AAV Transduction Research Articles

Multiyear Follow-up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A

BackgroundAdeno-associated virus (AAV)–mediated gene therapy is under investigation as a therapeutic option for persons with hemophilia A. Efficacy and safety data include 3 years of follow-up after a single administration of AAV5-hFVIII-SQ.MethodsWe report durable efficacy, long-term safety, and clinical and biologic results in 15 adults with severe hemophilia A (factor VIII level, ≤1 IU per deciliter) who had received a single infusion of AAV5-hFVIII-SQ at various dose levels. We evaluated the factor VIII level, annualized rate of bleeding events, use of factor VIII, safety, expression kinetics, and biologic markers of AAV transduction for up to 3 years.ResultsThree years after infusion, two participants (one who had received 6×1012 vector genomes [vg] per kilogram of body weight and one who had received 2×1013 vg per kilogram) had factor VIII expression of less than 1 IU per deciliter, as assessed on chromogenic assay. Seven participants (who had received 6×1013 vg per kilogram) had a median factor VIII expression of 20 IU per deciliter; the median number of annualized treated bleeding events was 0, and the median use of exogenous factor VIII was reduced from 138.5 infusions to 0 infusions per year. Bleeding in all target joints (major joints with ≥3 bleeding events within 6 months) in this cohort resolved (≤2 bleeding events within 12 months). Two years after infusion, six participants (who had received 4×1013 vg per kilogram) had a median factor VIII expression of 13 IU per deciliter; the median annualized rate of bleeding events was 0, and the median use of factor VIII was reduced from 155.5 infusions to 0.5 infusions per year. Bleeding in target joints resolved in five of six participants. The factor VIII pharmacodynamic profiles reflected cellular turnover in the blood and molecular events leading to episomal DNA stabilization for persistent expression, findings that are consistent with previous observations in two model systems. Transgene-derived human factor VIII (hFVIII) protein activity mirrored native hFVIII in hemostatic ability. No inhibitor development, thromboses, deaths, or persistent changes in liver-function tests were observed.ConclusionsGene therapy with AAV5-hFVIII-SQ vector in participants with hemophilia A resulted in sustained, clinically relevant benefit, as measured by a substantial reduction in annualized rates of bleeding events and complete cessation of prophylactic factor VIII use in all participants who had received 4×1013 vg per kilogram or 6×1013 vg per kilogram of the gene therapy. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795; EudraCT number, 2014-003880-38.)

Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors.

Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better “communication” between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids.

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy.

GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells invitro. These findings are further validated invivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. Invitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both invitro and invivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

Analysis of hepatic and retinal cell microRNAome during AAV infection reveals their diverse impact on viral transduction and cellular physiology

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell’s own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (−2.24 fold for AAV2 and −3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126–128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142–158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.

Reducing off target viral delivery in ovarian cancer gene therapy using a protease-activated AAV2 vector platform

Gene therapy is a promising strategy for treating metastatic epithelial ovarian cancer (EOC). However, efficient vector targeting to tumors is difficult and off-target effects can be severely detrimental. Most vector targeting approaches rely on surface receptors overexpressed on some subpopulation of cancer cells. Unfortunately, there is no universally expressed cell surface biomarker for tumor cells. As an alternative, we developed an adeno-associated virus (AAV) based “Provector” whose cellular transduction can be activated by extracellular proteases, such as matrix metalloproteinases (MMP) that are overexpressed in the tumor microenvironments of the most aggressive forms of EOC. In a non-tumor bearing mouse model, the Provector demonstrates efficient de-targeting of healthy tissues, especially the liver, where viral delivery is <1% of AAV2. In an orthotopic HeyA8 tumor model of EOC, the Provector maintains decreased off-target delivery in the liver and other tissues but with no loss in tumor delivery. Notably, approximately 10% of the injected Provector is still detected in the blood at 24 h while >99% of injected AAV2 has been cleared from the blood by 1 h. Furthermore, mouse serum raised against the Provector is 16-fold less able to neutralize Provector transduction compared to AAV2 serum neutralizing AAV2 transduction (1:200 vs 1:3200 serum dilution, respectively). Thus, the Provector appears to generate less neutralizing antibodies than AAV2. Importantly, serum against AAV2 does not neutralize the Provector as well as AAV2, suggesting that pre-existing antibodies against AAV2 would not negate the clinical application of Provectors. Taken together, we present an EOC gene delivery vector platform based on AAV with decreased off-target delivery without loss of on-target specificity, and greater immunological stealth over the traditional AAV2 gene delivery vector.

Therapeutic hFIX Activity Achieved after Single AAV5-hFIX Treatment in Hemophilia B Patients and NHPs with Pre-existing Anti-AAV5 NABs

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Polarized AAVR expression determines infectivity by AAV gene therapy vectors.

Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina

Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency.

Serotype survey of AAV gene delivery via subconjunctival injection in mice.

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

AAV8 virions hijack serum proteins to increase hepatocyte binding for transduction enhancement

It has been demonstrated that human serum albumin (HSA) directly interacts with AAV virions and enhances AAV transduction. Several other proteins have also been identified a potential for enhancing AAV8 liver transduction. In our study, LDL or transferrin could enhance transduction in vitro and in vivo. We also found that any combination of two or three of these proteins (HSA, LDL, and transferrin) increased AAV8 transduction in Huh7 cells and in mice liver, which was similar to albumin alone. Pre-incubation of HSA with AAV8 virions prevented AAV8 virions from binding to other proteins. Furthermore, these serum protein receptors didn’t impact AAV8 transduction but blocked the transduction enhancement from AAV8-serum protein complexes. These results indicate that serum proteins are hijacked by AAV8 vectors to increase hepatocyte binding, which shares same binding site. Importantly, the results could help us design an optimal formulation for effective AAV vector delivery in future clinical trials.

Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.

Induction of Virus-specific Immune Tolerance in A Chronic Hepatitis B Virus Mouse Model

Abstract Hepatitis B virus (HBV) infection causes severe public health problem. There are estimated 250 million people being chronically infected by HBV and are at an increased risk of developing end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma. It is well recognized that the chronicity of HBV infection is due to the impaired immune responses to HBV and thus not able to eliminate HBV from the infected hepatocytes. However, the immune mechanisms that lead to immune tolerance and HBV persistence have not been understood. A challenging question for HBV studies is lack of appropriate animal models due to the narrow host range of HBV. To address this challenge, we created a chronic HBV infection model in immunocompetent mice by transduction of adeno-associated virus serotype 8 (AAV8) vector which carries and these mice was transduced with carrying a replication-defective HBV genome with two point mutations in the polymerase(AAV/HBVp−). We showed that mice transduced with AAV/HBVp− produced HBV RNA and proteins, including serum HBV surface antigen and HBV e antigen as well as HBV core protein. The high levels of HBV RNA and proteins sustained for at least 1 year post AAV transduction. Viral transcripts and proteins were detected in the livers of the AAV-injected mice but not in other organs. DNA immunization of these AAV/HBVp− transduced mice failed to induce HBV-specific humoral and cellular immune responses, suggesting these mice developed immune tolerance toward HBV. Thus, the AAV/HBVp− transduced mouse model recapitulates many virological and immunological characteristics of chronic HBV in humans. This animal model could be useful for the development of new treatments and immune-based therapies for chronic HBV infections.

Direct interaction of human serum proteins with AAV virions to enhance AAV transduction: immediate impact on clinical applications

Direct interaction of human serum proteins with AAV virions to enhance AAV transduction: immediate impact on clinical applications

Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling.

Retinal ganglion cells (RGCs) do not normally regenerate injured axons, but die upon axotomy. Although IL-6-like cytokines are reportedly neuroprotective and promote optic nerve regeneration, their overall regenerative effects remain rather moderate. Here, we hypothesized that direct activation of the gp130 receptor by the designer cytokine hyper-IL-6 (hIL-6) might induce stronger RGC regeneration than natural cytokines. Indeed, hIL-6 stimulated neurite growth of adult cultured RGCs with significantly higher efficacy than CNTF or IL-6. This neurite growth promoting effect could be attributed to stronger activation of the JAK/STAT3 and PI3K/AKT/mTOR signaling pathways and was also observed in peripheral dorsal root ganglion neurons. Moreover, hIL-6 abrogated axon growth inhibition by central nervous system (CNS) myelin. Remarkably, continuous hIL-6 expression upon RGC-specific AAV transduction after optic nerve crush exerted stronger axon regeneration than other known regeneration promoting treatments such as lens injury and PTEN knockout, with some axons growing through the optic chiasm 6 weeks after optic nerve injury. Combination of hIL-6 with RGC-specific PTEN knockout further enhanced optic nerve regeneration. Therefore, direct activation of gp130 signaling might be a novel, clinically applicable approach for robust CNS repair.

P.262 - Prednisolone therapy reduces incidence of mononuclear T-cell infiltrates after AAV transduction of GALGT2 in the rhesus macaque

P.262 - Prednisolone therapy reduces incidence of mononuclear T-cell infiltrates after AAV transduction of GALGT2 in the rhesus macaque

30. Modulation of Intracellular Calcium Enhances AAV Transduction in the CNS

Recent preclinical studies and clinical trials utilizing recombinant AAV vectors have highlighted vector dose-related toxicity as a potential safety concern. Therefore, strategies to enhance AAV transduction may mitigate this toxicity, as they may allow for administration of lower vector doses. While some strategies rely on modification of the capsid to achieve such, pharmacological modulation of the cellular environment often has the benefit of enhancing AAV transduction independent of capsid serotype. Intracellular calcium is important in the life cycles of several viruses, such as Ebola virus and rotavirus. Several small molecule drugs and biologics exist that can modify intracellular calcium levels in a specific manner. In the current study, we sought to determine if modulation of intracellular calcium levels affects AAV transduction. Increasing intracellular calcium levels using either thapsigargin or ionomycin decreased AAV transduction by an order of magnitude. Conversely, decreasing intracellular calcium levels using the cell permanent calcium chelator BAPTA-AM increased transduction by 10 to 100-fold in a cell independent fashion. In addition, the effects of modification of intracellular calcium are not dependent on AAV serotype. Furthermore, in vivo studies performed in mice demonstrate that BAPTA-AM augments transduction by different AAV serotypes, when administered intracranially. In summary, our results support the preclinical evaluation of drugs and biologics that modulate intracellular calcium in the CNS and evaluate their potential for affecting AAV transduction. Results from ongoing studies in mouse models of disease will be presented.

539. Screening for Recombinant Adeno-Associated Viral Vectors That Selectively Transduce Hepatitis B Virus Infected Cells

Hepatitis B is a disease caused by infection with the hepatitis B virus (HBV), a small enveloped DNA virus which belongs to the Hepadnaviridae family. Even though a safe and effective vaccine is available, HBV represents the ninth most common cause of death worldwide with an estimated 350 million chronically infected individuals. HBV infection can cause cirrhosis and hepatocellular carcinoma and currently there are 7 approved drugs for the treatment of HBV infection in the USA. These drugs decrease the risk of liver damage from HBV by slowing but not eliminating viral replication in most patients, possibly related to the persistence of integrated HBV genomes. Gene therapy using recombinant adeno-associated viral (rAAV) vectors constitute a promising tool to combat HBV infection as rAAV vectors have the ability to transduce and establish long-term and stable transgene expression in liver cells. Preclinical studies have successfully utilized rAAV vectors to combat viral infections after delivering short-hairpin RNA (shRNA) expression cassettes for knocking down viral coding or host genes required for viral replication and spread. Because promiscuous targeting of host cellular genes in the majority of non-infected cells may be detrimental, specific targeting of HBV-infected cells adds a new layer of potential therapeutic targets and safety. Thus we set out to identify new AAV capsids that selectively transduce HBV-infected cells. To do this, we created new AAV shuffled capsid libraries and used these for multiple rounds of infection in HBV-infected hepatoma cells. Each library is co-infected with wild-type Adenovirus-5 (helper virus) allowing the rAAV genomes to replicate in the HBV-infected cells. Isolation of replicating rAAV over successive passages in cultured cells allows for a more stringent selection as all steps in AAV transduction must occur. We have varied the screens using various multiplicity of infections (MOIs) of the library and/or utilized a pre-clearing step using a HBV negative hepatoma cell line. Several selected capsids derived from multiple parental capsids were vectorized. One capsid (AAV-GK4) was found to transduce the infected cells approximately 6 times more robustly than uninfected cells (using an MOI of 100 and 1,000). Interestingly, the same result was observed with AAV-DJ, a variant previously selected on human hepatoma cells. Additional screens and candidates are pending further evaluation. The possibility of finding new rAAV capsids with an increased transduction efficiency and selective tropism for HBV-infected cells will provide a reagent or therapeutic that can deliver a transgene that will allow one to selectively modify or potentially kill infected but not the uninfected cells contained within the liver.

545. Syngeneic AAV Pseudo-Vectors Potentiates Full Vector Transduction

An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current study, we systemically examined how the AAV pseudo-vectors interact with the full AAV vectors in the transduction processes. Three different types of AAV pseudo-vectors were used: syngeneic AAV pseudo-vector (sAAV, purified from the full AAV vector preparation with the same reporter gene), null AAV pseudo-vector (nAAV, produced by using AAV packaging plasmid and adenoviral helper plasmid), and allogeneic AAV pseudo-vector (aAAV, purified from the AAV vector preparation with a genome different to reporter gene). All three AAV2 pseudo-vectors exhibited inhibition effects on full AAV2 vectors particle transduction in a dose dependent manner in vitro. AAV2 pseudo-vectors not only inhibited full AAV2 vector transduction but also decreased gene expression from full AAV8 vectors. However, sAAV2 pseudo-vectors exhibited less inhibition effects on the transduction efficiency of full AAV2 or AAV8 vectors compared with nAAV2 or aAAV2 pseudo-vectors in vitro. In contrast, all three kinds of AAV8 pseudo-vectors did not show such inhibition effects on full AAV8 and AAV2 vectors in vitro or in vivo. Moreover, the sAAV8 pseudo-vector enhanced its full AAV8 vector transduction while nAAV8 and aAAV8 pseudo-vector did not have similar effects. Nine fold sAAV8 pseudo-vectors increased AAV8-TTR-hF8-X5 transduction by 2~5-fold in two different hemophilia A mouse models as measured the coagulation activity of factor VIII by aPTT assays and antigen levels by Elisa. Further characterization showed that sAAV8 and aAAV8 pseudo-vectors contain DNAs of varying sizes. In general, these DNAs still have AAV ITR sequences and partial vector genomes near ITRs. It is likely that such partial genome could anneal to the full AAV genomes and facilitate the second strand DNA synthesis thus increasing full AAV transduction.