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366. The Hybrid AAV2.5 Vector Transduces Neurons Bordering the Third and Fourth Ventricles after Lateral Ventricular Infusion in Rats
AAV VECTORS II methylation may occur preferentially on discrete rAAV molecular structures found in transduced liver. Altogether, these results may impact on rAAV vector design by addressing these dynamic epigenetic modications in situ.
366. The Hybrid AAV2.5 Vector Transduces Neurons Bordering the Third and Fourth Ventricles after Lateral Ventricular Infusion in Rats
Dawn E. Bowles,2 Chengwen Li,1 Joseph E. Rabinowitz,3 R. Jude Samulski,1 Thomas J. McCown.1 1 UNC Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Department of Surgery, Duke University, Durham, NC; 3Department of Medicine, Thomas Jefferson University, Philadelphia, PA.
When contemplating the use of any AAV vector for site directed CNS gene therapy, the consequences of inadvertent vector leakage into the ventricular system should be considered, especially given the rapidly expanding repertoire of AAV serotypes and hybrids. The AAV2.5 vector was designed as a hybrid between AAV1 and AAV2 where 4 amino acids from AAV1 have been substituted (Q263A, N705A, V708A, T716N; AAV2 numbering), and one AAV1 amino acid (T265) has been inserted into the AAV2 capsid. These changes produced an AAV vector that exhibits peripheral properties characteristic of both AAV2 and AAV1, some of which provide certain advantages over AAV2. In order to determine the CNS properties of this AAV hybrid, self-complimentary AAV2.5-GFP was infused into the rat piriform cortex (1 microliter), and two weeks later the pattern of GFP expression was determined. Neurons composed the overwhelming majority of GFP positive cells, similar to the neuronal tropism of AAV2 or AAV1. However, quite different results were obtained after ventricular administration. When self-complimentary AAV2-GFP (10 microliters; 2.X 1012 viral particles/ml) was infused into the lateral ventricle of rats, only a few GFP positive ependymal cells were found in the area of infusion, and few if any neurons were transduced, either proximal or distal to the infusion site. In marked contrast, when self-complimentary AAV2.5 (10 microliters, 1 X 1012 viral particles/ml) was infused into the lateral ventricle, substantial numbers of GFP positive neurons were found in the hypothalamus along the rostral-caudal extent of the third ventricle, in the central gray surrounding the Sylvian aqueduct and in the vestibular nuclei near the fourth ventricle. More surprisingly, we found near total transduction of the subcommissural organ, a structure composed of specialized, nestin positive endothelial cells located within the dorsal third ventricle. Since functionally, the subcommissural organ has been implicated in the control of CSF ow and pressure, certainly inadvertent transduction could prove detrimental. These results emphasize the need to thoroughly characterize the tropism of novel AAV vectors, especially when considering specic CNS applications. (Supported by NINDS grant 063611) (A patent for AAV2.5 has been issued to UNC under the names of D. Bowles and R.J. Samulski, and rights have been retained by Asklepios Biopharmaceutical Inc., co-founded by R.J. Samulski).
367. Differential Lymphatic Transport of Chimeric and Naturally Occurring AAV Strains
Jana L. Phillips,1 Roland Cheung,1 Chengwen Li,1 Aravind Asokan.1,2 1 Gene Therapy Center, UNC Chapel Hill, Chapel Hill, NC; 2 Department of Genetics, UNC Chapel Hill, Chapel Hill, NC. Certain AAV serotypes, such as AAV8 and 9, exhibit the ability to transduce systemic organs and peripheral tissues following intravenous administration. In the current study, we tested the hypothesis that viral transport through lymphatic vasculature plays a critical, albeit differential role in systemic dissemination of AAV S142
vectors. Following a subcutaneous injection of 1x1010 viral particles into the hind footpad, AAV8 and AAV9 transduced major systemic organs, particularly the liver with efciency similar to that seen following an intravenous injection. In contrast, AAV2 remained in the localized area of injection, likely due to propensity for binding heparan sulfate in subcutaneous tissue. AAV2i8, which does not bind heparan sulfate, demonstrated selective and systemic transduction in muscle tissue, but not the liver. Interestingly, AAV2i8, AAV8 and AAV9, but not AAV2 vectors also transduced the draining popliteal lymph node (PLN) following footpad injection. Vector genome copies in the circulation were identical regardless of route of injection suggesting that AAV vectors transit rapidly from peripheral lymphatics to circulating blood. Our data suggests that certain AAV strains can travel through the lymphatic system, which might constitute a signicant mechanism for systemic dissemination.
368. Optimizing Liver Transduction of the Low Seroprevalent AAV rh32.33
Luk H. Vandenberghe, Ru Xiao, James M. Wilson. Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Liver serves as a target of gene transfer in the treatment of a variety of inherited and acquired diseases. Vector administered into the circulation can access hepatocytes through its fenestrated capillaries. Vectors based on adeno-associated viruses (AAVs) have yielded promising results in pre-clinical and clinical models of liver-directed gene therapy. However, we have shown that pre-existing neutralizing antibodies (NAB) are prevalent to most AAV types and that even trace levels can inhibit hepatocyte transduction in vivo. Moreover, the advantage of novel AAVs with increased hepatotropism to require lower therapeutic doses will likely not hold in the context of NAB as lower doses will be more readily neutralized. One solution may be a capsid variant we isolated from macaques called AAVrh32.33 which is rarely neutralized by human serum. This novel capsid is structurally distinct from most of the others we isolated and can efciently package vectors that are not inhibited by humoral responses to human AAVs although hepatocyte transduction is 5 to 10–fold lower than that achieved with AAV8 in naïve animals. We therefore evaluated AAVrh32.33 for liver gene therapy by modulating its expression prole through both genome and capsid modication. First, we packaged self complementary (sc) genomes encoding GFP or human a1-antitrypsin into rh32.33. Liver gene transfer efciency was evaluated in C57Bl/6 mice as well as C57Bl/6 RAG knockout mice following tail vein administration of AAV doses ranging up to 1012 GC. These studies demonstrated transduction of over 50% of hepatocytes without transaminase elevations following high dose scAAV rh32.33 vectors; transduction efciency of rh32.33 at this dose was equivalent to that of AAV8 at a 10-fold lower dose. Modications were introduced into the AAVrh32.33 capsid to preserve the desired serological prole (i.e., low neutralization by human sera), yet increase its hepatotropism and gene transfer efciency. In a rst strategy, individual surface exposed tyrosines were substituted for phenylalanine residues. These single amino-acid substitutions were not anticipate to substantially alter serological reactivity but increase gene transfer efciency based on published results with AAV2 and AAV8 in a similar strategy. Seven tyrosine variants expressing luciferase were evaluated for liver directed in C57Bl/6 mice but none were found to be more efcient than the wild type vector. In another approach, the N-terminal portion of the viral capsid (VP12u) that contains a phospholipase domain important for infectivity was fused with heterologous VP3 open reading frames. VP12u swapping among AAV serotypes can affect gene transfer kinetics and overall efciency. Here we, introduced AAV8 VP12u into rh32.33 in a reciprocal manner and demonstrate in C57Bl6 mice transduction to be increased over Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
366. The Hybrid AAV2.5 Vector Transduces Neurons Bordering the Third and Fourth Ventricles after Lateral Ventricular Infusion in Rats
Dawn E. Bowles,2 Chengwen Li,1 Joseph E. Rabinowitz,3 R. Jude Samulski,1 Thomas J. McCown.1 1 UNC Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Department of Surgery, Duke University, Durham, NC; 3Department of Medicine, Thomas Jefferson University, Philadelphia, PA.
When contemplating the use of any AAV vector for site directed CNS gene therapy, the consequences of inadvertent vector leakage into the ventricular system should be considered, especially given the rapidly expanding repertoire of AAV serotypes and hybrids. The AAV2.5 vector was designed as a hybrid between AAV1 and AAV2 where 4 amino acids from AAV1 have been substituted (Q263A, N705A, V708A, T716N; AAV2 numbering), and one AAV1 amino acid (T265) has been inserted into the AAV2 capsid. These changes produced an AAV vector that exhibits peripheral properties characteristic of both AAV2 and AAV1, some of which provide certain advantages over AAV2. In order to determine the CNS properties of this AAV hybrid, self-complimentary AAV2.5-GFP was infused into the rat piriform cortex (1 microliter), and two weeks later the pattern of GFP expression was determined. Neurons composed the overwhelming majority of GFP positive cells, similar to the neuronal tropism of AAV2 or AAV1. However, quite different results were obtained after ventricular administration. When self-complimentary AAV2-GFP (10 microliters; 2.X 1012 viral particles/ml) was infused into the lateral ventricle of rats, only a few GFP positive ependymal cells were found in the area of infusion, and few if any neurons were transduced, either proximal or distal to the infusion site. In marked contrast, when self-complimentary AAV2.5 (10 microliters, 1 X 1012 viral particles/ml) was infused into the lateral ventricle, substantial numbers of GFP positive neurons were found in the hypothalamus along the rostral-caudal extent of the third ventricle, in the central gray surrounding the Sylvian aqueduct and in the vestibular nuclei near the fourth ventricle. More surprisingly, we found near total transduction of the subcommissural organ, a structure composed of specialized, nestin positive endothelial cells located within the dorsal third ventricle. Since functionally, the subcommissural organ has been implicated in the control of CSF ow and pressure, certainly inadvertent transduction could prove detrimental. These results emphasize the need to thoroughly characterize the tropism of novel AAV vectors, especially when considering specic CNS applications. (Supported by NINDS grant 063611) (A patent for AAV2.5 has been issued to UNC under the names of D. Bowles and R.J. Samulski, and rights have been retained by Asklepios Biopharmaceutical Inc., co-founded by R.J. Samulski).
367. Differential Lymphatic Transport of Chimeric and Naturally Occurring AAV Strains
Jana L. Phillips,1 Roland Cheung,1 Chengwen Li,1 Aravind Asokan.1,2 1 Gene Therapy Center, UNC Chapel Hill, Chapel Hill, NC; 2 Department of Genetics, UNC Chapel Hill, Chapel Hill, NC. Certain AAV serotypes, such as AAV8 and 9, exhibit the ability to transduce systemic organs and peripheral tissues following intravenous administration. In the current study, we tested the hypothesis that viral transport through lymphatic vasculature plays a critical, albeit differential role in systemic dissemination of AAV S142
vectors. Following a subcutaneous injection of 1x1010 viral particles into the hind footpad, AAV8 and AAV9 transduced major systemic organs, particularly the liver with efciency similar to that seen following an intravenous injection. In contrast, AAV2 remained in the localized area of injection, likely due to propensity for binding heparan sulfate in subcutaneous tissue. AAV2i8, which does not bind heparan sulfate, demonstrated selective and systemic transduction in muscle tissue, but not the liver. Interestingly, AAV2i8, AAV8 and AAV9, but not AAV2 vectors also transduced the draining popliteal lymph node (PLN) following footpad injection. Vector genome copies in the circulation were identical regardless of route of injection suggesting that AAV vectors transit rapidly from peripheral lymphatics to circulating blood. Our data suggests that certain AAV strains can travel through the lymphatic system, which might constitute a signicant mechanism for systemic dissemination.
368. Optimizing Liver Transduction of the Low Seroprevalent AAV rh32.33
Luk H. Vandenberghe, Ru Xiao, James M. Wilson. Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Liver serves as a target of gene transfer in the treatment of a variety of inherited and acquired diseases. Vector administered into the circulation can access hepatocytes through its fenestrated capillaries. Vectors based on adeno-associated viruses (AAVs) have yielded promising results in pre-clinical and clinical models of liver-directed gene therapy. However, we have shown that pre-existing neutralizing antibodies (NAB) are prevalent to most AAV types and that even trace levels can inhibit hepatocyte transduction in vivo. Moreover, the advantage of novel AAVs with increased hepatotropism to require lower therapeutic doses will likely not hold in the context of NAB as lower doses will be more readily neutralized. One solution may be a capsid variant we isolated from macaques called AAVrh32.33 which is rarely neutralized by human serum. This novel capsid is structurally distinct from most of the others we isolated and can efciently package vectors that are not inhibited by humoral responses to human AAVs although hepatocyte transduction is 5 to 10–fold lower than that achieved with AAV8 in naïve animals. We therefore evaluated AAVrh32.33 for liver gene therapy by modulating its expression prole through both genome and capsid modication. First, we packaged self complementary (sc) genomes encoding GFP or human a1-antitrypsin into rh32.33. Liver gene transfer efciency was evaluated in C57Bl/6 mice as well as C57Bl/6 RAG knockout mice following tail vein administration of AAV doses ranging up to 1012 GC. These studies demonstrated transduction of over 50% of hepatocytes without transaminase elevations following high dose scAAV rh32.33 vectors; transduction efciency of rh32.33 at this dose was equivalent to that of AAV8 at a 10-fold lower dose. Modications were introduced into the AAVrh32.33 capsid to preserve the desired serological prole (i.e., low neutralization by human sera), yet increase its hepatotropism and gene transfer efciency. In a rst strategy, individual surface exposed tyrosines were substituted for phenylalanine residues. These single amino-acid substitutions were not anticipate to substantially alter serological reactivity but increase gene transfer efciency based on published results with AAV2 and AAV8 in a similar strategy. Seven tyrosine variants expressing luciferase were evaluated for liver directed in C57Bl/6 mice but none were found to be more efcient than the wild type vector. In another approach, the N-terminal portion of the viral capsid (VP12u) that contains a phospholipase domain important for infectivity was fused with heterologous VP3 open reading frames. VP12u swapping among AAV serotypes can affect gene transfer kinetics and overall efciency. Here we, introduced AAV8 VP12u into rh32.33 in a reciprocal manner and demonstrate in C57Bl6 mice transduction to be increased over Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy