Capsid Serotype Research Articles

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.

Adeno-Associated Virus-Based Gene Therapy for Inherited Disorders

Adeno-associated virus vectors are capable of long-term gene transfer without obvious adverse effects in a number of animal models. Over the last two decades, preclinical and early phase clinical trials in cystic fibrosis and alpha-1 antitrypsin deficiency were undertaken to test the feasibility of this approach. The results of those studies have been important since they have indicated that in vivo gene transfer is feasible and relatively safe. In addition, a number of key limitations to the current generation of AAV2 gene therapy vectors have been defined. The information about these limitations has been used to develop newer AAV vector approaches, based on new mutant and alternative serotype capsids and enhanced promoter systems. The evaluation of safety and efficacy of these newer agents is ongoing.

AAV2/5-mediated NGF gene delivery protects septal cholinergic neurons following axotomy

Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.

Transduction of Pancreatic Islets with Pseudotyped Adeno-Associated Virus: Effect of Viral Capsid and Genome Conversion

Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.

402. Comparison of Pseudotyped rAAV Vectors for Transduction of Primary Hematopoietic Stem and Progenitor Cells Assayed In Vitro and In Vivo

We evaluated a panel of AAV serotypes for their ability to transduce hematopoietic stem and progenitor cells of human and murine origin. rAAV-2 genomes encoding either dsRed1 or firefly luciferase under the control of the hybrid CBA promoter were packaged into capsids derived from AAV-1, AAV-5, AAV-7, AAV-8 and AAV-9 and purified by successive rounds of cesium chloride gradient centrifugation. Hematopoietic cells were transduced overnight at low cytokine concentrations, following which cells were assayed in vitro for transgene expression or transplanted into sublethally irradiated NOD/SCID mice. For murine marrow cells, AAV-7 and AAV-showed the highest levels of expression as compared with the other serotypes. For analysis of human cells, hematopoietic stem/progenitor cells were purified from cord blood, cytokine-primed peripheral blood and bone marrow samples obtained using IRB approved protocols. CD34 cells were immunomagnetically purified and transduced with rAAV-dsRed. Flow cytometric analyses revealed that AAV-7 consistently showed the highest level of transgene expression in vitro in CD34 cells derived from cord blood and bone marrow with levels ranging from 36-64% 24 hours after transduction. This was followed by AAV-8 and AAV-1, at 10-43% expression at 24 hours post-transduction, while the other serotypes exhibited lower levels of transgene expression. We next compared transduction of the CD34+CD3//Q-population, which is highly enriched for stem cells. CD34+CD3//Q-cells were flow sorted to purity prior to transduction with the different serotypes. Notably, this primitive population yielded higher levels of transduction than the more heterogeneous CD34 population, with approximately 60-70% of CD34+CD3//Q-cells from cord blood and cytokine primed peripheral blood showing dsRed expression 24 hours after transduction with AAV-7. As with CD34 cells, transduction with AAV-8 and AAV-1 were the next most efficient. We have previously demonstrated that although rAAV-2 transduction in vitro results in poor expression at early time points, possibly due to defined limitations in uncoating, nuclear entry and second strand synthesis, long term studies and in vivo assays demonstrate that rAAV-2 efficiently transduces primitive human hematopoietic stem cells capable of serial engraftment in immune deficient mice. Since in vitro assays may not reflect true stem cell activity, we initiated in vivo transplantation studies of stem cells transduced with different AAV serotypes to determine the relative long term engraftment and gene expression potentials of AAV genomes packaged in the different serotype capsids. Initial results following live imaging of luciferase-transduced recipients confirm both the early onset of gene expression with AAV-7 and AAV-8 and the hierarchy of transgene expression seen with the serotypes in vitro. Overall, our results suggest that rAAV genomes pseudotyped in AAV-7 and AAV-8 capsids are very promising for gene transfer into hematopoietic stem cells.

