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375. AAV2 Transduction Is Regulated by FAK and c-Src
BIOLOGY OF AAV AND VECTOR DEVELOPMENT 374. Epidermal Growth Factor Receptor Is a Receptor for Adeno-Associated Virus Serotype 6
John A. Chiorini,1 Melodie L. Weller,1 Panomwat Amornphimoltham,2 Michael Schmidt,1 J. Silvio Gutkind.2 1 Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD; 2Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD.
Fig. 1: Transduction efficiency of WT and Y-F mutants of AAV6 serotype vectors in primary human CD34+ cells at 5,000 vgs/cell. As is evident, the transduction efficiency of two of the tyrosine-mutant vectors [705F>Y731F] was significantly higher compared with the WT AAV6 vectors. Further studies with the optimal single and/ or multiple tyrosine-mutant scAAV6 vectors in a Hu/NOD-SCID xenograft mouse model in vivo are warranted to demonstrate the feasibility of the use of these novel vectors for achieving highefficiency transduction of HSCs as well as their potential use in gene therapy applications in hematological disorders.
373. Inhibition of Adeno-Associated Virus by the Host Factors APOBEC3A and APOBEC3B
Iñigo Narvaiza,1 Daniel C. Linfesty,1 Benjamin N. Greener,1 Yoshiyuki Hakata,2 Caroline E. Lilley,1 Nathaniel R. Landau,2 Matthew D. Weitzman.1 1 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA; 2Department of Microbiology, New York University School of Medicine, New York, NY. Adeno-Associated Virus (AAV) is a valuable vector for gene delivery. We have identified two members of the APOBEC3 family of proteins, APOBEC3A (A3A) and APOBEC3B (A3B), as host factors that restrict AAV replication. APOBEC3 proteins comprise a family of cytidine deaminases with inhibitory properties against viruses and retro-transposons. We recently demonstrated that APOBEC3A (A3A) is an active cytidine deaminase on single stranded DNA (ssDNA). Moreover, we showed that A3A, but not A3G, is a potent inhibitor of adeno-associated virus (AAV) and human LINE-1. Here we present results from experiments aimed at understanding APOBEC3 specificity and the mechanism responsible for AAV inhibition. AAV is a ssDNA virus that replicates exclusively in the nucleus. While A3A localizes in both nucleus and cytoplasm, A3B is restricted to the nucleus and A3G is cytoplasmic. Our results show that A3B also possesses activity against AAV, and that the C-terminal domain of A3B is sufficient for AAV inhibition. In contrast, we demonstrate that nuclear localization of A3G does not confer antiviral activity against AAV. Regarding the antiviral mechanism of A3A against AAV, we have generated A3A mutants and A3A/A3G chimeras, which suggest cytidine deaminase activity is not required for AAV inhibition. A3A mutants lacking in vitro deaminase activity are able to inhibit both recombinant AAV production and AAV replication. In addition, we have identified specific residues of A3A and A3B required for AAV inhibition, and shown that when these are introduced into A3G they confer antiviral activity against AAV to the C-terminus of A3G. In summary, we have found evidence for deaminase independent antiAAV activity, and have identified specific regions of A3A required for AAV inhibition. Furthermore, we believe that APOBEC3 proteins may have a potential impact in both AAV production and AAV-mediated gene delivery. S146
A critical step for the success of gene therapy is the efficient transfer of genes in a cell-type and tissue specific manner. Empirical studies have determined that AAV6 can efficiently transduce muscle, brain, lung and select tumors. Based on these findings, AAV6 has been proposed as a vector for phase 1 clinical trials for the treatment of cystic fibrosis and muscular dystrophy. However little is known about the cellular requirements for transduction with this vector, limiting further enhancement of its transduction activity and use in other applications. To better understand the overall mechanism of AAV transduction and to enhance its utility as a vector for gene transfer, we used a bioinformatics-based approach to identify genes that correlated with AAV6 transduction. Comparative gene analysis combined with pathway visualization software identified a statistically significant correlation between epidermal growth factor receptor (EGFR) expression and AAV6 transduction. Subsequent experiments confirmed this correlation and suggested that EGFR is necessary for vector internalization and likely functions as a receptor for this virus. To exploit this new understanding of AAV6 tropism, xenograft head and neck squamous cell carcinomas (HNSCC) presenting with aberrant EGFR expression were transduced with AAV6-CMV-luciferase through direct injection. Only tumors with elevated expression and membrane localization of EGFR were efficiently transduced by AAV6.
375. AAV2 Transduction Is Regulated by FAK and c-Src Paul M. Kaminsky,1 Ann C. Raddant,1 John F. Engelhardt.1 Anatomy and Cell Biology, University of Iowa, Iowa City, IA.
1
Previous studies have demonstrated that αVβ5 and α5β1 integrins are important co-receptors for AAV2 infection and endocytosis. However, the signaling events that regulate integrin-dependent endocytosis, following AAV2/integrin docking, remain unclear. A large body of literature links integrin activation to the recruitment and activation of numerous intracellular signaling proteins, such as focal adhesion kinase (FAK) and c-Src. These integrin effectors are known to bind and regulate Rac1 and phosphoinositide 3-kinase (PI3K), both of which have been shown to regulate rAAV2 endocytosis and vesicular trafficking. FAK is a non-receptor tyrosine kinase known to associate with the cytosolic domain of active β-integrins and induce intermolecular autophosphorylation at Y397. In the present, we sought to define the mechanisms that link integrin activation with downstream signaling pathways and hypothesized that FAK and c-Src play critical roles in this process. In support of our hypothesis, rAAV2 transduction of FAK KO fibroblasts was significantly lower than control FAK WT fibroblasts. FAK deficient fibroblasts also demonstrated reduced uptake of AAV2, suggesting that FAK influences viral endocytosis. Experiments in c-Src-deficient mouse embryonic fibroblasts (SYF cells: c-Src-KO, Yes-KO, and Fyn-KO) and c-Src complemented mouse embryonic fibroblasts (SYF+cSrc cells: c-Src overexpression, Yes-KO, and Fyn-KO) yielded similar trends, although the difference in transduction was significantly greater (∼4,000 fold) than that seen in FAK-deficient cells (4-8 fold). Interestingly, the presence of FAK and c-Src were required for AAV1 but not AAV5 transduction, suggesting some level of conservation between serotypes. Previous integrin studies support a model in which PKC functions in integrin activation and thus, FAK and c-Src recruitment. Consistent with Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
NEUROLOGIC & OPHTHALMIC I PKC involvement in FAK-mediated c-Src activation, PMA treatment of cells (reversible PKC agonist) prior to rAAV2 infection led to a significant induction in rAAV2 transduction in FAK WT, but not FAK KO cells. Similarly, PKC activated rAAV2 transduction in SFY+cSrc, but not SYF, cells. These findings support the involvement of PKC in FAK/c-Src dependent transduction and suggest that PKC lies upstream of FAK and c-Src. This observation is consistent with previous integrin studies that suggest PKC activity is necessary for efficient tyrosine phosphorylation of FAK. We hypothesize that PKC activity facilitates integrin activation, thereby enhancing signal transduction at the plasma membrane and viral endocytosis. These experiments have begun to outline a basic endocytic model linking integrin activation to the initiation of intracellular signaling cascades via PKC and FAK/c-Src that are required for rAAV2 infection. This information can ultimately be used identify signaling events which may augment rAAV transduction for gene therapy applications.
376. Characterization of DNA Integrity for Recombinant AAV Vector with Oversize Genome Biao Dong,1 Hui Lu,1 Weidong Xiao.1 1 Children’s Hospital of Philadelphia, Philadelphia, PA.
The application of recombinant associated virus (rAAV) in gene therapy is limited by its packaging capacity at 4.7 kb. Recent studies suggested that rAAV could be able to direct persistent transgene expression beyond 105% of normal vector packaging limits. However, it remains unclear on the mechanism leading to transgene expression from large rAAV vector. The two main hypotheses are that rAAV can package extra nucleotides into its capsid, or the partially packaged rAAV sequences may complement each other to restore full expression cassette. To investigate this issue, we constructed a serial of rAAV vectors with a viral genome 2.8, 3.9, 5.4, 6.4 and 7.2 Kb, respectively. In order to evaluate the efficacy of functional expression cassette formation and monitor the deletion in the packaged rAAV sequence, a plasmid replication origin and an ampicillin resistant marker were also included in the vector sequence. By Southern dot blot analysis, we were able to confirm that rAAVs could be produced using these vector plasmids. However, the vector yields decreased approximately 10-100 fold for larger vectors (5.4, 6.4 and 7.2 Kb) as compared with those smaller ones (2.8 and 3.9 Kb). Alkaline Southern blot hybridization suggested that the packaged genomes for large vectors were truncated, while those for small ones were intact. In the cells transduced by the above vectors, circularized rAAV monomer could be rescued 24 hr after infection. In mice receiving the above vectors, circularized rAAV genomes were also recovered at day 21 post vector administration. Restriction enzyme mapping and DNA sequencing confirmed that most of the packaged virus genomes were less than 4.7 Kb regardless of the initial vector size. It is very common to observe vectors with deletion at least one AAV ITR. There were no full size rAAV genomes could be recovered by this way for vector genomes larger than 5.4 Kb. Our studies suggested that the frequency for formation of full size vector is very low when the vector size is over the packaging limit.
