448 using AAV5 vectors to reduce the risk of AAV vector mobilization

448 Using AAV5 Vectors To Reduce the Risk of AAV Vector Mobilization Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S174 BIOLOGY OF AAV AND VECTOR[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

S174

BIOLOGY OF AAV AND VECTOR DEVELOPMENT II

Biology of AAV and Vector Development II

446 AAV-rDNA Mediated Site-Specifi c

Integration in Human Cell and Safety Analysis in

Long-Term AAV-rDNA Treated Mice

Zhongya Wang,1 Jyoti Mathur,2 Leszek Lisowski,2 Mark Kay,2

1 Oregon Stem Cell Center, Oregon Health & Sciences University,

Portland, OR; 2 Pediatrics and Genetics, Stanford University, Palo

Alto, CA.

Recombinant adeno-associated virus (AAV) vectors remain

mostly episomal and hence are lost in dividing cells over time

We have previously reported new vectors, AAV-rDNAs, which

contain ribosomal DNA homology fl anking the expression cassette

These vectors are able to mediate site-specifi c integration 10 times

more frequently than regular AAV vectors in vivo In a Hereditary

Tyrosinemia I (HT1) mice model, Fah-/- mice were rescued with

10-30 fold lower vector dose (1x109 vg for AAV8-rDNA-Fah) than

the identical vector without rDNA homology In the hemophilia B

mice model, more than 20% of hFIX levels persisted at even after

2/3 partial hepatectomy and two subleathal CCl4 treatments, which

produce massive liver damage and induce liver regeneration, while

the control AAV-stuffer-hFIX mice only had 1-5% of hFIX left

after the same treatments As integrating vectors, AAV-rDNAs will

persist in the host genome for lifetime or until the host cells die An

important issue to be addressed before clinical application is if the

vector will cause pathological change to the host while integrating

in the genome To address this question, high doses of

AAV-rDNA-hFAH (up to 2x10e12 for serotype 8 and 3x10e11 for serotype

2) were administrated to WT mice After 12 to 14 months, these

mice were sacrifi ced for further analysis There was no detectable

pathological appearance or tumors in the liver and other organs

from the AAV-rDNA-hFah long term treated mice except that 2/6

had lymphoma hFah PCR was performed on genomic DNA from

these lymphoma samples to see if AAV-rDNA-hFah integration was

involved in the tumorgenesis process Importantly, the hFah gene

was undetectable in these lymphoma samples by two independent

hFah PCRs, suggesting that AAV-rDNA-hFah is not involved in the

tumorgenesis Large scale analysis of the integration sites is being

performed to further pursue the mechanisms and safety concerns

of AAV-rDNA mediated integration Another important issue to be

addressed is if AAV-rDNAs also work in human cells Two new

constructs, pAAV-hrDNA-neo and pAAV-neo, were made to produce

the AAV virus of serotype DJ Human fi broblasts were transduced

with different MOIs of AAV-hrDNA-neo and AAV-neo After G418

selection, plates infected with AAV-hrDNA-neo had about 10 times

more clones than plates infected with the same dose of AAV-neo

Southern blotting and PCR were applied to detect the integration

sites The presence of junction fragments in the population of G418

resistant cells derived from AAV-hrDNA-neo infection suggests a

high frequency of site-specifi c integration These results indicate

AAV-rDNAs also mediate higher frequency of integration in human

cells and that a large percentage of integration events are site-specifi c

in ribosomal DNA Fah-/-/Rag2-/-/Il2rg-/- mouse repopulated with

human hepatocytes will be used to test whether AAV-hrDNA also

functions in human cells in vivo Together, these results suggest

that AAV-rDNAs can integrate and provide therapeutic level gene

expression without any pathological damage to the host, suggesting

a promising future for this new vector

447 Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelia

Wuping Li,1 Liqun Zhang,2 Jarrod S Johnson,1 Raymond J Pickles,2 Jude R Samulski.1

1 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2 Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC.

Recombinant adeno-associated virus type 2 (AAV-2) vectors have been used to deliver cystic fi brosis transmembrane regulator (CFTR) gene to airway of cystic fi brosis (CF) patients in clinical trials Although the vectors were found to be safe and well tolerated,

no signifi cant effi cacy was achieved due to the lack of effi cient transduction in human airway epithelia To improve the effi ciency

of AAV transduction in these cells, we applied a chimeric AAV

library, generated by DNA shuffl ing in vitro, to ciliated human airway

epithelial cells (HAE), to enrich and select for AAV variants with enhanced transduction effi ciency Virions that successfully infected HAE were amplifi ed with helper adenovirus, and the recovered chimeric AAV pool was subjected to further rounds of screening on HAE After fi ve successive screening cycles, we have identifi ed two novel AAV variants (dubbed chimeric HAE-1 and HAE-2), which are comprised of capsid components from serotypes AAV1, AAV6, & AAV9 The transduction effi ciencies of these two novel AAV variants

in airway epithelia were compared to numerous AAV serotypes (1, 2, 5, 6, & 9) While AAV 6 scored best of the natural isolates, HAE variants were three times higher for airway transduction using reporter gene (GFP & Luc) expression Binding assays determined that this enhancement was post entry, and these observations were further collaborated by co-administration of proteasome inhibitors or anthracycline compounds that enhanced chimeric vector transduction effi ciencies even higher Structural analysis determined that two key amino acids (584 and 598) of HAE-1 where responsible for increased transduction while HAE-2, the better chimeric capsid, involved 13

aa changes Finally chimeric vector carrying mini-CFTR expression cassette (4.9kb), exhibited a signifi cant increase in forskolin-inducible chloride transport (up to 23-31%) following apical inoculations to human CF airway epithelia These studies provide a fi rst step in testing unique AAV variants that may lead to successful clinical gene delivery in human CF airway epithelia

448 Using AAV5 Vectors To Reduce the Risk of AAV Vector Mobilization

F Curtis Hewitt,1,2 Chengwen Li,1 Steven J Gray,1 Shelley Cockrell,4 Michael Washburn,2 R Jude Samulski.1,2,3

1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3 Department of Pharmacology, University of North Carolina

at Chapel Hill, Chapel Hill, NC; 4 Interdisciplinary Biomedical Graduate Program, University of Pittsburgh, Pittsburgh, PA.

Current adeno-associated virus (AAV) gene therapy vectors package a transgene fl anked by the terminal repeats (TRs) of AAV2 Although these vectors are replication defi cient, wild type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a TR2-fl anked transgene in trans during superinfection

by a helper virus, leading to “mobilization” of the vector genome from treated cells in a wt AAV capsid More importantly, it appears likely that the majority of currently characterized AAV serotypes (as well

as the majority of new novel isolates) are capable of rescuing and replicating AAV2 vector templates To investigate this possibility,

we fl anked a GFP transgene with type 2 and the most divergent AAV serotype, type 5 TRs (TR2 or TR5) Consistent with AAV clades and previous reports, AAV5 specifi cally replicated TR5 vectors while

Molecular Therapy Volume 17, Supplement 1, May 2009

BIOLOGY OF AAV AND VECTOR DEVELOPMENT II

AAV serotypes 2 and 6 replicated TR2-fl anked vectors To exploit

this specifi city we created a TR5 vector production system for Cap1-5

which produced roughly equivalent titers and transduction potential

compared to the TR2 vectors currently used Next, we showed that

persisting, presumably integrated, rAAV genomes fl anked by TR2s

or TR5s were mobilized in vitro after addition of the cognate AAV

Rep (as well as Rep6 for TR2) and adenoviral helper Finally, we

showed that a cell line containing a stably integrated wt AAV2

genome resulted in mobilization of a TR2 fl anked vector but not a

TR5 fl anked vector upon adenoviral superinfection Based on this data

and the relative prevalence of wt AAV serotypes in the population, we

propose TR5 vectors have a signifi cantly lower risk of mobilization

and should be considered for clinical use

449 Transcytosis of AAV8 and AAV9 across

Endothelial Barrier

Bo He,1 Zhenhua Yuan,1 Chunping Qiao,1 Victoria Madden,2

Dhiren Thakker,1 Juan Li,1 Xiao Xiao.1

1 Molecular Pharmaceutics, Eshelman Pharmacy School,

University of North Carolina at Chapel Hill, Chapel Hill, NC;

2 Department of Pathology and Laboratory Medicine, University of

North Carolina at Chapel Hill, Chapel Hill, NC.

