Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo.

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.     

Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral second-strand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to approximately 16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.     

Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency

We have documented that a cellular chaperone protein, FKBP52, when phosphorylated at tyrosine and/or serine/threonine (Ser/Thr) residues, interacts with the D-sequence in the inverted terminal repeats of the adeno-associated virus 2 (AAV) genome, inhibits the viral second-strand DNA synthesis, and leads to inefficient transgene expression from recombinant AAV vectors in certain cell types. We have also demonstrated that FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and that deliberate overexpression of TC-PTP leads to more efficient viral second-strand DNA synthesis, and increased transgene expression. However, the identity of the putative Ser/Thr protein phosphatase that dephosphorylates FKBP52 at Ser/Thr residues has remained elusive. Using known inhibitors of Ser/Thr phosphatases, we have now identified protein phosphatase 5 (PP5) to be a candidate enzyme. Deliberate overexpression of PP5 in 293 cells, which does not influence cellular growth, leads to approximately 5-fold increase in the transduction efficiency of conventional single-stranded AAV vectors, but no significant enhancement in the transduction efficiency of self-complementary AAV vectors, suggesting that PP5 plays a role in AAV second-strand DNA synthesis. Electrophoretic mobility-shift assays show that in cells overexpressing PP5, the extent of the complex formation between FKBP52 and the AAV D-sequence is significantly reduced. These studies suggest that PP5-mediated dephosphorylation of FKBP52 at Ser/Thr residues augments viral second-strand DNA synthesis and enhances AAV transduction efficiency, which has implications in the optimal use of these vectors in human gene therapy.     

Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.     

High-efficiency Transduction and Correction of Murine Hemophilia B Using AAV2 Vectors Devoid of Multiple Surface-exposed Tyrosines

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30–80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.     

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.     

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.     

The Pleiotropic Effects of Natural AAV Infections on Liver-directed Gene Transfer in Macaques

Adeno-associated viral (AAV) vectors hold great potential for liver-directed gene therapy. Stable and high levels of transgene expression have been achieved in many murine models. Systemic delivery of AAV vectors in nonhuman primates (NHPs) that are natural hosts of AAVs appear to be challenging due to the high prevalence of pre-existing neutralizing antibodies (NAbs). This study evaluates the performance of AAV8, hu.37, and rh.8 vectors expressing green fluorescent protein (GFP) from a liver-specific promoter in rhesus macaques. Two of the animals that received AAV8 showed transduction of 24 and 40% of hepatocytes 7 days after systemic vector delivery. Importantly, expression was detected in several animals after 35 days despite the elevation of liver enzymes and development of transgene-specific T cells in liver. Pre-existing low levels of NAbs profoundly impacted the outcome of gene transfer and redirected vector DNA to spleen. We developed a sensitive in vivo passive transfer assay to detect low levels of NAbs to these novel AAV serotypes. Other strategies need to be developed to reduce immune response to the transgene in order to maintain long-term gene expression.     

Patterns of gene expression from in utero delivery of adenoviral-associated vector serotype 1

Adenoviral-associated viral vectors (AAV) have shown significant promise for efficient gene delivery to multiple tissues. Studies of different serotypes of AAV revealed different expression patterns provided by gene delivery in postnatal mice. Previous in utero gene delivery studies of AAV serotype 2 (AAV2) demonstrated efficient gene expression in certain fetal tissues depending on route of administration. We studied the pattern of gene expression from AAV serotype 1 (AAV1) using intramuscular, intraperitoneal, and intravascular routes of administration in embryonic day 16 C57BL/6 mice. Limb skeletal muscle transduction was only achieved with AAV1 by intramuscular administration. The levels of gene expression were 20-fold higher than a comparable administration of AAV2. Diaphragm muscle transduction by AAV1 was achieved at the highest level by intraperitoneal administration, and to a lesser degree by intravascular administration. All delivery routes resulted in transgene expression in the lung. Our results indicate that AAV1 can offer higher transgene expression in fetal skeletal muscle than AAV2 with intramuscular administration. The transgene expression pattern in different tissues, which depends on vector serotype and route of administration, will need to be considered in planning therapeutic studies for specific disorders.     

Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors.

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.     

Enhanced transduction of mouse salivary glands with AAV5-based vectors

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.     

Enhancing transduction of the liver by adeno-associated viral vectors

A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 x 10(7) vg per mouse (4 x 10(8) vg kg(-1)) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4+/-0.6 to 9+/-2 microg ml(-1) in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10-20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.     

Muscle‐directed gene therapy for hemophilia B with more efficient and less immunogenic AAV vectors

Summary. Background: Adeno‐associated viral vector (AAV)‐mediated and muscle‐directed gene therapy is a safe and non‐invasive approach to treatment of hemophilia B and other genetic diseases. However, low efficiency of transduction, inhibitor formation and high prevalence of pre‐existing immunity to the AAV capsid in humans remain as main challenges for AAV2‐based vectors using this strategy. Vectors packaged with AAV7, 8 and 9 serotypes have improved gene transfer efficiencies and may provide potential alternatives to overcome these problems. Objective: To compare the long‐term expression of canine factor IX (cFIX) levels and anti‐cFIX antibody responses following intramuscular injection of vectors packaged with AAV1, 2, 5, 7, 8 and 9 capsid in immunocompetent hemophilia B mice. Results: Highest expression was detected in mice injected with AAV2/8 vector (28% of normal), followed by AAV2/9 (15%) and AAV2/7 (10%). cFIX expression by AAV2/1 only ranged from 0 to 5% of normal levels. High incidences of anti‐cFIX inhibitor (IgG) were detected in mice injected with AAV2 and 2/5 vectors, followed by AAV2/1. None of the mice treated with AAV2/7, 2/8 and 2/9 developed inhibitors or capsid T cells. Conclusions: AAV7, 8 and 9 are more efficient and safer vectors for muscle‐directed gene therapy with high levels of transgene expression and absence of inhibitor formation. The absence of antibody response to transgene by AAV7, 8 and 9 is independent of vector dose but may be due to the fact that these three serotypes are associated with high level distribution to, and transduction of, hepatocytes following i.m. injection.     

Comparison of adenoviral and adeno-associated viral vectors for pancreatic gene delivery in vivo

Although effective gene therapy vectors have been developed for organ systems such as the liver, an effective delivery vector to the pancreas in vivo has remained elusive. Of the currently available viral vectors, adenovirus and adeno-associated virus (AAV) are two of the most efficient at transducing nondividing cells. We have constructed recombinant adenovirus (AdVLacZ), adeno-associated virus serotype 2 (AAV2LacZ), and pseudotyped adeno-associated virus serotype 5 and 8 (AAV5LacZ, AAV8LacZ) carrying the LacZ reporter, and compared the transduction efficiency of these four vectors in the pancreas of mice in vivo. We showed that adenovirus, AAV2, and AAV8 are capable of transducing the pancreas in vivo, but with different expression kinetics, efficiencies of transduction, and persistence. AdVLacZ-transduced pancreas exhibited maximum LacZ expression at 1 week postdelivery, with greater than 90% of expression lost at 4 weeks. AAV2LacZ-transduced pancreas displayed peak LacZ levels at 4 weeks postdelivery, with no significant decrease in expression for up to 8 weeks. AAV8LacZ was at least 10-fold more efficient than AAV2LacZ in transducing the pancreas in vivo, with significant levels of expression detectable at 1 week, whereas AAV5LacZ did not result in any detectable transgene expression at all tested time points. All three vectors primarily transduced pancreatic acinar cell types, with limited transduction of pancreatic endocrine cells. AdVLacZ elicited a significant leukocyte infiltration early after delivery into the pancreas, whereas none of the AAV vectors elicited a significant leukocyte response. None of the tested vectors caused significant changes in serum amylase or blood glucose levels, suggesting that they do not significantly alter pancreatic function. These vectors will be useful for studying novel gene delivery based treatments in animal models for diabetes and other pancreatic disorders.     

Genomic stability of self-complementary adeno-associated virus 2 during early stages of transduction in mouse muscle in vivo

Studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) as self-complementary duplex strand (sc) results in early transgene expression, possibly eliminating rate-limiting second-strand synthesis. In the present study, we evaluated the molecular organization, stability of the sc AAV genome, and transgene expression in the quadriceps muscle of C57BL/6J mice in vivo as compared with single-stranded (ss) AAV. Studies were carried out with rAAV encoding green fluorescent protein (GFP) or human carcinoembryonic antigen (CEA) either as single-stranded or self-complementary duplex strand structures, encapsidated in AAV-2 capsids. Mice were injected with 10(11) particles of the respective viruses and the vector-injected muscles were harvested 1 week, 2 weeks, 3 weeks, or 2 months later. Tissues were processed for total DNA isolation for the analyses of vector genomic configuration and copy number, and for immunostaining of transgene expression. ELISA was done on serum samples to quantitate CEA-specific humoral immune response as a correlate of transgene expression. Results of Southern blot and PCR analyses indicated more disintegration of the monomeric ss AAV DNA in vivo compared with linear sc AAV DNA. The results also indicated efficient conversion of the self-complementary duplex-stranded vector genome to dimer during early time points. As expected, transgene expression was detected at early time points with self-complementary duplex-stranded vector and persisted stably. However, the advantage of higher transgene expression from sc AAV was balanced over time by the single-stranded vector. These data demonstrate that sc AAV provides better stability for transgene structure during the initial stages of transduction and may have better utility in AAV gene therapy in situations, which mandate early transgene expression.     

Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, double-stranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAV-mediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.     

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.     

Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8⁺ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.