377. Leveraging Capsid Design and Pharmacological Modulation To Enhance Trafficking Dynamics of AAV
Jarrod S. Johnson,1 David J. Dismuke,1 R. Jude Samulski.1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill.
1
Adeno-associated virus (AAV) serotypes are being tailored for numerous applications, yet our knowledge of how capsid components and environmental parameters influence infection is still in its infancy. At the cellular level, the capsid must trigger and survive a cascade of events that include internalization into an endocytic compartment, Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
endosomal processing, endosomal escape, nuclear targeting, and uncoating. However, the majority of capsids do not successfully deliver their transgenes to the nucleus. Subcellular trafficking of the most thoroughly characterized serotype, AAV2, is reported to be highly inefficient, due to either capsid deficiencies, cellular defense mechanisms, or both. Thus, we hypothesized transduction could be improved by manipulating capsid residues that direct trafficking in concert with leveraging physical and pharmacological stressors to influence cellular processes that impact infection. A putative nuclear localization signal and a phospholipase domain are harbored in the N-terminus of capsid protein VP1. These regions are normally contained inside the capsid, but are thought to translocate to the capsid exterior during subcellular trafficking to aid in endosomal escape and nuclear targeting. To more clearly understand how these capsid components and cellular pathways control trafficking of AAV2, we have tested a battery of mutant virions in vitro and in vivo while exploring the effects of pharmacologically modulating cellular pathways involved in protein sorting, degradation, and metabolism. Using immunofluorescence detection of intact capsids and biochemical fractionation assays, we tracked AAV2 and mutant virions to different subcellular destinations. We were able to clearly visualize their localization by rendering infected cells in 3-dimensions with volume imaging software. In accord with their trafficking patterns, mutant virions show differential transduction responses to drug treatments, exhibiting negligible, partial, or in some cases substantial transduction enhancement. Additionally, we have identified new residues of importance in VP1 and uncovered that AAV2 is subject to a specific cellular process that restricts virion access to the nucleus. We report that by targeting this pathway with pharmacological inhibitors or with siRNA-mediated knockdown of pathway components, transduction can be significantly improved. Moreover, we demonstrate that exploitation of certain physical or pharmacological stressors can have pronounced effects on AAV transduction in vivo, through visualizing bioluminescent gene delivery in mice. Ultimately, with these studies we have gained insight into how capsid residues direct access to the nucleus, and have explored how manipulating cellular parameters impacts infection in a complex environment.
Neurologic & Ophthalmic I 378. Mechanisms of Microglia Reconstitution after Hematopoietic Stem Cell Transplantation in the Brain of Leukodystrophic Mice
Alessia Capotondo,1,2 Letterio S. Politi,3 Angelo Quattrini,4 Giuseppe Scotti,3 Luigi Naldini,1,2 Alessandra Biffi.1 1 HSR-TIGET, San Raffaele Scientific Institute, Milan, Italy; 2VitaSalute University, Milan, Italy; 3Neuroradiology Unit, San Raffaele Scientific Institute, Milan, Italy; 4Neurology Unit, San Raffaele Scientific Institute, Milan, Italy. Metachromatic (MLD) and Globoid Leukodystrophy (GLD) are two Lysosomal Storage Disorders (LSD) due to the inherited deficiency of arylsulfatase A and Galactocerebrosidase deficiency, leading to the accumulation of un-degraded substrates within the nervous tissue. We previously showed that transplantation of genecorrected Hematopoietic Stem and Progenitor Cells (HSPC) in presymptomatic and symptomatic myeloablated MLD mice prevents and corrects neurologic disease manifestations. The efficacy of this treatment relies on microglia replacement by the progeny of the transplanted cells, which becomes an effective source of the functional enzyme in the affected nervous system, restores the normal microglia scavenger activity and reduces neuroinflammation. However, the actual contribution of donor cells to microgliosis in affected brains, the origin of Central Nervous System (CNS) migrating cells and the importance of acute damage to the blood brain barrier in eliciting S147
John A. Chiorini,1 Melodie L. Weller,1 Panomwat Amornphimoltham,2 Michael Schmidt,1 J. Silvio Gutkind.2 1 Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD; 2Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD.
Fig. 1: Transduction efficiency of WT and Y-F mutants of AAV6 serotype vectors in primary human CD34+ cells at 5,000 vgs/cell. As is evident, the transduction efficiency of two of the tyrosine-mutant vectors [705F>Y731F] was significantly higher compared with the WT AAV6 vectors. Further studies with the optimal single and/ or multiple tyrosine-mutant scAAV6 vectors in a Hu/NOD-SCID xenograft mouse model in vivo are warranted to demonstrate the feasibility of the use of these novel vectors for achieving highefficiency transduction of HSCs as well as their potential use in gene therapy applications in hematological disorders.
373. Inhibition of Adeno-Associated Virus by the Host Factors APOBEC3A and APOBEC3B
Iñigo Narvaiza,1 Daniel C. Linfesty,1 Benjamin N. Greener,1 Yoshiyuki Hakata,2 Caroline E. Lilley,1 Nathaniel R. Landau,2 Matthew D. Weitzman.1 1 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA; 2Department of Microbiology, New York University School of Medicine, New York, NY. Adeno-Associated Virus (AAV) is a valuable vector for gene delivery. We have identified two members of the APOBEC3 family of proteins, APOBEC3A (A3A) and APOBEC3B (A3B), as host factors that restrict AAV replication. APOBEC3 proteins comprise a family of cytidine deaminases with inhibitory properties against viruses and retro-transposons. We recently demonstrated that APOBEC3A (A3A) is an active cytidine deaminase on single stranded DNA (ssDNA). Moreover, we showed that A3A, but not A3G, is a potent inhibitor of adeno-associated virus (AAV) and human LINE-1. Here we present results from experiments aimed at understanding APOBEC3 specificity and the mechanism responsible for AAV inhibition. AAV is a ssDNA virus that replicates exclusively in the nucleus. While A3A localizes in both nucleus and cytoplasm, A3B is restricted to the nucleus and A3G is cytoplasmic. Our results show that A3B also possesses activity against AAV, and that the C-terminal domain of A3B is sufficient for AAV inhibition. In contrast, we demonstrate that nuclear localization of A3G does not confer antiviral activity against AAV. Regarding the antiviral mechanism of A3A against AAV, we have generated A3A mutants and A3A/A3G chimeras, which suggest cytidine deaminase activity is not required for AAV inhibition. A3A mutants lacking in vitro deaminase activity are able to inhibit both recombinant AAV production and AAV replication. In addition, we have identified specific residues of A3A and A3B required for AAV inhibition, and shown that when these are introduced into A3G they confer antiviral activity against AAV to the C-terminus of A3G. In summary, we have found evidence for deaminase independent antiAAV activity, and have identified specific regions of A3A required for AAV inhibition. Furthermore, we believe that APOBEC3 proteins may have a potential impact in both AAV production and AAV-mediated gene delivery. S146
A critical step for the success of gene therapy is the efficient transfer of genes in a cell-type and tissue specific manner. Empirical studies have determined that AAV6 can efficiently transduce muscle, brain, lung and select tumors. Based on these findings, AAV6 has been proposed as a vector for phase 1 clinical trials for the treatment of cystic fibrosis and muscular dystrophy. However little is known about the cellular requirements for transduction with this vector, limiting further enhancement of its transduction activity and use in other applications. To better understand the overall mechanism of AAV transduction and to enhance its utility as a vector for gene transfer, we used a bioinformatics-based approach to identify genes that correlated with AAV6 transduction. Comparative gene analysis combined with pathway visualization software identified a statistically significant correlation between epidermal growth factor receptor (EGFR) expression and AAV6 transduction. Subsequent experiments confirmed this correlation and suggested that EGFR is necessary for vector internalization and likely functions as a receptor for this virus. To exploit this new understanding of AAV6 tropism, xenograft head and neck squamous cell carcinomas (HNSCC) presenting with aberrant EGFR expression were transduced with AAV6-CMV-luciferase through direct injection. Only tumors with elevated expression and membrane localization of EGFR were efficiently transduced by AAV6.
375. AAV2 Transduction Is Regulated by FAK and c-Src Paul M. Kaminsky,1 Ann C. Raddant,1 John F. Engelhardt.1 Anatomy and Cell Biology, University of Iowa, Iowa City, IA.
1
Previous studies have demonstrated that αVβ5 and α5β1 integrins are important co-receptors for AAV2 infection and endocytosis. However, the signaling events that regulate integrin-dependent endocytosis, following AAV2/integrin docking, remain unclear. A large body of literature links integrin activation to the recruitment and activation of numerous intracellular signaling proteins, such as focal adhesion kinase (FAK) and c-Src. These integrin effectors are known to bind and regulate Rac1 and phosphoinositide 3-kinase (PI3K), both of which have been shown to regulate rAAV2 endocytosis and vesicular trafficking. FAK is a non-receptor tyrosine kinase known to associate with the cytosolic domain of active β-integrins and induce intermolecular autophosphorylation at Y397. In the present, we sought to define the mechanisms that link integrin activation with downstream signaling pathways and hypothesized that FAK and c-Src play critical roles in this process. In support of our hypothesis, rAAV2 transduction of FAK KO fibroblasts was significantly lower than control FAK WT fibroblasts. FAK deficient fibroblasts also demonstrated reduced uptake of AAV2, suggesting that FAK influences viral endocytosis. Experiments in c-Src-deficient mouse embryonic fibroblasts (SYF cells: c-Src-KO, Yes-KO, and Fyn-KO) and c-Src complemented mouse embryonic fibroblasts (SYF+cSrc cells: c-Src overexpression, Yes-KO, and Fyn-KO) yielded similar trends, although the difference in transduction was significantly greater (∼4,000 fold) than that seen in FAK-deficient cells (4-8 fold). Interestingly, the presence of FAK and c-Src were required for AAV1 but not AAV5 transduction, suggesting some level of conservation between serotypes. Previous integrin studies support a model in which PKC functions in integrin activation and thus, FAK and c-Src recruitment. Consistent with Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
NEUROLOGIC & OPHTHALMIC I PKC involvement in FAK-mediated c-Src activation, PMA treatment of cells (reversible PKC agonist) prior to rAAV2 infection led to a significant induction in rAAV2 transduction in FAK WT, but not FAK KO cells. Similarly, PKC activated rAAV2 transduction in SFY+cSrc, but not SYF, cells. These findings support the involvement of PKC in FAK/c-Src dependent transduction and suggest that PKC lies upstream of FAK and c-Src. This observation is consistent with previous integrin studies that suggest PKC activity is necessary for efficient tyrosine phosphorylation of FAK. We hypothesize that PKC activity facilitates integrin activation, thereby enhancing signal transduction at the plasma membrane and viral endocytosis. These experiments have begun to outline a basic endocytic model linking integrin activation to the initiation of intracellular signaling cascades via PKC and FAK/c-Src that are required for rAAV2 infection. This information can ultimately be used identify signaling events which may augment rAAV transduction for gene therapy applications.
376. Characterization of DNA Integrity for Recombinant AAV Vector with Oversize Genome Biao Dong,1 Hui Lu,1 Weidong Xiao.1 1 Children’s Hospital of Philadelphia, Philadelphia, PA.
The application of recombinant associated virus (rAAV) in gene therapy is limited by its packaging capacity at 4.7 kb. Recent studies suggested that rAAV could be able to direct persistent transgene expression beyond 105% of normal vector packaging limits. However, it remains unclear on the mechanism leading to transgene expression from large rAAV vector. The two main hypotheses are that rAAV can package extra nucleotides into its capsid, or the partially packaged rAAV sequences may complement each other to restore full expression cassette. To investigate this issue, we constructed a serial of rAAV vectors with a viral genome 2.8, 3.9, 5.4, 6.4 and 7.2 Kb, respectively. In order to evaluate the efficacy of functional expression cassette formation and monitor the deletion in the packaged rAAV sequence, a plasmid replication origin and an ampicillin resistant marker were also included in the vector sequence. By Southern dot blot analysis, we were able to confirm that rAAVs could be produced using these vector plasmids. However, the vector yields decreased approximately 10-100 fold for larger vectors (5.4, 6.4 and 7.2 Kb) as compared with those smaller ones (2.8 and 3.9 Kb). Alkaline Southern blot hybridization suggested that the packaged genomes for large vectors were truncated, while those for small ones were intact. In the cells transduced by the above vectors, circularized rAAV monomer could be rescued 24 hr after infection. In mice receiving the above vectors, circularized rAAV genomes were also recovered at day 21 post vector administration. Restriction enzyme mapping and DNA sequencing confirmed that most of the packaged virus genomes were less than 4.7 Kb regardless of the initial vector size. It is very common to observe vectors with deletion at least one AAV ITR. There were no full size rAAV genomes could be recovered by this way for vector genomes larger than 5.4 Kb. Our studies suggested that the frequency for formation of full size vector is very low when the vector size is over the packaging limit.
377. Leveraging Capsid Design and Pharmacological Modulation To Enhance Trafficking Dynamics of AAV
Jarrod S. Johnson,1 David J. Dismuke,1 R. Jude Samulski.1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill.
1
Adeno-associated virus (AAV) serotypes are being tailored for numerous applications, yet our knowledge of how capsid components and environmental parameters influence infection is still in its infancy. At the cellular level, the capsid must trigger and survive a cascade of events that include internalization into an endocytic compartment, Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
endosomal processing, endosomal escape, nuclear targeting, and uncoating. However, the majority of capsids do not successfully deliver their transgenes to the nucleus. Subcellular trafficking of the most thoroughly characterized serotype, AAV2, is reported to be highly inefficient, due to either capsid deficiencies, cellular defense mechanisms, or both. Thus, we hypothesized transduction could be improved by manipulating capsid residues that direct trafficking in concert with leveraging physical and pharmacological stressors to influence cellular processes that impact infection. A putative nuclear localization signal and a phospholipase domain are harbored in the N-terminus of capsid protein VP1. These regions are normally contained inside the capsid, but are thought to translocate to the capsid exterior during subcellular trafficking to aid in endosomal escape and nuclear targeting. To more clearly understand how these capsid components and cellular pathways control trafficking of AAV2, we have tested a battery of mutant virions in vitro and in vivo while exploring the effects of pharmacologically modulating cellular pathways involved in protein sorting, degradation, and metabolism. Using immunofluorescence detection of intact capsids and biochemical fractionation assays, we tracked AAV2 and mutant virions to different subcellular destinations. We were able to clearly visualize their localization by rendering infected cells in 3-dimensions with volume imaging software. In accord with their trafficking patterns, mutant virions show differential transduction responses to drug treatments, exhibiting negligible, partial, or in some cases substantial transduction enhancement. Additionally, we have identified new residues of importance in VP1 and uncovered that AAV2 is subject to a specific cellular process that restricts virion access to the nucleus. We report that by targeting this pathway with pharmacological inhibitors or with siRNA-mediated knockdown of pathway components, transduction can be significantly improved. Moreover, we demonstrate that exploitation of certain physical or pharmacological stressors can have pronounced effects on AAV transduction in vivo, through visualizing bioluminescent gene delivery in mice. Ultimately, with these studies we have gained insight into how capsid residues direct access to the nucleus, and have explored how manipulating cellular parameters impacts infection in a complex environment.
Neurologic & Ophthalmic I 378. Mechanisms of Microglia Reconstitution after Hematopoietic Stem Cell Transplantation in the Brain of Leukodystrophic Mice
Alessia Capotondo,1,2 Letterio S. Politi,3 Angelo Quattrini,4 Giuseppe Scotti,3 Luigi Naldini,1,2 Alessandra Biffi.1 1 HSR-TIGET, San Raffaele Scientific Institute, Milan, Italy; 2VitaSalute University, Milan, Italy; 3Neuroradiology Unit, San Raffaele Scientific Institute, Milan, Italy; 4Neurology Unit, San Raffaele Scientific Institute, Milan, Italy. Metachromatic (MLD) and Globoid Leukodystrophy (GLD) are two Lysosomal Storage Disorders (LSD) due to the inherited deficiency of arylsulfatase A and Galactocerebrosidase deficiency, leading to the accumulation of un-degraded substrates within the nervous tissue. We previously showed that transplantation of genecorrected Hematopoietic Stem and Progenitor Cells (HSPC) in presymptomatic and symptomatic myeloablated MLD mice prevents and corrects neurologic disease manifestations. The efficacy of this treatment relies on microglia replacement by the progeny of the transplanted cells, which becomes an effective source of the functional enzyme in the affected nervous system, restores the normal microglia scavenger activity and reduces neuroinflammation. However, the actual contribution of donor cells to microgliosis in affected brains, the origin of Central Nervous System (CNS) migrating cells and the importance of acute damage to the blood brain barrier in eliciting S147