371 Systemic Gene Expression after Intravenous Injection of Adeno Associated Virus 2/9 to Fetal and Neonatal Mice Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society o[.]
AAV VECTORS II which increased the transduction efficiency approximately ten-fold compared with the wild-type (WT) AAV2 vector Combining the best performing S662V mutant with T491V further enhanced the transduction efficiency by approximately 8-fold Taken together, these data suggest that high-efficiency transduction of moDCs by capsid-modified AAV vectors is indeed feasible, which supports the potential utility of these vectors for future human DC vaccine studies 369 Two Complementary Methods To Identify AAVS1 Integrants Peter Ward,1 Christopher Walsh.1 Medicine/Tisch Cancer Center, Mount Sinai School of Medicine, New York, NY Wild-type Adeno-associated virus (wtAAV) has been found integrated in a site-specific manner into the long-arm of chromosome 19 (a site designated as AAVS1) Integration is mediated by the viral encoded Rep protein Studies suggest that integration into this region is not harmful to the host and that transgene expression is favored Therefore transgene-targeted integration at the AAVS1 site would facilitate a safe and efficient gene transfer for ex vivo use Using current LM-PCR-based methods, however, integrated wild-type and recombinant AAV (rAAV) genomes are commonly found not at AAVS1 but rather at other sites We tested the hypothesis that this failure to detect integrants at AAVS1 was due to LM-PCR selection bias by employing two PCR methods that avoid detection bias HepG2 cells were infected with rAAV2-GFP and co-infected with wt AAV2 to supply Rep in trans Sorted cells were single-cell cloned and cultured for several weeks DNA was extracted and AAV-genome junctions amplified by LM-PCR Sequencing was performed without bacterial cloning Twenty-six clones that retained fluorescence were analyzed We could assign integration locales to only of the 26 clones using this method; of the clones were located at the AAVS1 site Failure of the PCR polymerase to cross the ITR seemed to explain most of the failures to determine integration loci in the remaining clones To circumvent this problem we used an inverse PCR method, again without bacterial cloning Five clones not previously defined yielded integration sequences with of the integrants located at AAVS1 Analysis of the remaining 13 clones indicated integration within other integrated AAV sequences in several cases Of note, we found that site-specific integration of AAV-GFP genomes can be achieved without co-integration of the wt AAV genome In summary, we have developed a PCR algorithm for easily determining the integration of rAAV transgenes With these methods, we found that the majority of integrants were within the AAVS1 region This makes AAVS1 sitespecific integration a viable option for targeted ex vivo gene therapy 370 Optimization of Kidney-Targeted Gene Delivery for Cystinosis Using AAV Celine J Rocca,1 Frank Harrison,1 Brian Yeagy,1 R Jude Samulski,2 Stephanie Cherqui.1 Molecular and Experimental Medecine, The Scripps Research Institute, La Jolla, CA; 2Gene Therapy Center, University of North Carolina at Chapel Hill, NC A wide range of monogenic kidney disorders has been identified and so far no gene therapy has been developed The main goal of our project is to develop an efficient kidney-targeted gene delivery system using recombinant Adeno-Associated Viruses (rAAV) As a proof of concept, we are using the cystinosis mouse model, the Ctns-/- mice Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders and characterized by intracellular accumulation of cystine The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin Affected individuals typically present with proximal tubulopathy (Fanconi syndrome) before one year of age and progressive loss of glomerular Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy function and finally progress to end-stage renal failure The Ctns/- mice develop renal dysfunction similar to the patients as soon as months of age and chronic renal failure by the age of 15 months Thus, they represent an excellent model for chronic kidney diseases Our preliminary results showed that the optimal route of injection for AAV in the kidney is a retrograde renal vein injection Using AAV serotype expressing Luciferase or GFP, we have respectively demonstrated that luciferase was detected within the injected kidneys and the intensity of the expression was stable for at least months, and that GFP positive cells were observed in the explanted kidneys In vitro, we have shown that the level of cystine content was significantly reduced in transduced Ctns-/- fibroblasts after transduction with AAV2-CTNS However, the prevalence of neutralizing antibodies in the human population for AAV2 is very high and would probably impact its efficiency for gene delivery Therefore, our goal is to optimize kidney-targeted gene delivery via renal vein injection by testing several rAAV serotypes that have the potential of transducing renal cells and a low prevalence of neutralizing antibodies in human We are currently performing injections of rAAV5, 6, and coding for either the luciferase or the GFP We are also testing different promoters that could lead to a more specific and efficient renal cells transduction such as the Parathyroid Hormone Receptor (PTHR) kidney specific promoter P1 The optimal dose is also being determined The reporter gene expression will be visualized and quantified by using confocal microscopy and quantitative PCR for GFP, and IVIS imaging system for luciferase Once the optimal conditions will be defined, we propose to test this approach based on renal vein injection of rAAV-CTNS as a minimally invasive procedure for treating the renal dysfunction in cystinosis Renal function will be measured by blood and urine analyses and renal structure by histology Cystine content and CTNS expression will be measured in the kidney at different time points during a one-year period In parallel, we are also testing our different AAV systems (serotype/promoter) in vitro in tubular proximal tubular cells from cystinosis patients If successful, this strategy will be able to be used in many monogenic hereditary nephropathies 371 Systemic Gene Expression after Intravenous Injection of Adeno-Associated Virus 2/9 to Fetal and Neonatal Mice Ahad A Rahim,1 Andrew M Wong,2 Klemens Hoefer,2 Suzanne M Buckley,1 Citra N Mattar,3 Jerry K Chan,3 Jonathan D Cooper,2 Simon N Waddington.1 Gene Transfer Technology Group, UCL Institute for Women’s Health, London, United Kingdom; 2Pediatric Storage Disorders Group, KCL Institute of Psychiatry, London, United Kingdom; Department of Obstetrics and Gynaecology, National University of Singapore, Singapore A variety of monogenic diseases present pathology in a range of organs throughout the body An example is Gaucher disease where affected tissue includes the liver, spleen, nervous system, bones and haematological abnormalities Although enzyme replacement therapy can be used to effectively treat the visceral manifestations of Gaucher disease, some tissues, such as the bones, remain refractive to therapies and the lifelong treatment of the disease is expensive Furthermore, enzyme replacement therapy is not available for other more rare diseases We evaluated the tropism of single-stranded (ss) and self-complimentary (sc) AAV2/9-GFP after intravenous injection to fetal (E15) and neonatal (P1) mice One month post-injection, the administered animals were culled and the visceral tissues examined for GFP expression by fluorescence stereoscopic microscopy, immunohistochemistry, immunofluorescence, scanning confocal microscopy and qPCR analysis We report efficient gene delivery to various tissues, organs and cell types using both ss- and sc-AAV2/9 e.g liver, lung, heart, spleen, skin, kidney, intestine, bladder, muscle and vasculature Interestingly, bone and cells within the bone marrow S145 ADENOVIRUS AND OTHER DNA VIRUS VECTORS: BIOLOGY AND VECTOR DESIGN were also transduced Intravenous administration of scAAV2/9 to late gestation fetal macaques also produced widespread gene delivery and confirmed our observations in the mice We, and others, have shown that systemic administration of AAV2/9 crosses the blood-brain barrier and mediates efficient gene delivery to the nervous system The data presented here highlights the promise that this vector shows for treating systemic diseases that are perinatal lethal and affect both the brain and the viscera 372 Targeted Mutagenesis of Ubiquitin-Binding Lysine Residues on the Adeno-Associated Virus (AAV)2 Capsid Improves Its Transduction Efficiency Nishanth Gabriel, Ramya Duraiswamy, Rupali A Gadkari, Sudha Govindarajan,3 Banumathi Ramakrishna,4 Arun Srivastava,5 N Srinivasan,3 Alok Srivastava,1,2 Giridhara Rao Jayandharan.1,2 Dept of Hematology, Christian Medical College, Vellore, India; Centre for Stem Cell Research, Vellore, India; 3Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India It is now well recognized that hepatic gene transfer of high doses of AAV vectors predispose to a robust adaptive immune response, from the data available from hemophilia clinical trials Thus there is a need to develop novel strategies which will allow lower doses of vectors to be used to achieve sustained phenotypic correction and limit vector related immune-toxicities Previous studies have demonstrated the utility of mutating surface-exposed tyrosine residues on AAV2 capsid which possibly protects the vector particles from ubiquitin-mediated proteasome degradation and resulted in a significant increase in transgene expression (Zhong et al, 2008) We hypothesized that mutations at lysine residues of AAV2 common capsid region, VP3, which are direct targets for host ubiquitin ligases, will improve its transduction efficiency Our in silico analysis using an ubiquitination prediction software (UbPred, http://www.ubpred org/) identified seven lysine residues (K39, K137, K143, K161, K490, K527 and K532) on AAV2 capsid which could be potentially ubiquitinated Lysine>Arginine mutations in AAV2 Rep/Cap coding plasmid was carried out and highly purified stocks of a recombinant self-complementary AAV2 vectors expressing EGFP [scAAV-CBaEGFP] were generated in each of the seven mutant plasmids The physical particle titres of lysine mutant vectors was comparable to wild-type (WT) scAAV vectors (∼0.5-1 X 10^12 vgs/mL), suggesting that these mutations not affect the structure or packaging ability of mutant capsids scAAV vectors containing WT or each of the seven lysine mutant capsids were then evaluated for their transduction potential in vitro Approximately X 104 HeLa or HEK293 cells were mock- infected or infected with AAV at different multiplicities of infection (MOI, 500, 2000 or 5000 vgs/cell) Forty-eight hours post-infection, transgene (EGFP) expression was measured by fluorescence microscopy and by flow-cytometry Our results (Fig 1) demonstrate that one of the seven mutants tested, the K532R vector, significantly increased gene expression in both HeLa (18X) and HEK 293 (9X) cells in vitro in comparison to WT-AAV2 This increased transduction efficacy of the K532R vector was consistent across three different MOIs tested, with an average increase of 10- fold over the WT vector Further ongoing studies in normal and hemophilia B mice will demonstrate if the AAV2 K532R vector can also improve hepatic gene transfer, in vivo Figure 1: Transduction efficiency of AAV2 lysine mutants (MOI 2000) in HeLa and HEK293 cells in vitro The relative fold-increase in gene expression is shown as inserts Adenovirus and Other DNA Virus Vectors: Biology and Vector Design 373 Sustained and Safe Inhibition of Hepatitis B Virus Replication In Vivo Using HelperDependent Adenovirus Vectors To Deliver Antiviral RNAi Expression Cassettes Carol Crowther,1 Mohube B Mowa,1 Abdullah Ely,1 Patrick Arbuthnot.1 Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Joahannesburg, South Africa Hepatitis B virus (HBV) is hyperendemic to southern Africa, east and south east Asia where there are approximately 350 million chronically infected individuals Chronic carriers have an increased risk of developing potentially fatal complications of cirrhosis and hepatocellular carcinoma Licensed HBV treatments rarely eliminate the virus from infected individuals, and improvement of HBV therapy remains a priority We have previously demonstrated that CMV and U6 (Pol II/III) expression cassettes that generate artificial antiviral RNA interference (RNAi) activators can be used to inhibit HBV gene expression in vivo Nevertheless, achieving safe and efficient delivery of these anti-HBV RNAi sequences remains an important objective Recombinant adenoviruses (Ads) are amongst the most efficient hepatotropic gene delivery vehicles, but a drawback of their use is transient transgene expression and toxicity resulting from induction of host immune responses To limit vector immunostimulation, we have generated RNAi-activating anti-HBV gutless helper-dependent (HD) Ads Efficacy against HBV replication was tested in HBV transgenic mice, which stringently simulate the human condition of chronic HBV infection Two days after intravenous administration of 5×109 recombinant HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced Markers of HBV replication were decreased by approximately 95% in animals receiving the HD Ads and this effect was sustained for weeks without adverse effects Compared to unmodified first generation anti-HBV Ads, inhibition of viral replication was significantly more sustained Moreover, acute hepatotoxicity and proinflammatory cytokine release following administration of HD Ads was attenuated HD Ad DNA was present at high concentrations in the livers of mice at termination of the investigation, which indicates that diminished efficacy was a result of switching off of transgene expression rather than elimination of the vector from hepatocytes Alternative transcription control elements, reduction of immunostimulation by pretreatment with dexamethasone and polymer modification are being investigated to improve delivery of anti-HBV RNAi activators by these vectors 374 Scavenger Receptor A (SR-A) and Scavenger Receptor Expressed on Endothelial Cells I (SREC-I) Are Receptors of HelperDependent Adenoviral Vectors Pasquale Piccolo,1 Francesco Vetrini,2 Pratibha Mithbaokar,1 Nathan C Grove,2 Donna Palmer,2 Philip Ng,2 Nicola BrunettiPierri.1,3 Telethon Institute of Genetics and Medicine, Naples, Italy; 2Dept Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Dept Pediatrics, Federico II University, Naples, Italy Helper dependent adenoviral vectors (HDAds) can mediate longterm, high level transgene expression from transduced hepatocytes S146 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... inserts Adenovirus and Other DNA Virus Vectors: Biology and Vector Design 373 Sustained and Safe Inhibition of Hepatitis B Virus Replication In Vivo Using HelperDependent Adenovirus Vectors To Deliver...ADENOVIRUS AND OTHER DNA VIRUS VECTORS: BIOLOGY AND VECTOR DESIGN were also transduced Intravenous administration of scAAV2/9 to late gestation fetal macaques also produced widespread gene. .. livers of mice at termination of the investigation, which indicates that diminished efficacy was a result of switching off of transgene expression rather than elimination of the vector from hepatocytes