Adeno-Associated Virus (AAV) based vectors have shown great

promise for human gene therapy In the previous studies of systemic

gene delivery into muscle, we have demonstrated that AAV8 was

the most effi cient vector for crossing blood vessel barrier to attain

systemic gene transfer and prolonged transduction in skeletal and

cardiac muscles Other studies further revealed that AAV9 vector

could also effi ciently cross vascular endothelial cell barriers The

potential mechanisms, however, remain elusive Elucidation of AAV

transendothelial movement pathways will facilitate the improvement

of AAV systemic gene delivery In this study, we hypothesized that

transcytosis maybe the major pathway involved in AAV8 and AAV9

crossing the microcapilaries of the vasculature In transcytosis

experiment, we employed Caco-2 cells, which are originated form

intestine epithelium and commonly used to study transcellular

transport of macromolecule drugs We also employed human

umbilical vein endothelial cells (Huvec) as a model cell system to test

the capability of AAV8, AAV9 and AAV2 penetrating cell monolayer

on transwell membrane The viral DNA in the medium of basolateral

chamber was measured by real-time PCR Cross-section of fi xed cells

was examined under transmission electron microscope (TEM), which

showed direct evidence of transcytosis that AAV8 and AAV9, but

not AAV2, particles underwent: 1) endocytosis with membrane pit

formation on the apical side of cell monolayer; 2) intracellular vesicles

traffi cking in the cytoplasm, and 3) exocytosis on the basolateral

side of cell monolayer The real-time PCR results demonstrated that

AAV9 and AAV8 had much better transcytosis capability than AAV2

in both Huvec and Caco-2 cell models We detected 5.5x105 vector

genome(vg) of AAV9, 4.4x105vg of AAV8 and 1.1x105 vg of AAV2,

respectively, when 2x108 vg particles were loaded on the Huvec cell

monolayer This pattern is in general agreement with the results of

in vivo systemic gene delivery Together, the in vitro fi ndings along

with our previous studies suggest that AAV8 and AAV9 vectors

appear to exhibit effi cient transcytosis capability to cross the blood

vessel endothelial barriers, which may contribute to their superiority

in systemic gene delivery to the muscle, heart and other tissues and

organs

450 Overcoming Barriers to AAV Mediated Retinal Transduction from the Vitreous

Deniz Dalkara,1,3 Kate D Kolstad,1,2 Ryan R Klimczak,1,2 Meike Visel,1 Natalia Caporale,1,2 John G Flannery,1,2 David V Schaffer.1,3

1 HWNI, UCB, Berkeley, CA; 2 MCB, UCB, Berkeley, CA; 3 ChemE, UCB, Berkeley, CA.

Adeno-associated viral gene therapy has shown great promise

in treating inherited and acquired retinal disorders, with three clinical trials in progress this year Mutations in genes specifi c to photoreceptors and the retinal pigment epithelium (RPE) comprise the great majority of defects leading to inherited blindness; however, the majority of AAV serotypes, such as AAV1 and AAV5, can infect these cell types with subretinal delivery The one exception is AAV2, which can infect the inner retina from the vitreous Unfortunately, retinal detachment occurs during subretinal injection, causing cellular changes that impact the microenvironment and survival of retinal neurons and can also affect the return of vision The purpose of this study is to understand and overcome barriers to AAV mediated retinal transduction from the vitreous while minimizing any deleterious effect

on retinal function To examine physical barriers to viral infection,

we have fl uorescently labeled virions of numerous relevant AAV serotypes (1, 2, 5, 8 and 9) and followed their distribution in the retina after intravitreal injection AAV serotypes 2 and 9 show accumulation

at the vitreoretinal junction while AAV8 exhibits only weak binding at the same location We did not observe accumulation of labeled AAV1 and 5 capsids at the vitreoretinal junction, suggesting a lack of receptor binding sites for these serotypes at the inner limiting membrane (ILM) We hypothesized that the inner limiting membrane could pose

a barrier to AAV-mediated gene delivery, and we therefore attempted

to utilize a non-specifi c protease to mildly digest the ILM We were able to substantially enhance viral access to multiple retinal cell types

as judged by robust GFP expression after intravitreal injection of each serotype The use of enzymatic treatment had the greatest effect

on AAV5 transduction, producing the most remarkable expression profi le Specifi cally, GFP could be detected in a variety of retinal cell types including retinal ganglion cells, Müller glia, inner nuclear layer cells, photoreceptors, and the RPE In addition, the protease treatment, at a low dose, has no deleterious effect on retinal function as demonstrated by electroretinogram and visual cortex cell population responses to visual stimuli presented to enzyme treated eyes and untreated contralateral eyes To our knowledge, this is the fi rst time broad, effi cient outer retinal cell transduction has been achieved through an intravitreal injection We anticipate this fi nding will help avoid the limitations, risks, and damage associated with subretinal injections currently necessary for gene therapy in the clinic

451 Comparison of Transduction Effi ciency of Tyrosine-Mutant AAV Vectors in Muscle

Chunping Qiao,1 Hui Zheng,1 Zhenhua Yuan,1 Jianbin Li,1 Li Zhong,2 Arun Srivastava,2 Juan Li,1 Xiao Xiao.1

1 Department of Molecualr Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, NC; 2 Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL.

Mutations of the surface-exposed tyrosine in AAV2 vectors led to dramatic improvement of transduction effi ciency both in vitro and

in vivo The increased transduction effi ciency of tyrosine-mutant AAV2 vector is due to the lack of capsid ubiquitination and improved intracellular traffi cking to the nucleus In this study, we were interested

in studying the transduction effi ciency of different serotypes of tyrosine-mutant AAV vectors in muscle The identifi cation of high effi cient AAV vectors in muscle tissue will result in better therapeutic effect for muscle-directed gene therapy We investigated three AAV serotypes, namely AAV8, 9 and 6, with two tyrosine residue mutation for each AAV serotype Expression of luciferase reporter gene under the control of cytomegalovirus (CMV) promoter packaged by different