305. Development of a qPCR Based Assay for Assessing Integrity of Oversized AAV Vectors

AAV VECTOR DEVELOPMENT one of several large genomes in human cells. As a control, we demonstrated that modifications to expand the AAV genome did not affect the ability to package a standard-sized genome. Different insert sizes (100bp, 500bp, 1kb and 2kb) were cloned into the noncoding region of the AAV genome downstream of CAP and prior to the ITR. Each of the four shuttle plasmids were vectorized and tested to confirm that they could still package a routine-sized genome (rAAV-ApoE/hAAT-eGFP, 4.7kb) as efficiently as AAV2. Controlverified plasmid libraries were used to produce large preparations of actively replicating AAV libraries. Huh7 cells were infected with AAV libraries at a range of MOIs and subsequently with adenovirus for AAV replication. Presence of adenovirus induces cell lysis and release of AAVs into the media. Media is collected 3 days later and used for western blot analysis of VP1/2/3. To avoid cross-packaging, the sample with the lowest detectable level of VP is used for the next round of selection. Selective pressure was applied for several screen rounds and the resultant capsid variants were fully Sanger sequenced and vectorized. Capsids are now being used to generate large capacity AAV-GFP vectors. Each lot will be titered by dot blot and those that produce virus at <1e10 vg/mL will not be considered further for use as a clinical candidate. Alkaline Southern blots are also being performed to verify an improvement in packaged genome size pre- and post-transduction. Cellular transduction efficiencies will be assessed on a variety of human cell lines from numerous organs and liver cancer models. The use of AAV capsid libraries may provide a way to overcome packaging size limitations inherent to current AAV vectors.

304. The Inhibitory Effect of Various Transfection Agents on rAAV Vector-Mediated Transgene Expression Both In Vitro and In Vivo

Yuan Wang,1,2 Lina Wang,1 George V. Aslanidi,3,4 Changquan Ling,1,2 Arun Srivastava,3,4 Chen Ling.3,4 1 Department of Traditional Chinese Medicine, Second Military Medical University, Shanghai, China; 2Shanghai University of Traditional Chinese Medicine, Shanghai, China; 3Division of Cellular and Molecular Therapy, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL. The ability of transfection agents (TAs) to introduce DNA into cells has provided a powerful means to study the function and control of mammalian genes, and in some instances to examine the roles of specific protein(s) in recombinant adeno-associated viral (rAAV) vector-mediated transduction. A wide variety of TAs are available, including cationic polymers, cationic lipids, dendrimers, and calcium phosphate-base agents, all of which have been introduced to study various steps in the life cycle of rAAV vectors. For example, we evaluated the role of a cellular serine/threonine protein phosphatase, protein phosphatase 5 (PP5), in rAAV2 vector-mediated gene transfer using liposome-based lipids (Gene Ther., 14(6): 545-550, 2007). Similarly, Nonnenmacher et al. (Cell Host Microbe, 10(6): 563-576, 2011) utilized cationic polymers to deliver various genes into HeLa cells and concluded that rAAV2 vectors use the pleiomorphic CLIC/ GEEC pathway as the major endocytic infection route. Liu et al. (Gene Ther., 20(3): 308-317, 2013), on the other hand, transfected HEK293 cells with DsRed-Rab5, -Rab7 or -Rab11 expression plasmids by the calcium phosphate precipitation method and concluded that late endosomes might not be involved in the rAAV8 transduction. Yet, there have been no systematic studies comparing the effects of various TAs on rAAV vector-mediated transgene expression. Here, we report that, in the absence of recombinant plasmids, all TAs, inhibited rAAV2 vector transduction efficiency in various cell lines in vitro. For example, non-liposomal lipid formulations inhibited the transduction efficiency by >1.8-fold, lipid-based liposomes by >1.2fold, and calcium phosphate-based agents by >3-fold. Interestingly, cationic polymers and dendrimers inhibited viral-mediated transgene Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

expression by >10-fold. Similar results were obtained in the presence of plasmids, except that non-liposomal lipids inhibited the transduction efficiency by >4-fold. In addition, we observed that the inhibitory effect of the TAs was largely limited to pre-treatment of cells, or treatment within the first 24 hours of viral transduction, suggesting that the intracellular trafficking step of vector particles was being affected. We also compared the effects of cationic polymers and lipid-based liposomes on rAAV vector transduction efficiency in mice in vivo and observed that at 4-weeks post-vector administration, liposomes had little effect on the transduction efficiency of rAAV2 or rAAV8 vectors, whereas cationic polymers significantly inhibited liver-targeted transgene expression by both serotype vectors. Taken together, these studies underscore the need to exercise caution in interpreting transgene expression data obtained following rAAV vector-mediated transduction, especially when additional transgene products are delivered by TAs.

