ensure cell-type-specific gene expression 27. The inverted terminal repeats on

both ends of the vectors are structures of AAV that are 145 base pairs long and are

necessary for replication of the genome 26. Simian virus (SV) 40 polyadenylation

(poly(A)) signal is an RNA sequence that adds poly(A) tails to mRNA sequences to allow

for maturation28. This process is essential for eventual translation and expression of the

transgenic material of the recombinant viral vector28.

Figure 1: (A) AAV2-CBo-hRPE65, 4070 bp, and (B) AAV2-CBSB-hRPE65, 3921 bp.

The two vectors differ by 152 bp at the 5' end of the CMV immediate early enhancer (long vs. normal). ITR describes the AAV2 inverted terminal repeat, followed by the

CMV immediate early enhancer. This is preceded by chicken B-actin (CBA) promoter, Exon 1, intron, and Exon 2. The transgene, human RPE65 cDNA, follows Exon 2. The

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design is concluded by the presence of SV40 polyadenylation signal before the opposite ITR. 17

The growing field of gene therapy along with increased advances in molecular

biology and medicine has signaled a need for in silico design of viral vectors to be used

in clinical trials. In silico, or ‘performed by computer’, is a necessary step for vector

design that occurs before ex vivo and in vivo studies of affects on animal models or

human tissue/subjects can take place 2. The growing field of bioinformatics, management

information systems for molecular biology, has made this possible on two levels 2. Firstly,

the growth of basic biological information databases has supplied researchers and

programmers alike with readily available and accessible sequence data 2. Secondly,

programs and tools are now being developed with specific goals in mind (e.g. building

phylogenic trees showing relationships between organisms) that make use of the

exponentially growing databases 2. Vector design in silico is now possible with the

development of programs that use sequence information input to design novel,

recombinant viral vectors. Genome Compiler is one such program and was used in this

thesis project to design AAV vectors with aforementioned features for the treatment of

different forms of LCA.

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Materials and Methods

The three vector design programs that were chosen to be tested were Benchling,

Genome Compiler, and Gene Designer. It was necessary to assess the program’s relative

strengths and weaknesses on the following criteria: interface, usability, robustness,

aesthetics of design, and workflow. This evaluation ensured the best program was chosen

to design quality vector(s) with relative ease. To accomplish this, test vectors were

assembled in silico to be used as benchmarks for the robustness and aesthetics of design.

The act of assembly itself was the inherent measure for interface, usability, and

workflow. After three recombinant adeno-associated viral vectors were assembled, it was

found that Genome Compiler had strengths in all the criteria desired, most notably in

usability and interface. It was therefore chosen as the program to be used in designing

vectors for the thesis project.

The Genome Compiler program was installed in conjunction with Adobe Air on a

MacBook Pro laptop running OS X Mountain Lion. Genome Compiler makes use of a

workshop style interface that was used to construct and manipulate vectors from DNA

sequences. The National Center for Biotechnology Information (NCBI) search tool

incorporated in the program was used to import desired sequence information. In cases

were a certain sequence or element could not be found easily the genInfo identifier (GI)

number was copied from the NCBI page directly and searched for in Genome Compiler.

After importing or uploading from NCBI the program sorts the sequences by type (e.g.

Viral DNA) into folder in the library under Materials. It is ideal that DNA sequences be

found using NCBI search; if this search only results in mRNA sequence data, it can be

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‘translated’ to a DNA sequence automatically in the program. Genome Compiler utilizes

a drag-and-drop workflow that allows the user to easily build on desired templates.

Figure 2: Genome Compiler main workshop area. NCBI search tool, library sorting, and drag and drop features shown. Projects are built in the blank area by incorporating desired elements into imported templates.

The initial strategy used to build the AAV vector template (viral DNA and certain

features used by Jacobson et al) started with the complete viral genotype of AAV

serotype 2. The inverted terminal repeats (ITRs) of AAV could then be located in the

DNA sequence, labeled, and used as markers to build between in Genome Compiler.

Human genes that responded to different LCA types could then be inserted using the final

template. The exclusion of viral DNA outside the ITRs, however, led to a revised strategy

of importing sequences independently and piecing them together in Genome Compiler.

The final result would be the inverted terminal repeat of adeno-associated virus serotype

2 followed by cytomegalovirus (CMV) immediate early (ie) enhancer, chicken beta actin

(CBA) promoter, simian virus (SV) 40 polyadenylation (poly(A)) signal, and the reverse

complement of the ITR sequence. This template could then be used to insert human DNA

in silico for each LCA type between the promoter and poly(A) signal.

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The ITR of AAV-2 nucleotide sequence was searched for in NCBI outside of

Genome Compiler. This method proved to be more useful; the title of the nucleotide file

often times did not specifically describe the relevance of the sequence. The ITR sequence

used was a synthetic construct under a certain patent number (Sequence 1 from Patent

WO0192551); it was therefore necessary to import this sequence rather than attempting

to locate it by name alone in Genome Compiler. The accession number for this file was

used to run a standard nucleotide BLAST, or basic local alignment search tool. This

patent sequence for the ITR of AAV-2 was searched against the ‘others’ database (non-

human and non-mouse sequences) optimizing for highly similar sequences (megablast).

The BLAST compared the nucleotide sequence of the ITR synthetic construct against

viral DNA sequences in the database and reported query covers for highly similar

sequences. It was found that in comparison to ‘Adeno-associated virus 2 right terminal

sequence’ there was a 100% match in identities as seen in Figure 3 below. This did not

confirm, however, that the synthetic ITR sequence was of the correct length. The known

length of 145 bases did not correspond to the 198 bases of the synthetic ITR sequence.

This determined that the nucleotide sequence needed to be truncated by 53 bases after

importation to Genome Compiler. A simple deletion of these bases was performed and

the sequence was labeled ‘Inverted terminal repeat of AAV serotype 2’.

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Figure 3: BLAST result comparing query to ‘Adeno-associated virus 2 right terminal sequence’. There was a 145/145 (100%) match between the two sequences suggesting the

nucleotide sequence is highly relevant and can be imported into Genome Compiler

The next sequence to be imported into the program was the CMV ie enhancer

sequence. The NCBI search resulted in synthetic constructs under patent numbers much

like the search for the ITR sequence; the same strategy and approach was implemented in

importing the sequence to Genome Compiler. A BLAST search was used aligning the

selected nucleotide file for a CMV ie synthetic construct against the ‘others’ database for

highly similar sequences (megablast). The results of this search needed to be more

thoroughly investigated than those of the ITR search; enhancer regions can often be

modified and/or combined with different promoters as a single synthetic construct. Three

different CMV enhancer synthetic construct sequences of varying lengths were used as

queries against the others database. The compiling of the information (query cover,

statistical significance, and identity matches) confirmed the most relevant sequence to

import to Genome Compiler. It was dragged into the workspace immediately following

the AAV-2 ITR sequence.

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The inclusion of the chicken beta actin (CBA) promoter involved the same

process as importing the enhancer sequence. BLAST searches confirmed the relevancy of

the CBA promoter sequence found in NCBI’s nucleotide database. To further confirm the

results, a program called SnapGene, a bioinformatics tool for designing PCR primers,

was used to import the available CBA sequence in the programs’ database to Genome

Compiler for comparative purposes. The comparison showed no differences in the CBA

sequences imported from NCBI and SnapGene’s databases. The sequence was labeled in

Genome Compiler and placed immediately following this CMV ie enhancer sequence. As

previously mentioned, enhancers and promoters are often grouped/built together in

synthetic biology. The CMV ie enhancer / CBA promoter combination is not an

uncommon one and has been used in many studies for viral vector construction. To

further verify sequence relevance, the combined nucleotide sequence of the CMV ie

enhancer and CBA promoter was searched against the others database for highly relevant

sequences in BLAST. The results of this search confirmed that the same nucleotide

sequence appears in many other sequences of the database that respond to synthetic

constructs used for viral vectors or other purposes.

The final sequence that needed to be imported to Genome Compiler was the

simian virus (SV) 40 poly(A) signal. The NCBI search resulted in a nucleotide file of the

complete genome of SV 40. This complete genome file contained certain keywords that

differentiated the sections of the genome. It was not clear that a certain nucleotide

sequence responded to the poly(A) signal, prompting the need for further investigation.

The potentially correct sequence was copied to PolyApred, a program that predicts

polyadeylation signal sequence relevance based on signature sequences scores. Poly(A)

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signals have signature sequences (e.g. AATAAA) that can simply be searched for by the

program and matched. The positive match in PolyApred was confirmed by a subsequent

BLAST search of the sequence against the others database for highly relevant sequences.

The SV40 poly(A) signal sequence was imported into Genome Compiler and placed

immediately following the CBA promoter.

To complete the AAV-2 vector template, the final step was taken to add the ITR

to the opposite of the sequence. In Genome Compiler the reverse complement of the

previously imported ITR sequence was copied and placed immediately following the

SV40 poly(A) signal. This action completed the template that was then used to build

recombinant vectors for treating different forms of LCA with human DNA that responded

to each type. Figure 4 shows the final template along with an inserted ‘gap’ section

between the promoter and poly(A) signal to be replaced by human DNA.

Figure 4: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278

bp), gap (gene of interest for LCA type), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV

serotype 2 (145 bp). 945 bp of exogenous material is present. The final step involved importing human genes that correspond to different forms

of LCA; sixteen forms of LCA are listed by OMIM. LCA 2 and the RPE65 gene was

excluded as this was the form of LCA and gene targeted by Jacobson et al, whose vector

designs were used as a model in this study. Other forms of LCA were excluded due to

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size of the gene; previous studies and research has suggested genes that are relatively

large are incompatible with the small AAV genome. The threshold of 4.7 kb of

exogenous material was established29. The enhancer, promoter, and poly(A) signal

together were 945 bp in length, leaving 3.755 kb for human DNA to be incorporated in

the final designs. This size limitation included the following types of LCA and their

respective genes: LCA1 (GUCY2D), LCA3 (SPATA7), LCA4 (AIPL1), LCA9

(NMNAT1), LCA11 (IMPDH1), LCA13 (RDH12), LCA15 (TULP1), and LCA16

(KCNJ13). NCBI searches were utilized to import the human genes into Genome

Compiler corresponding to the different forms of LCA. The nucleotide sequences were

then incorporated into the AAV-2 vector template between the promoter and poly(A)

sequences. The results were eight AAV-2 vectors built in silico for treating different

forms of LCA.

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Results

Figure 5: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus

immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278 bp), Homo sapiens retinal guanylyl cyclase 1 (GUCY2D) gene (3.641 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of

inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 4.586 kb of exogenous material is present. GUCY2D is the gene that defines LCA 1.

Figure 6: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278 bp), Homo sapiens spermatogenesis-associated 7 (SPATA7) gene (1.935 kb), Simian

virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.880 kb of exogenous

material is present. SPATA7 is the gene that defines LCA 3.


immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278 bp), Homo sapiens aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene (2.247 kb),

Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 3.192 kb of exogenous material is present. AIPL1 is the gene that defines LCA 4.

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bp), Homo sapiens nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) gene (3.781 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp),

reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 4.726 kb of exogenous material is present. NMNAT1 is the gene that defines LCA 9.


immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278 bp), Homo sapiens IMP (inosine 5'-monophosphate) dehydrogenase 1 (IMPDH1) gene

(2.526 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 3.471 kb of exogenous material is present. IMPDH1 is the gene that defines LCA 11.


bp), Homo sapiens retinol dehydrogenase 12 (RDH 12) gene (1.934 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted

terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.879 kb of exogenous material is present. RDH12 is the gene that defines LCA 13.

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bp), Homo sapiens tubby-related protein 1 (TULP1) gene (1.980 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.925 kb of exogenous material is

present. TULP1 is the gene that defines LCA 15.


bp), Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 13 (KCNJ13) gene (3.376 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2

(145 bp). 4.321 kb of exogenous material is present. KCNJ13 is the gene that defines LCA 16.

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Discussion

Of the sixteen different types of Leber congenital amaurosis, eight adeno-

associated viral vectors were designed. The LCA types excluded due to the payload

capacity of AAV were all within 1.2 kb of permitted exogenous material with the

exception of LCA 10 (CEP290 gene). The 4.7 kb limit for inclusion of foreign DNA is

not definitive; while the use of larger genes for AAV gene therapy have lacked efficacy,

this does not suggest AAV should be abandoned for slightly larger gene sizes.

Additionally, the relatively large sizes of the CBA promoter and CMV ie enhancer limit

the amount of human DNA to be incorporated and could be substituted to increase gene

capacity in AAV. Genome Compiler could be used to design the following LCA types

beyond the set payload limitation of 4.7 kb: LCA 5 (LCA 5 gene), LCA 6 (RPGRIP1),

LCA 7 (CRX), LCA 8 (CRB1), LCA 12 (RD3), and LCA 14 (LRA). The genes that

correspond to these types of LCA range from 3.95 kb to 4.909 kb. The CMV ie enhancer,

CBA promoter, and SV 40 poly(A) signal present in the design vectors was 945 bp in

total length. The use of shorter sequences could bring this total down to approximately

500 bp. This step alone would allow for packaging of RPGRIP1, CRB1, and RD3 to treat

the respective LCA forms of LCA 6, LCA 8, and LCA 12. The remaining three forms

(LCA 5, LCA 7, and LCA 14) could be designed and used as upper limitation testers for

AAV in a laboratory setting.

The genetic heterogeneity of LCA describes 16 known genes associated with

retinal dystrophies. The respective percentages of each LCA type in the population are

currently unknown; it is unclear whether or not the designed AAV vectors for eight

different types of LCA represents a great percentage of the population. It is estimated that

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LCA 10 (CEP290 gene) accounts for approximately 21% of all LCA cases. CEP290 is a

relatively large gene (7.97 kb) and well beyond the small payload capacity of AAV. LCA

10 is the sole form of LCA that would have predictably low success if a gene therapy

approach was taken using AAV as a viral vector packaged with human CEP290.

Adenoviruses and retroviruses have much larger exogenous packing capacity due to their

sizes. CEP290 could be packaged in a viral vector of one of these two categories and

designed in silico using Genome Compiler. As mentioned previously, there is an inherent

concern of safety (pathogenicity and immunogenicity) associated with these types of

viruses and their usage in gene therapy treatments. The concerns of safety and efficacy of

a proposed adenoviral or retroviral design could be addressed in animal model studies.

The sheltered treatment area of the eye and retina suggest limited adverse affects in using

these types of viruses. Additionally, the efficacy of such a study could be weighed against

using AAV to treat LCA types with genes above the 4.7 kb packaging capacity. This

would require additional animal model testing with an adenoviral or retroviral approach

for delivery of human LCA 5 gene, CRX gene, and LRAT gene responding to LCA types

5, 7, and 14 respectively. These vectors could be designed in silico using Genome

Compiler.

The primary future consideration for designing AAV vectors for different types of

LCA is to order the designs to be made synthetically. This process can be carried out in

Genome Compiler by ordering designs to be constructed and delivered as gene therapy

treatments. The study performed by Jacobson et al could be used for the basis of animal

model proof of concept research. The results of this study suggested high efficacy and

safety that led to human clinical trials for treatment of LCA 2 (RPE65 gene). These

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results could act as a benchmark for the following qualities: initial visual improvement,

retinal function, and duration of improved vision after treatment. Visual improvement can

be measured using standard procedures for proper function testing in dogs, the ideal

model for testing gene therapy treatment for LCA. Retinal and ocular improvement can

be viewed visually with histological comparison and staining. Safety could be measured

using standards for biodistrubution and immunogenicity related to dosage level. The

amount of AAV vector to be administered may vary for each LCA type, despite the

design being identical outside the human DNA incorporated. The results of a collective

study would confirm the validity of designed AAV vectors for each LCA type. The

potential success of such a study would pave the way for human clinical trials for those

afflicted with a wide variety of gene mutations related to different types of LCA.

The design program utilized in this thesis project, Genome Compiler, had great

strengths in interface and ability to simply import and organize relevant sequences. The

program could, however, be improved and optimized for gene therapy viral vector

design. As gene therapy becomes more relevant in molecular medicine, there is a need to

improve in silico design to expedite the process to eventual synthetic construction of

therapeutics. Genome Compiler could implement viral templates including features such

as inverted terminal repeats (ITRs) to manipulate in the process of gene therapy design.

Common features such as the chicken beta actin (CBA) promoter could be loaded into the

program and available for simple selection and incorporation into designs. NCBI search

within Genome Compiler is often difficult due to the lacking function of opening

sequence files. Synthetic construct sequences are often listed under patent numbers and

not the relevant name; it is necessary to using the NCBI website for further investigation

26

prior to importing the sequence. The addition of an in-house BLAST tool would allow

users to analyze the relevancy of sequences by identity matching before incorporation

into vector designs. These small changes would improve Genome Compiler as a gene

therapy design tool and potential improve the robustness and viability of a potentially

constructed viral vector.

27

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