499. Species-Specific Differences in the Polarity of rAAV5 and rAAV2 Transduction of Mouse and Human Airway Epithelia

Top of pageAbstract Species-specific differences in the tropism of viral vectors between mice and humans have made it difficult to anticipate the most appropriate adeno-associated virus (AAV) serotypes for clinical trials. In the context of developing gene therapies for cystic fibrosis lung disease, human air-liquid interface (ALI) models of airway epithelium have been useful as an intermediate surrogate for evaluating the human tropisms of various rAAV serotypes. In this model, we have previously shown that apical infection with rAAV2 and rAAV5 vectors have very similar transduction efficiencies. Additionally, in the presence of proteasome inhibitors, transduction is enhanced |[sim]|1000-fold with both serotypes following apical infection. In contrast to these findings in the human ALI model, in vivo rAAV5 transduction is 50-100-fold greater than that of rAAV2 in mouse lung. These differences could be the result of model system dependencies or species-specific differences in the tropism of AAV2 and AAV5 for airway epithelium in mice and humans. To resolve whether species-specific differences (human vs. mouse) or model system-specific differences (in vitro ALI culture vs. in vivo lung) are the cause of the discrepancies in rAAV5 and rAAV2 transduction in these various studies, we have compared transduction of these rAAV serotypes in mouse and human airway epithelia in ALI culture using pseudotyped luciferase rAAV vectors. Differentiated mouse airway epithelia in ALI culture gave electrophysiologic characteristics similar to those previously reported for murine tracheal epithelium, but distinct from that of human ALI cultures. Following apical infection of mouse airway epithelia in ALI culture, rAAV2/5 gave |[sim]|100-fold greater levels of transduction than infection with rAAV2/2. These results were similar to the differences in transduction between these two serotypes seen in vivo in mouse lung. These findings demonstrate that ALI cultures of mouse airway epithelium retain species-specific differences in comparison to human airway epithelium, which is reflected in a greater apical tropism for rAAV5 as compared to rAAV2 capsid serotypes. Interestingly, basolateral infection of mouse ALI cultures also retained a greater transduction efficiency (10-fold) with rAAV2/5, as compared to rAAV2/2. These differences in basolateral tropisms were reversed for human ALI cultures. Viral transduction for both rAAV2/2 and rAAV2/5 from the apical membrane of both mouse and human ALI cultures was enhanced 2-3 logs by the addition of proteasome modulating agents Doxorubicin and LLnL at the time of infection. Basolateral infection in the absence of proteasome modulating was always more effective than apical infection regardless of the species. However, the addition of proteasome modulating agents appeared to normalize transduction from the apical membrane to maximal levels regardless of the serotype or species. These findings suggest that rAAV2 and rAAV5 vectors have different transduction efficiencies for mouse and human airway epithelium due to serotype-specific differences in post-entry blocks modulated by the proteasome pathway.

118. Self-Complementary AAV Vectors Pseudotyped with Either Serotype 5 or 8 Capsids Give Therapeutic Human Factor IX Levels at Significantly Reduced Doses in Non-Human Primates

We have created a novel self-complementary adeno-associated virus (scAAV) vector for Factor IX (hemophilia B) gene therapy, and have tested this vector with multiple serotype capsids in both murine and Rhesus macaque models. A small, liver-specific transcriptional cassette enabled inclusion of the 1386 bp of human Factor IX coding sequence in a single terminal resolution site deleted scAAV vector. At both early and late time points after transduction, self-complementary vector genomes purified from murine liver show striking differences from those produced by conventional single-stranded vectors, and these differences are consistent with the predicted asymmetric hairpin structure of scAAV. Using vectors pseudotyped with serotype 8 capsids (scAAV2/8) in mice, our new vector provides 30|[ndash]|60 fold greater circulating Factor IX (138 |[mu]|g/ml from 1011 particles) than our previous, conventional single-stranded AAV2/8 vector. This enhancement was observed relatively uniformly across a dose range of 109 to 1011 genome copies/mouse, in agreement with previous observations of the non-saturable behavior of AAV2/8 vectors in liver transduction. scAAV2/5 vectors produced |[sim]|50 fold lower levels of human Factor IX than scAAV2/8 in mice, and with somewhat delayed kinetics.

507. Modeling Adeno-Associated Virus 2 Capsid for the Identification of Targeting Ligands for Enhanced Gene Transfer to Polarized Human Airway Epithelial Cells

Top of pageAbstract Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of CFTR gene. Despite intensive studies on genetic, biochemical and physiological basis of this debilitating disease, pharmacokinetic interventions have only been palliative. Newer therapeutic approaches based on molecular medicine, including gene therapy, offer better hope for the treatment of CF. Adeno-associated viral vectors (AAV) are capable of stable expression with low immunogenicity. Although optimal delivery and expression of CFTR gene in human airway by AAV can phenotypically correct the disease, gene transfer efficiency is highly limited due to pathophysiological barriers in these patients. Despite the fact that alternate AAV serotype vectors have shown better transduction compared to AAV2-based vectors, global gene transfer efficiency in human airway epithelium and lung has been dismal. Towards engineering better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV by genetic capsid modification using the well-characterized AAV2 serotype. Based on recent elucidation of AAV2 crystal structure and other targeting studies, we genetically incorporated high-affinity peptide ligands to human airway epithelium on the HI loop region of AAV2 capsid protein at amino acid 587. Six independent mutant AAV were constructed, packaged and purified containing heterologous peptide ligands previously reported to have high affinity binding to known and unknown receptors on human airway epithelial cells. The vectors were tested on non-human airway and human airway epithelial cells for enhanced infectivity. One of the mutant vectors, molecularly decorated with a peptide THALWHT, not only showed highest transduction of human airway epithelial cells but also indicated significant transduction (>105 compared to wild-type capsid) upon polarization of these cells. Interestingly, this modified vector was also able to infect these cells independently of heparan sulfate proteoglycan receptor. Ongoing in vivo characterization of this vector in preclinical CF models may lead to its therapeutic utility, and/or can be used to modify other AAV serotype capsids for enhanced gene transfer to human airway epithelium.

Enhancing rAAV vector expression in the lung

Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (Cbeta) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the Cbeta promoter, and the Cbeta promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the Cbeta-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 x 10(6) relative light units (RLU)/g tissue and 2.7 x 10(6) RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful.

Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

Promoters and serotypes: targeting of adeno‐associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo

The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes.

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector.

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.

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.

12. A New Class of Hybrid Adeno-Associated Viral Vectors with Non-Homologous ITRs Improves Directional Recombination and Dual-Vector Reconstitution of Large Transgenes

The relatively small package capacity (less than 5 kb) of adeno-associated virus vectors has been effectively doubled with the development of dual vector heterodimerization approaches. However, the efficiency of such dual vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatamerization. In the present study, we sought to evaluate the mechanisms by which ITR sequences mediate intermolecular recombination of AAV genomes with the goal of engineering more efficient vectors capable of reconstituting large transgenes delivered by a dual vector trans-splicing approach. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at either ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly reduced capacity to form circular intermediates in infected cells, as compared to homologous AV2:2 and AV5:5 ITR vectors, despite their similar capacity to express an encoded EGFP transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a head-to-tail fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor) for which each delivered one exon of a beta-galactosidase minigene flanked by donor and acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their efficiency to reconstitute ?-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual vector sets had a significantly enhanced trans-splicing efficiency (6-10 fold, depending on the capsid serotype) as compared to homologous ITR vectors. Molecular studies of viral genome structure suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provide new strategies to manipulate viral genome conversion products and direct intermolecular recombination events required for efficient dual AAV vector reconstitution of transgene.

99. Long-Term Expression of Soluble TNFR:Fc Protein Following Multiple Administrations of AAV-TNFR:Fc Vector Pseudotyped with Capsids of Alternate Serotypes

The use of recombinant adeno-associated serotype (rAAV2) as a vector for the liver has been hindered by the relatively low efficiency of stable transduction of hepatocytes (approximately 5%). Alpha-1 antitrypsin(AAT) deficiency is a common single gene disorder in which a mutation in the AAT gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs and a toxic effect on the hepatocyte where it is produced. Therefore, optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction in order to be effective for both liver and lung disease. Recently, rAAV genomes have been successfully pseudotyped with capsids from novel serotypes (eg. rAAV7 and AAV8), and AAV8 was shown to be quite efficient for hepatic transduction (Gao et al. PNAS 2002; 99:11854-59). We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express AAT in both a greater percentage of cells and in greater overall amounts. 1 × 1010 particle doses of serotypes 1, 2, 5 and 8 vectors carrying the human alpha-1-antitrypsin (hAAT) gene were injected in C57Bl/6 female mice at six weeks of age. Circulating hAAT in mouse serum was measured by a human-specific sandwich ELISA. RESULTS: In the serotype comparison studies, rAAV8 vectors express significantly sooner after injection than serotypes 1 or 5. This was demonstrated in samples from 1 week post-injection where serotypes 1 and 5 had no detectable hAAT expression, while the rAAV8 group had a mean level of 574 ug/ml and the rAAV2 group had a mean level of 2 ug/ml hAAT. By week 3, rAAV8 injected animals had circulating hAAT levels 30 times higher than AAV2(607 ug/ml versus 22 ug/ml). Subsequently, a dose escalation study was performed with doses of 1 × 109, 1×1010 and 1×1011 particles of the rAAV8-hAAT vector. At three weeks post-portal vein injection, the animals showed a clear dose-response with mean serum levels of 917, 7,065 and 806,449 ng/ml, respectively in the three groups. These results confirm the impression that rAAV8 may be a more promising gene vector for therapy of liver disorders. Further studies will be necessary to quantify the percent transduction of hepatocytes with rAAV8 and the functional effect in a liver disease model, as well as to evaluate the safety and biodistribution of the rAAV8-hAAT vector.

990. Transduction of Pancreatic Islets with Pseudotyped Adeno-Associated Virus: Effect of Viral Capsid

Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be pseudotyped with other serotype capsids to assemble infectious virions. Using this pseudotyping strategy, AAV2 genomes carrying the GFP reporter gene were encapsidated into AAV1, AAV2, and AAV5 capsids. The utility of cross-packaged AAV1, AAV2, and AAV5 capsid-mediated transduction to murine and human pancreatic islets was evaluated. These pseudotyped rAAV vectors all showed some ability to transduce both human and murine pancreatic islets. The transduction efficiency of rAAV vector was dependent on the cross-packaged capsid. Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islet transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. The vector capsids permit species specific transduction. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. Pseudotyped rAAV transduced islet grafts maintained normal function in vitro, reversed diabetes in vivo, and expressed transgenic product persistently in vivo. These studies provide a methodologic framework for evaluating the function of pseudotyped rAAV.

VP2 Gene Based Phylogenetic Relationship of Indian Isolates of Bluetongue Virus Serotype 1 and Other Serotypes from Different Parts of the World

Bluetongue virus (BTV), a member of genus Orbivirus, family Reoviridae, is non-enveloped with double shelled structure and 10 segmented double stranded RNA genome. The RNA segment L2 encodes an outer capsid serotype specific viral protein VP2. BTV serotype 1 (BTV-1) specific novel primer pair, forward primer (1240-1271 bp) and reverse primer (1844-1813 bp), was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1. The 604 bp PCR product of VP2 gene of BTV-1 Avikanagar (A), Chennai (C) and Sirsa 3 (S3) Indian isolates were cloned in pPCR-Script Amp SK (+) vector and transformed into XL10-Gold Kan ultracompetent Epicurian coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. BTV-1A, C and S3 isolates revealed 99% nucleotide sequence identity within 1304-1844 bp region of VP2 gene. The partial VP2 gene sequences (1240-1844 bp region) revealed that BTV-1 Indian isolates were 89% identical with Australian (AUS) BTV-1 isolates while the identity with South African (SA) BTV-1 isolate was 75%. Phylogenetically, three BTV-1 Indian isolates formed one group which is closely related to BTV-1AUS isolates followed by BTV-1SA, BTV-2, 9, 23, 13, 17, 10 and 11 isolates from different parts of world. Based on partial VP2 gene sequences, it is concluded that Indian isolates of BTV-1 are closely related to BTV-1AUS isolates than BTV-1SA and other serotypes.

Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands.

The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1alpha receptor (FGFR1alpha), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1alpha-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.

Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.