305. Development of a qPCR Based Assay for Assessing Integrity of Oversized AAV Vectors

Heikki T. Turunen,1 Ru Xiao,1 Eva Plovie-Buys,1 Luk H. Vandenberghe.1 1 Gene Transfer Vector Core, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA. In vivo gene transfer using AAV is emerging as a platform technology for the treatment of several forms of acquired and inherited disorders. One of the main limitations to it broad application is the genome size constrained imposed by the architecture of the viral particle. Efforts to map maximum genome capacity have ranged from 4.75kb up to 8.9kb. Data suggest that preparations exceeding wild type genome size are impacted in terms of genomic and structural integrity as is reflected in depressed yields and heterogeneity. While several biochemical and molecular methods are available for qualitatively assessing this issue, quantitative assays have to date not been developed. AAV genome packaging is initiated from either of the two identical ITR sequences flanking the genome. With normal sized genomes packaging proceeds in 3’ to 5’ direction until the opposite ITR is reached, whereas with oversized genomes packaging is terminated before the opposite ITR is reached, resulting in fragmented genomes lacking the distal ITR. As the ITRs are identical, a 1:1 ratio of (+) and (-) strand genomes is expected. However, we have detected a transgene sequence derived bias in genome packaging resulting in unequal ratios of (+) and (-) strands. Based on this observation we developed a TaqMan qPCR assay to study packaging of oversized genomes for rAAV vectors. As PCR is a template strand independent reaction, normal sized rAAV genomes give equal titers from any part of the genome regardless of the ratio of (+) and (-) genomes as templates. We performed TaqMan qPCR targeting promoter (5’) and polyA (3’) sequences for a normal sized (4.6 kbp) rAAV vector, and equal titers were detected, as expected. We then modified the construct to make it oversized (6.8 kbp) while retaining positions and sequences for the qPCR assay targets. Vectors were produced and titered identically to the normal sized vectors, and the 5’ qPCR gave 4 times higher signal than the 3’ qPCR. Similar results were obtained with AAV2, AAV5 and AAV8 capsids. Effects of the ITRs or non-packaged cisplasmid sequences to the strand-specific packaging were ruled out by confirming ITR integrity by sequencing, and by inverting the transgenic construct sequence between the ITRs and repeating the assay. Therefore, identical titers obtained from the two qPCR assays indicate that the whole genome has been packaged, whereas different titers indicate fragmented templates and thus oversized genomes. We are currently studying the effect of genome size to the ratio of (+) and (-) strands and developing an assay to study packaging of S117

ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS II oversized AAV genomes. In addition, strand specific packaging of AAV genomes is a novel observation and may have implications in vector production.

306. Generating Novel AAV Capsid Mutants for Large Genome Packaging Through Protein Libraries and Directed Evolution

Heikki T. Turunen,1 Luk H. Vandenberghe.1 1 Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA. One of the major shortcomings of the AAV as a gene therapy vector is its small size, allowing efficient packaging of vector constructs of only up to ~5 kbp in size. Although ultimately limited by the physical dimensions of the viral capsid, we hypothesize that by improving the efficiency of the viral genome packaging process a significant increase in AAV genetic payload carrying capacity can be achieved. Here, we have generated an approach combining protein libraries and directed evolution to generate novel AAV8 vectors with hopes to identify mutations enabling more efficient packaging of larger genomes. Wild type AAV8 genome was modified with silent point mutations to introduce restriction enzyme sites to facilitate insertion of library inserts. In addition, a cassette providing resistance to puromycin antibiotic was added to make the genome oversized and to allow selection for viruses carrying said oversized genomes if required. Libraries were generated to regions in Rep, Cap and AAP predicted to be involved in the genome packaging process. For each library seven amino acids were targeted with NNK mutations, generating libraries with total variability of 327, or ~3.4E10. However, due to the inherent inefficiency of restriction enzyme mediated insertion of library fragments into AAV8 genome, libraries with variability of 1E7-3E7 were achieved. Recombinant AAV8 vectors were produced by transfecting HEK-293 cells with AAV8 vectors including library mutation fragments, with adenovirus helper plasmid or wild type adenovirus. Vectors were purified and used to transduce fresh HEK293 cells treated with adenovirus helper. The cycling was repeated similarly for up to 10 cycles, with samples collected after each cycle. Samples were titered with qPCR, and library containing regions were amplified by PCR and subcloned back into the AAV8 genome to enrich library fragments allowing generation of viable vectors. No viable vectors were obtained from the Rep library. Rep is highly conserved throughout all AAV serotypes, and likely even slight changes caused by the majority of library mutations result in a non-functional protein, thus preventing amplification during cycling. However, decent titers were obtained from the Cap library, and several viable mutants have been isolated. Preliminary analyses have revealed mutants that are produced and are able to transduce cells with efficiencies comparable to wild type AAV8. All mutants have been sequenced and are analyzed to find patterns resulting in functional or non-functional viruses. Should positive patterns be detected, vectors with such targeted mutations will be made and assessed for production and transduction. Assays for packaging larger genomes are currently underway.

307. Engineering Recombinant AAV Vectors for More Efficient and Restricted Gene Expression in the CNS after Systemic Administration

Benjamin E. Deverman,1 Bryan Simpson,1 Piers Pravdo,1 Abhik Banerjee,1 Paul H. Patterson.1 1 Biology and Biological Engineering, California Institute of Technology, Pasadena, CA.

extend these findings, we are developing delivery vehicles capable of providing long-term, controllable expression of LIF, or other transgenes, that is widespread within, yet restricted to, the CNS. Recombinant adeno-associated virus 9 (rAAV9) is an attractive candidate for such a vector as it can deliver genes to neurons and glia throughout the CNS when injected intravenously (IV). For this systemic rAA9-based delivery approach to succeed in the adult, however, significant improvements in CNS transduction efficiency and selectivity are needed. In addition, it would be optimal to be able to modulate or turn off transgene expression post-delivery. We are making these enhancements by (i) using directed evolution/in vivo screening to develop AAV9-based capsids that more efficiently transduce CNS astrocytes, (ii) developing rAAV genomes that use gene regulatory elements (promoter/enhancer elements and miRNA binding sites) to restrict expression to CNS astrocytes and (ii) optimizing dox-inducible regulatory elements to provide control over transgene expression post-delivery. These optimized vectors should be useful as vehicles for the efficient and selective delivery of cytokines, trophic factors or other therapeutic cargo to the CNS for the treatment of neurological disorders.

Adenovirus Vectors and Other DNA Virus Vectors II 308. Natural Antibodies Inhibit Liver Transduction with Adenovirus Vectors in Mice

Qi Qiu,1 Zhili Xu,1 Jie Tian,1 Rituparna Moitra,1 Andrew P. Byrnes.1 1 Division of Cellular and Gene Therapies, FDA CBER, Bethesda, MD. Natural antibodies are a relatively understudied component of the innate immune system that can inhibit the efficiency of gene therapy. It is well established that liver transduction with intravenously-injected Ad5 vectors is higher in antibody-deficient mice than in wild-type mice. We have previously shown that normal wild-type mice have pre-existing natural IgM antibodies that can bind to Ad5 vectors and inhibit liver transduction. In the current work, we examined the relationship between IgM concentration and liver transduction by Ad5 vectors. We also asked how differences in IgM concentration between C57BL/6 and BALB/c mice are related to differences in liver transduction between these two mouse strains. Results: Liver transduction was evaluated after i.v. injection of Ad5 vectors into knockout (KO) mice that have abnormally low or high levels of natural antibodies. We found greatly elevated liver transduction in Rag-1 KO mice (no IgM) and CD19-cre mice (low IgM), as compared to liver transduction in control mice of the same background (C57BL/6). In contrast, mice with elevated IgM concentration (Ptpn6f/f;CD19-cre) had greatly reduced liver transduction. These results demonstrate an inverse correlation between IgM concentration and liver transduction. When Kupffer cells were depleted prior to vector injection, liver transduction was significantly enhanced and the concentration of IgM no longer influenced liver transduction. This result indicates that the inhibitory effects of IgM on liver transduction are mediated through Kupffer cells. When comparing different mouse strains, we found that BALB/c mice had higher IgM concentration and lower liver transduction than C57BL/6 mice. Crosses between these two strains confirmed that natural IgM concentration is negatively correlated with liver transduction. Conclusions: We conclude that high natural IgM concentration has a major negative effect on the ability of Ad5 vectors to transduce liver.

We previously demonstrated that delivery of the cytokine leukemia inhibitory factor (LIF) to the brain by recombinant adenovirus enhances oligodendrocyte progenitor cell proliferation and remyelination in a mouse model of multiple sclerosis (MS). To S118

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy