- Email: [email protected]
13. Peri-Nuclear Accumulation of Adeno-Associated Virus: A Novel Barrier Limiting AAV Transduction?
AAV VIRUS & VECTOR BIOLOGY mutation) were divided into 3 libraries, each of which also contained 15 each of control AAV clones derived from wild type AAV9 and a heparin-binding mutant AAV2R585E. The results showed that amino acids important for intact viral particle formation reside in different capsid aa positions but cluster at inter-protein interfaces and some are found on the capsid surface around the five-fold symmetry axis. An in vitro infection and virus binding assay using CHO Lec2 cells that express terminal galactose, a primary receptor for AAV9, revealed that multiple surface-exposed amino acids from different capsid regions are responsible for virus binding and cluster around the three-fold symmetry axis. Interestingly, we also identified the amino acids that do not play a role in binding but are critical for post-binding processing of the viral capsids. An in vivo transduction study in mice identified several alanine mutants that substantially decrease transduction in the liver but not in other tissues including the heart and brain. Interestingly, we found that an amino acid stretch (aa 562-567) containing a triacidic cluster (EEE) is important for the transduction in the brain, heart and liver, but not so in the kidney and spleen. In parallel to this study, we are also performing a comprehensive hexapeptide scanning using AAV2R585E and have created 125 mutants. This approach demonstrated that the AAV9’s three-fold capsid protrusion contains a dominant neutralizing epitope. Thus, our new approach holds enormous potential for studying AAV capsid biology and provides intellectual basis for developing novel AAV vectors with the most desirable biological properties.
11. The 3D Structure of Adeno-Associated Virus Serotype 9 and Its Unique Properties
Michael A. DiMattia,1 Hyun-Joo Nam,1 Kim Van Vliet,1 Antonette Bennett,1 Brittney L. Gurda,1 Robert McKenna,1 Norman H. Olson,2 Robert S. Sinkovits,2 Mark Potter,1 George Aslanidi,1 Sergei Zolotukhin,1 Nicholas Muzyczka,1 Timothy S. Baker,2 Mavis Agbandje-McKenna.1 1 University of Florida, Gainesville, FL; 2University of CaliforniaSan Diego, La Jolla. The three-dimensional structure of Adeno-associated virus 9 (AAV9), an AAV serotype with enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood brain barrier compared to other AAV serotypes, has been determined by cryoelectron microscopy and three-dimensional image reconstruction and X-ray crystallography to 9.7 and 2.8-Å resolution, respectively. The AAV9 capsid structure is very similar to those of other AAVs, e.g. AAV2 and AAV8, conserving the surface topology described for these viruses, including depressions at each icosahedral twofold symmetry axis and surrounding each fivefold axis, three separate protrusions surrounding each threefold axis, and a channel at each fivefold axis. However, the structure differs in three of the nine capsid surface variable regions (VRs, previously described when AAV2 and AAV4 were compared), VRI, VRII, and VRIV, compared to AAV2 and AAV8. VRI differences modify the raised region of the capsid surface between the twofold and fivefold depressions, VRII causes conformational differences at the fivefold channel, and the VRIV difference produces smaller threefold protrusions in AAV9 that are less “pointed” compared to AAV2 and AAV8. Significantly, residues in several of the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
12. Microfluidic Live-Neuron Tracking of AAV9 Axonal Transport within the Endosomal System Michael J. Castle,1,2 Erika L. F. Holzbaur,1 John H. Wolfe.1,2 1 University of Pennsylvania, Philadelphia, PA; 2Children’s Hospital of Philadelphia, PA.
While many Adeno-Associated Virus (AAV) vector serotypes efficiently transduce nervous tissue, gene transfer is rarely observed beyond the injection site. It is thus difficult to achieve widespread gene transfer to the brain using AAV vectors, impeding the treatment of neurological disorders that require global correction. However, some AAV serotypes have been shown to undergo axonal transport to distal brain regions following intraparenchymal injection, effectively distributing gene transfer more widely in the brain. Unfortunately, knowledge of the cellular mechanisms underlying the axonal transport of AAV is limited, hindering the development of novel AAV vectors and treatment strategies that are designed to utilize this transport to enhance distribution of gene transfer. This study aims to investigate the axonal trafficking of AAV9, a serotype that is strongly transported in both the anterograde and retrograde directions in vivo. A microfluidic system was developed that allows for specific application of AAV to either the cell bodies or axon endings of cultured E18 rat cortical neurons, facilitating the direct examination of anterograde and retrograde AAV transport. Dye-conjugated AAV9 particles were developed for live-cell imaging in this system. Within 1h of application to the axon ending, AAV9 is trafficked into a non-motile Early Endosome compartment, a low-velocity but retrograde-directed Lysosome compartment, and a Late Endosome compartment that moves retrograde at speeds typical of dynein-mediated transport. Transfection of p150-CC1, a fragment of the p150Glued dynactin subunit which acts as a dominant negative dynein inhibitor, significantly inhibits retrograde movement of AAV9, confirming that dynein mediates this transport. By 4h after either axon or cell infection, colocalization with the Lysosome, the Recycling Endosome, and the Synaptic Vesicle compartments is increased. After entry at the cell body, AAV9 is transported anterograde at high velocities and in a highly-directed manner. This transport is significantly inhibited by transfection with Kif3A-HL, a dominant negative inhibitor of the Kinesin II complex, but not Kif5C-HL, a dominant negative inhibitor of the Kinesin I complex, indicating that anterograde AAV9 transport is mediated by Kinesin II. Surprisingly, anterograde-directed AAV9 does not colocalize with post-Golgi vesicles, suggesting that synaptic vesicle-associated AAV9 is not the anterograde-directed population. Pre-treatment of cultures with Neuraminidase, which cleaves membrane-bound sialic acid to galactose, thereby enhancing AAV9 transduction in some systems, increased the number of retrogradedirected AAV9 particles by ∼50% and anterograde-directed particles by ∼100%. Thus, Neuraminidase treatment may be an effective way to enhance the spread of AAV9 gene transfer in vivo. Work is ongoing to further classify the intracellular compartments involved in the axonal transport of AAV9, as well as to compare the transport and trafficking of other AAV serotypes in this system.
13. Peri-Nuclear Accumulation of AdenoAssociated Virus: A Novel Barrier Limiting AAV Transduction?
Ping-Jie Xiao,1,2 Chengwen Li,1 Lluis Samaranch,4 Steve Gray,1 Adrian P. Kells,4 Krystof S. Bankiewicz,4 Richard Jude Samulski.1,3 1 Gene Therapy Center, University of North Carolina at Chapel Hill, NC; 2Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC; 3Department of Pharmacology, University of North Carolina at Chapel Hill, NC; 4 Department of Neurological Surgery, University of California San Francisco, San Francisco, CA. Adeno-associated virus (AAV) is a promising gene therapy vector since low toxicity, long-term gene expression, and diseases correction S5
AAV VIRUS & VECTOR BIOLOGY have been observed in animal models and human clinical trials. In order to transduce a cell in vitro, recombinant AAV (rAAV) has to start with receptor-mediated cell surface binding and internalization, followed by endosomal trafficking, escape, and eventually release the genetic material in the nucleus. However, little is known about its trafficking behavior in animal tissues. Using a sensitive singleparticle imaging method, we observed significant peri-nuclear accumulation of AAV2 in liver at 10 hours after intrahepatic viral injection. Using cultured human cell lines, we observed similar perinuclear accumulation of AAV2 particles 4-6 hours after infection. By screening a variety of pharmacological reagents, we found that this peri-nuclear localization of AAV2 is dependent on intact microtubule network, but not affected by Golgi integrity or actin filaments. Disruption of this peri-nuclear localization of AAV2 leads to 2-4 fold increase in the level of transgene expression. Our quantitative 3D microscopy demonstrated that more AAV2 particles were observed in the nucleus after such disruption, indicating peri-nuclear accumulation may be a rate-limiting step for AAV2 nuclear entry. Adenovirus (Ad) co-infection studies carried out in our lab suggested that Ad may facilitate the escape of AAV2 from the peri-nuclear localization as one of the mechanism to enhance AAV2 transduction. More recently, in mouse studies, we also observed enhanced transgene expression after administration of anti-microtubule drugs in multiple tissues/organs. And all the above results support the hypothesis that peri-nuclear accumulation of AAV is a novel barrier for efficient AAV transduction.
after i.m. and i.n. delivery. The vector genome biodistribution study confirmed that AAV genome was restricted in the muscle and lung after i.m and i.n delivery of CLvD8, while AAV genome was detected in other organs after AAV9 delivery by the same routes. These data implied that the four amino acid residues mutated in CLVD8 capsid protein may play a role in crossing vascular barrier by AAV9. To identify the critical residue(s), four vectors containing the single mutation for each of these four amino acids were constructed and packaged for luciferase expressing vector genome. The vectors were evaluated in C57BL/6 mice after i.v., i.m. and i.n. administration. The data showed that two single mutants out of four shared similar transduction pattern with CLvD8. To further address our hypothesis, using newly established crystal structure of AAV9 VP3 as the model, we will compare the capsid structure of CLvD8 and the single mutated vectors with that of AAV9, which may lead to identifying the critical functional domains contributing to its crossing vascular barrier.
14. Critical Amino Acid Redisues Contribute to Crossing Vascular Barrier
Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain and retina. The reason for their exquisite tropism for these cells has long remained elusive. Work performed by different laboratories has shown that, in cultured cells, one of the major, rate-limiting determinants of AAV permissivity relates to the way in which cellular DNA Damage Response (DDR) proteins process viral genomes once these reach the nucleus. In particular, work from our and others’ laboratories has demonstrated that, once internalized into the nucleus of cycling cells, AAV genomes physically interact with members of the MRN (Mre11, Rad50, Nbs1) complex. In eukaryotic cells, MRN controls the DDR by sensing DNA damage and governing the activation of the ataxia-telangiectasia mutated (ATM) kinase; besides being a key component in the homology-directed repair, MRN also participates in the repair through classical and alternative NHEJ pathways. Multiple evidence indicates that the interaction between AAV genomes and cellular DDR proteins restricts AAV transduction in several cultured cell types. Precisely how this information, obtained mostly in cultured cells, relates to the exquisite permissivity to AAV transduction of post-mitotic cells in vivo still remains to be understood. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DDR and that this markedly correlates with increased permissivity to AAV transduction. In particular, expression of Mre11, Rad50, Nbs1, which bind the incoming AAV genomes, fades in cardiomyocytes at approximately two weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincides with increased AAV permissivity. MicroRNA-24, which is upregulated upon myoblast and cardiomyocyte differentiation, markedly induces AAV permissivity by downmodulating the MRN protein Nbs1. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction. To provide further, direct evidence that down-regulation of MRN complex leads to increased permissivity to AAV transduction in vivo, we knocked-down the MRN complex in juvenile liver by intraportal vein injection of cationic lipid formulations containing either the siRNAs against Mre11, Rad50 or Nbs1, or microRNA-24. Significantly higher transduction was observed in the livers that received the tested small RNAs compared to those transfected with a non-targeting siRNA. Taken together,
Li Zhong,1,2 Shaoyong Li,1 Mengxin Li,1 Jun Xie,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Jason Goetzmann,5 Terence Flotte,1,3 Guangping Gao.1,4 1 Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 2Hematology/Oncology, Depart. of Medicine, University of Massachusetts Medical School, Worcester; MA, 3Pediatrics, University of Massachusetts Medical School, Worcester; MA, 4Depart. of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester; MA, 5New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA. The AAV9 vector has the ability to cross the vascular barrier and efficiently transduce cardiac and skeletal muscle fibers. AAV9 vector can even overcome the blood brain barrier following i.v. injection, holding great potential to target CNS for treatment of a variety of neuromuscular and degenerative disorders. Recently, the terminal galactose in the cell surface β-galactose glycans has been identified as an AAV9 receptor. However, the correlations between the capsid structure and viral activity for traversing the vascular barrier by AAV9 vectors are unclear. In an attempt to elucidate this enigma, we isolated natural variants of AAV9 from chimpanzee tissues and studied their functions. The CLvD8, a variant isolated from chimpanzee liver RNA, containing four amino acid mutations in the VP3 protein, displays the inability to cross vascular barrier, which serves a useful model in our study. This vector was evaluated in C57BL/6 mice for nLacZ, luciferase and α1-anti-trypsin (AAT) gene transduction after intravenous (i.v.), intramuscular (i.m.), and intra-nasal (i.n.) administration. The study to compare AAT expression suggested that AAV9 significantly outperforms CLvD8. However, difference between these two vectors for the nLaZ and luciferase transduction in liver, muscle and lung is not as much as AAT expression. Especially, in the lung, both AAV9 and CLvD8 led to similar nLacZ and luciferase transduction after i.n. delivery and CLvD8 primarily target alveoli as does AAV9. In live image for luciferase expression, AAV9 led to strong transduction in other organs outside of the administration sites after i.m. and i.n. delivery up to 17 weeks. However, the CLvD8mediated luciferase expression was restricted in the local tissues S6
15. In Vivo Knock-Down of MRN Complex Members Improves Liver and Cardiac Transduction with AAV Vectors
Jasmina Lovric,1 Miguel Mano,1 Lorena Zentilin,1 Ana Eulalio,1 Serena Zacchigna,1 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italy.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
11. The 3D Structure of Adeno-Associated Virus Serotype 9 and Its Unique Properties
Michael A. DiMattia,1 Hyun-Joo Nam,1 Kim Van Vliet,1 Antonette Bennett,1 Brittney L. Gurda,1 Robert McKenna,1 Norman H. Olson,2 Robert S. Sinkovits,2 Mark Potter,1 George Aslanidi,1 Sergei Zolotukhin,1 Nicholas Muzyczka,1 Timothy S. Baker,2 Mavis Agbandje-McKenna.1 1 University of Florida, Gainesville, FL; 2University of CaliforniaSan Diego, La Jolla. The three-dimensional structure of Adeno-associated virus 9 (AAV9), an AAV serotype with enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood brain barrier compared to other AAV serotypes, has been determined by cryoelectron microscopy and three-dimensional image reconstruction and X-ray crystallography to 9.7 and 2.8-Å resolution, respectively. The AAV9 capsid structure is very similar to those of other AAVs, e.g. AAV2 and AAV8, conserving the surface topology described for these viruses, including depressions at each icosahedral twofold symmetry axis and surrounding each fivefold axis, three separate protrusions surrounding each threefold axis, and a channel at each fivefold axis. However, the structure differs in three of the nine capsid surface variable regions (VRs, previously described when AAV2 and AAV4 were compared), VRI, VRII, and VRIV, compared to AAV2 and AAV8. VRI differences modify the raised region of the capsid surface between the twofold and fivefold depressions, VRII causes conformational differences at the fivefold channel, and the VRIV difference produces smaller threefold protrusions in AAV9 that are less “pointed” compared to AAV2 and AAV8. Significantly, residues in several of the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
12. Microfluidic Live-Neuron Tracking of AAV9 Axonal Transport within the Endosomal System Michael J. Castle,1,2 Erika L. F. Holzbaur,1 John H. Wolfe.1,2 1 University of Pennsylvania, Philadelphia, PA; 2Children’s Hospital of Philadelphia, PA.
While many Adeno-Associated Virus (AAV) vector serotypes efficiently transduce nervous tissue, gene transfer is rarely observed beyond the injection site. It is thus difficult to achieve widespread gene transfer to the brain using AAV vectors, impeding the treatment of neurological disorders that require global correction. However, some AAV serotypes have been shown to undergo axonal transport to distal brain regions following intraparenchymal injection, effectively distributing gene transfer more widely in the brain. Unfortunately, knowledge of the cellular mechanisms underlying the axonal transport of AAV is limited, hindering the development of novel AAV vectors and treatment strategies that are designed to utilize this transport to enhance distribution of gene transfer. This study aims to investigate the axonal trafficking of AAV9, a serotype that is strongly transported in both the anterograde and retrograde directions in vivo. A microfluidic system was developed that allows for specific application of AAV to either the cell bodies or axon endings of cultured E18 rat cortical neurons, facilitating the direct examination of anterograde and retrograde AAV transport. Dye-conjugated AAV9 particles were developed for live-cell imaging in this system. Within 1h of application to the axon ending, AAV9 is trafficked into a non-motile Early Endosome compartment, a low-velocity but retrograde-directed Lysosome compartment, and a Late Endosome compartment that moves retrograde at speeds typical of dynein-mediated transport. Transfection of p150-CC1, a fragment of the p150Glued dynactin subunit which acts as a dominant negative dynein inhibitor, significantly inhibits retrograde movement of AAV9, confirming that dynein mediates this transport. By 4h after either axon or cell infection, colocalization with the Lysosome, the Recycling Endosome, and the Synaptic Vesicle compartments is increased. After entry at the cell body, AAV9 is transported anterograde at high velocities and in a highly-directed manner. This transport is significantly inhibited by transfection with Kif3A-HL, a dominant negative inhibitor of the Kinesin II complex, but not Kif5C-HL, a dominant negative inhibitor of the Kinesin I complex, indicating that anterograde AAV9 transport is mediated by Kinesin II. Surprisingly, anterograde-directed AAV9 does not colocalize with post-Golgi vesicles, suggesting that synaptic vesicle-associated AAV9 is not the anterograde-directed population. Pre-treatment of cultures with Neuraminidase, which cleaves membrane-bound sialic acid to galactose, thereby enhancing AAV9 transduction in some systems, increased the number of retrogradedirected AAV9 particles by ∼50% and anterograde-directed particles by ∼100%. Thus, Neuraminidase treatment may be an effective way to enhance the spread of AAV9 gene transfer in vivo. Work is ongoing to further classify the intracellular compartments involved in the axonal transport of AAV9, as well as to compare the transport and trafficking of other AAV serotypes in this system.
13. Peri-Nuclear Accumulation of AdenoAssociated Virus: A Novel Barrier Limiting AAV Transduction?
Ping-Jie Xiao,1,2 Chengwen Li,1 Lluis Samaranch,4 Steve Gray,1 Adrian P. Kells,4 Krystof S. Bankiewicz,4 Richard Jude Samulski.1,3 1 Gene Therapy Center, University of North Carolina at Chapel Hill, NC; 2Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC; 3Department of Pharmacology, University of North Carolina at Chapel Hill, NC; 4 Department of Neurological Surgery, University of California San Francisco, San Francisco, CA. Adeno-associated virus (AAV) is a promising gene therapy vector since low toxicity, long-term gene expression, and diseases correction S5
AAV VIRUS & VECTOR BIOLOGY have been observed in animal models and human clinical trials. In order to transduce a cell in vitro, recombinant AAV (rAAV) has to start with receptor-mediated cell surface binding and internalization, followed by endosomal trafficking, escape, and eventually release the genetic material in the nucleus. However, little is known about its trafficking behavior in animal tissues. Using a sensitive singleparticle imaging method, we observed significant peri-nuclear accumulation of AAV2 in liver at 10 hours after intrahepatic viral injection. Using cultured human cell lines, we observed similar perinuclear accumulation of AAV2 particles 4-6 hours after infection. By screening a variety of pharmacological reagents, we found that this peri-nuclear localization of AAV2 is dependent on intact microtubule network, but not affected by Golgi integrity or actin filaments. Disruption of this peri-nuclear localization of AAV2 leads to 2-4 fold increase in the level of transgene expression. Our quantitative 3D microscopy demonstrated that more AAV2 particles were observed in the nucleus after such disruption, indicating peri-nuclear accumulation may be a rate-limiting step for AAV2 nuclear entry. Adenovirus (Ad) co-infection studies carried out in our lab suggested that Ad may facilitate the escape of AAV2 from the peri-nuclear localization as one of the mechanism to enhance AAV2 transduction. More recently, in mouse studies, we also observed enhanced transgene expression after administration of anti-microtubule drugs in multiple tissues/organs. And all the above results support the hypothesis that peri-nuclear accumulation of AAV is a novel barrier for efficient AAV transduction.
after i.m. and i.n. delivery. The vector genome biodistribution study confirmed that AAV genome was restricted in the muscle and lung after i.m and i.n delivery of CLvD8, while AAV genome was detected in other organs after AAV9 delivery by the same routes. These data implied that the four amino acid residues mutated in CLVD8 capsid protein may play a role in crossing vascular barrier by AAV9. To identify the critical residue(s), four vectors containing the single mutation for each of these four amino acids were constructed and packaged for luciferase expressing vector genome. The vectors were evaluated in C57BL/6 mice after i.v., i.m. and i.n. administration. The data showed that two single mutants out of four shared similar transduction pattern with CLvD8. To further address our hypothesis, using newly established crystal structure of AAV9 VP3 as the model, we will compare the capsid structure of CLvD8 and the single mutated vectors with that of AAV9, which may lead to identifying the critical functional domains contributing to its crossing vascular barrier.
14. Critical Amino Acid Redisues Contribute to Crossing Vascular Barrier
Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain and retina. The reason for their exquisite tropism for these cells has long remained elusive. Work performed by different laboratories has shown that, in cultured cells, one of the major, rate-limiting determinants of AAV permissivity relates to the way in which cellular DNA Damage Response (DDR) proteins process viral genomes once these reach the nucleus. In particular, work from our and others’ laboratories has demonstrated that, once internalized into the nucleus of cycling cells, AAV genomes physically interact with members of the MRN (Mre11, Rad50, Nbs1) complex. In eukaryotic cells, MRN controls the DDR by sensing DNA damage and governing the activation of the ataxia-telangiectasia mutated (ATM) kinase; besides being a key component in the homology-directed repair, MRN also participates in the repair through classical and alternative NHEJ pathways. Multiple evidence indicates that the interaction between AAV genomes and cellular DDR proteins restricts AAV transduction in several cultured cell types. Precisely how this information, obtained mostly in cultured cells, relates to the exquisite permissivity to AAV transduction of post-mitotic cells in vivo still remains to be understood. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DDR and that this markedly correlates with increased permissivity to AAV transduction. In particular, expression of Mre11, Rad50, Nbs1, which bind the incoming AAV genomes, fades in cardiomyocytes at approximately two weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincides with increased AAV permissivity. MicroRNA-24, which is upregulated upon myoblast and cardiomyocyte differentiation, markedly induces AAV permissivity by downmodulating the MRN protein Nbs1. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction. To provide further, direct evidence that down-regulation of MRN complex leads to increased permissivity to AAV transduction in vivo, we knocked-down the MRN complex in juvenile liver by intraportal vein injection of cationic lipid formulations containing either the siRNAs against Mre11, Rad50 or Nbs1, or microRNA-24. Significantly higher transduction was observed in the livers that received the tested small RNAs compared to those transfected with a non-targeting siRNA. Taken together,
Li Zhong,1,2 Shaoyong Li,1 Mengxin Li,1 Jun Xie,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Jason Goetzmann,5 Terence Flotte,1,3 Guangping Gao.1,4 1 Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 2Hematology/Oncology, Depart. of Medicine, University of Massachusetts Medical School, Worcester; MA, 3Pediatrics, University of Massachusetts Medical School, Worcester; MA, 4Depart. of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester; MA, 5New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA. The AAV9 vector has the ability to cross the vascular barrier and efficiently transduce cardiac and skeletal muscle fibers. AAV9 vector can even overcome the blood brain barrier following i.v. injection, holding great potential to target CNS for treatment of a variety of neuromuscular and degenerative disorders. Recently, the terminal galactose in the cell surface β-galactose glycans has been identified as an AAV9 receptor. However, the correlations between the capsid structure and viral activity for traversing the vascular barrier by AAV9 vectors are unclear. In an attempt to elucidate this enigma, we isolated natural variants of AAV9 from chimpanzee tissues and studied their functions. The CLvD8, a variant isolated from chimpanzee liver RNA, containing four amino acid mutations in the VP3 protein, displays the inability to cross vascular barrier, which serves a useful model in our study. This vector was evaluated in C57BL/6 mice for nLacZ, luciferase and α1-anti-trypsin (AAT) gene transduction after intravenous (i.v.), intramuscular (i.m.), and intra-nasal (i.n.) administration. The study to compare AAT expression suggested that AAV9 significantly outperforms CLvD8. However, difference between these two vectors for the nLaZ and luciferase transduction in liver, muscle and lung is not as much as AAT expression. Especially, in the lung, both AAV9 and CLvD8 led to similar nLacZ and luciferase transduction after i.n. delivery and CLvD8 primarily target alveoli as does AAV9. In live image for luciferase expression, AAV9 led to strong transduction in other organs outside of the administration sites after i.m. and i.n. delivery up to 17 weeks. However, the CLvD8mediated luciferase expression was restricted in the local tissues S6
15. In Vivo Knock-Down of MRN Complex Members Improves Liver and Cardiac Transduction with AAV Vectors
Jasmina Lovric,1 Miguel Mano,1 Lorena Zentilin,1 Ana Eulalio,1 Serena Zacchigna,1 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italy.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy