- Email: [email protected]
861. AAV Gene Delivery for the Cornea
AAV VECTORS: VECTOR DEVELOPMENT Although AAV-2, contrary to AAV-1, transduced the striatum, our data suggests that AAV-8 is able to transduce the striatum even more efficiently. Even though the exact amounts of transduced cells still have to be quantified by stereology, our data strongly suggests that the number of GFP expressing cells in the CA1 en CA3 regions of the hippocampus is much higher in the brains injected with AAV-8 than in the brains injected with AAV-1 and AAV-2. Besides that, our data strongly suggests that AAV-8 has not only the capacity to efficiently transduce different structures of the CNS but also the capacity to transduce different cell types. In conclusion, intraventricular injection of neonatal mice with AAV-8 results in widespread delivery of GFP to the CNS. Moreover, AAV-8 transduces a higher number of (different) cells and different structures of the CNS compared to AAV-1 and AAV-2.
859. Enhancing Neurotropism with Multiple Mutations in the AAV-2 Capsid Jianfeng Xu,1 Caroline Bass,1 Chunyan Ma,1 Ernest F. Terwilliger.1 1 Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA. There is keen interest in developing practical strategies for targeting gene delivery to specific neuron populations. Unfortunately, the distinctive features of neurons, and their complex interconnections, impose stringent requirements on any potential gene delivery system. For a number of reasons, recombinant gene vectors derived from Adeno Associated Virus offer a strong set of starting candidates for this type of application. Unfortunately, standard vectors derived from AAV-2 do not disperse well after injection in the brain or spinal cord and are limited in their capacity for retrograde transport. Two distinct properties crucial for viruses targeting any type of neurons - a neuron-specific binding affinity, and a mechanism for keying into an efficient retrograde transport pathway after uptake are absent in AAV. Other viruses, including rabies and herpes viruses, and even some pathogenic parvoviruses, exhibit highly efficient uptake and retrograde transport along neural axons. In some cases the capsid features conferring these properties have been identified, enabling attempts at re-engineering these properties into more innocuous vectors. This is assisted in the case of AAV-2 by the availability of detailed X-ray crystallographic data. Our strategy for modifying AAV-2 was two-tiered. First, peptides derived from the sequence of a potent NMDA receptor antagonist were incorporated in the capsid. Second, to enhance retrograde transport, we attempted to model a new affinity for the cellular Dynein Motor Complex (DMC) into the virus. Recent studies have identified specific DMC components which associate with particular classes of cargo proteins, including some neurotropic viruses. Indicator genes were next packaged in capsids incorporating either class of mutation alone, both types together, or in the standard capsid. This virus set was then evaluated across a panel of cell types. Much higher gene transfer efficiencies were achieved in several neuronal cell types, including differentiated PC-12 cells and sequestered axons from cultured dorsal root ganglia, by vectors bearing both types of mutation, compared with viruses bearing either alone or the standard capsid. In these cell systems the effects of the mutations were strongly synergistic. This enhanced gene transfer could be competed with excess NMDA antagonist, or with agents that suppressed the DMC. Verification that an inserted peptide conferred binding to the DMC was obtained by coimmunoprecipitation of the predicted DMC peptide together with the mutant capsid protein using an AAV capsid antibody, but not with the standard AAV-2 capsid. These results hold strong promise for the development of practical S334
and convenient methodologies to target gene vectors to specific neuron populations, and provide strong motivation for further work. In addition, the more general strategy of combining distinct mutations affecting unrelated properties in order to generate chimeric vectors with unique new affinities should find applications in targeting other cell types and tissues.
860. Tissue Distribution of Expression Using AAV8-Based Vectors after Intramuscular Injection and Other Routes of Delivery Tsuyoshi Ogura,1 Hiroaki Mizukami,1 YuanYuan Zhang,1 Takashi Okada,1 Akihiro Kume,1 Seiji Madoiwa,2 Jun Mimuro,2 Yoichi Sakata,2 Keiya Ozawa.1 1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan; 2Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan. AAV8-based vectors are shown to be especially efficient in transducing liver and muscle and currently considered as one of the most promising vector system. In this study, we investigated tissue distribution of expression using AAV8-based vectors after intramuscular (IM) injection and other routes of delivery. For this purpose, AAV8-based vectors carrying luciferase gene (Luc) under CAG promoter was administered into tibialis anterior (TA) muscles of C57BL/6 mice. In vivo bioluminescence imaging analysis using IVIS system revealed robust transgene expression not only in lower limbs (around TA) but also in the upper abdominal region (suggesting liver). Analysis by ex vivo bioluminescence imaging after sacrifice confirmed expression in liver as well as TA muscles. To verify whether these findings are unique to AAV8, AAV1- and AAV2based vectors were tested in the same manner. When AAV1- or AAV2-Luc was injected within TA muscles, Luc expression was confined to the injected sites. The level of transgene expression observed in liver after AAV8-Luc injection was comparable to that observed in lower limbs after AAV1-Luc injection. To substantiate ectopic expression after intramuscular injection, vector genomes within the tissues were quantitated at early time points. Vector genome copies were sharply decreased in muscle and increased in liver at 24 hours after injection in the case of AAV8-based vectors. When AAV8-Luc vector was administered intravenously (IV) or intraperitoneally (IP), luciferase expression was confined to liver. A sex-related difference in luciferase expression in liver was observed regardless of the route of administration. In addition to adults, we compared tissue distribution of expression in neonate by the routes of administration of AAV8-Luc vector. Surprisingly, liver specific Luc expression was observed only after IV injection; in IM and IP administration, transgene expression was confined to lower limbs and peritoneum, respectively. Moreover, sex-related difference in transgene expression was not observed in any tissues in neonatal injection. These data suggest remarkable affinity of AAV8-based vectors to liver, and will be useful in designing strategies for gene therapy using AAV8-based vectors in adult and neonate.
861.
AAV Gene Delivery for the Cornea
Jia Liu,1 Sonal S. Tuli,2 David C. Bloom,1 Gregory S. Schultz,3 William W. Hauswirth,1,2 Alfred S. Lewin.1 1 Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL; 2Ophthalmology, University of Florida, College of Medicine, Gainesville, FL; 3Obstetrics and Gynecology, University of Florida, College of Medicine, Gainesville, FL. Purpose: Gene therapy approach has attracted significant interest in curing chronic and potentially recurrent corneal diseases. Adeno-associated Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
AAV VECTORS: VECTOR DEVELOPMENT virus (AAV) is non-pathogenic to the host and has the advantages of long term delivery of genes to both dividing and non dividing cells. The goal of this study includes (1) to compare gene transfer efficiency of different serotypes of AAV vectors in the rabbit cornea, (2) to locate transgene expression in different cell populations of corneal tissue. Methods: An AAV2 vector expressing GFP driven by the chicken- β-actin promoter and CMV enhancer was packaged in 5 different capsid types (1, 2, 5, 7, 8). Both eyes of NZ white rabbits were infected with the same serotype or pseudo type. Equal numbers of AAV particles (1011 vector genomes) were diluted in buffer and applied to the cornea for 10 minutes following excimer ablation of the corneal epithelium to 25 microns. Seven days were allowed for gene expression before corneas were removed. The corneas were fixed in 4% paraformaldehyde before cryosectioning at 10um thickness. One sample was analyzed at every third section. Immunostaining using biotinylated antibody against GFP and an alkaline phosphatase detection as well as a peroxidase detection system were employed. Bright field and fluorescence micrographs were taken for each section using a morphometric microscope, and the level of GFP immunostaining was measured using MCID software. Results: By 7 days all the serotypes of AAV led to expression of the transgene in all layers of the corneal tissue. Epithelial cells were heavily stained compared with stroma and endothelial cells. AAV type 1 was 20 -30% more efficient in gene delivery in comparison with other serotypes, followed by AAV type 8 which exhibited the highest level of penetration to deep layers of the cornea. Conclusions: Among all the serotypes that have been currently tested AAV type 1 and 8 can provide long term and highly efficient transduction. These experiments point the way for delivery of potential therapeutic genes to the cornea ex vivo for the transplant and in vivo. Dr. Hauswirth — AGTC
862. Adipose Tissue Transduction Using AAV8Based Vectors: Inadvertent Gene Transfer into Liver 1
1
2
YuanYuan Zhang, Tsuyoshi Ogura, Jun Mimuro, Takashi Okada,1 Akihiro Kume,1 Yoichi Sakata,2 Keiya Ozawa,1 Hiroaki Mizukami.1 1 Division of Genetic Therapeutics, Jichi Medical School, Minamikawachi, Tochigi, Japan; 2Division of Cell and Molecular Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan. We have shown that intra-adipose tissue injection using AAV1 vectors resulted in tissue-specific transgene expression in db/db mice. To attain a more efficient expression, we tried the same method using AAV8-based vector (AAV8-CMV-Epo). Remarkably higher plasma Epo concentration was observed. When a vector encoding human Factor IX (AAV8-CMV-hFIX) was used at a dose of 2 x 1011 vg/body, more than 10% equivalent of normal human concentration was observed and considered as therapeutic level. In order to confirm tissue specific expression, transduced adipose tissues were surgically removed and plasma Factor IX concentration was pursued one week later. Surprisingly, approximately two-third of plasma concentration remained after total elimination of target tissue. Copy number of hFIX mRNA within the tissue correlated well with the plasma Factor IX concentration, supporting the contribution of both tissues. In vivo imaging analysis by IVIS system suggested transgene expression within liver when AAV8 vector encoding luciferase was used for adipose tissue transduction. Organ specific expression analyzed by ex vivo imaging confirmed robust expression in liver rather than adipose tissue. These data suggest extreme tropism of AAV8-based vectors to liver, and we should be careful about overestimating results when tissues other than liver were targeted. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
863. Directed Evolution of Adeno-Associated Virus (AAV) Vectors for Human Gene Therapy Luca Perabo,1,2 Jan Endell,1 Susan King,1 Daniela Goldnau,1 Kerstin Lux,1 Michael Hallek,1,2,3 Hildegard Buening.1,2 1 Gene Centre, LMU - University of Munich, Munich, Germany; 2 Clinic I for Internal Medicine, University of Cologne, Cologne, Germany; 3GSF - National Center for Research and Environment, Munich, Germany. The success of viral vectors in gene therapy will depend on our ability to solve a number of problems, the most important regarding safety and efficiency of the vector. Overcoming these obstacles requires appropriate molecular engineering of the viral particles. We performed combinatorial manipulation of the viral capsid by error prone PCR to generate mutants with optimized phenotype. To demonstrate the potential of this technology, we randomized the capsid of adeno-associated virus of type 2 (AAV-2), scattering point mutations throughout the capsid gene. The obtained library of viral particles was panned for the selection of viral clones that escape neutralization by human antibodies. We identified several capsid variants that efficiently transduce target cells in vitro in the presence of human serum concentrations that completely neutralize wild type AAV-2. These clones could be used as gene therapy vectors for the treatment of patients with AAV-2 preexisting immunity. The mutants identified in this study unveil undiscovered properties of the viral capsid. This suggests that combinatorial engineering overcomes the limitations of rational design posed by incomplete understanding of the infectious process, offering at the same time a powerful tool to dissect basic viral biology by reverse genetics.
864. Adsorption of Adeno-Associated Virus to Commonly Used Catheter Materials Daisy P. Cross,1 Erica TenBroek,1 Cygni (Howie) Chan,1 Laura Christoferson,1 Orhan Soykan,1 Daniel C. Sigg.2 1 Corporate Science and Technology, Medtronic, Inc., Minneapolis, MN; 2CRM Therapy Delivery, Medtronic, Inc., Minneapolis, MN. Published literature has suggested that various materials that are commonly used in injection catheters (e.g., stainless steel, nitinol) inactivate adenovirus (Adv), a viral vector that is widely used in the field of gene therapy. No such information is available for recombinant adeno-associated virus (rAAV). rAAV has a number of advantages over Adv, most notably, that rAAV is associated with more sustained gene expression. Therefore, we specifically investigated the stability of rAAV when exposed to stainless steel or nitinol catheters. Hollow lumen catheters (stainless steel: 2 catheters, 3 replicates total; nitinol: 2 catheters, 6 replicates total) were filled with rAAV serotype 2 encoding for enhanced green fluorescent protein (rAAV2eGFP) at a concentration of 1 x 1011 genomic particles/mL, then incubated at 37°C. At various time points after filling (0-30 min), virus solution was ejected out of the catheter and applied to cultured cells. Incubation with polypropylene was used as the negative control. EGFP expression was assessed via flow cytometry, and viral titers were assessed via ELISA. rAAV-mediated gene expression appeared to be compromised after contact with various catheter materials in a time-dependent fashion. After 30 min, nitinol reduced rAAV-mediated gene expression to ∼60% of the original activity, and stainless steel to ∼50%. The number of eluted rAAV particles also decreased in a time-dependent fashion. Our data indicates that, unlike findings with Adv, the decreased rAAV-mediated gene expression after material contact is due to adsorption of the rAAV protein capsids to the materials, as opposed to inactivation of the rAAV itself.
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859. Enhancing Neurotropism with Multiple Mutations in the AAV-2 Capsid Jianfeng Xu,1 Caroline Bass,1 Chunyan Ma,1 Ernest F. Terwilliger.1 1 Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA. There is keen interest in developing practical strategies for targeting gene delivery to specific neuron populations. Unfortunately, the distinctive features of neurons, and their complex interconnections, impose stringent requirements on any potential gene delivery system. For a number of reasons, recombinant gene vectors derived from Adeno Associated Virus offer a strong set of starting candidates for this type of application. Unfortunately, standard vectors derived from AAV-2 do not disperse well after injection in the brain or spinal cord and are limited in their capacity for retrograde transport. Two distinct properties crucial for viruses targeting any type of neurons - a neuron-specific binding affinity, and a mechanism for keying into an efficient retrograde transport pathway after uptake are absent in AAV. Other viruses, including rabies and herpes viruses, and even some pathogenic parvoviruses, exhibit highly efficient uptake and retrograde transport along neural axons. In some cases the capsid features conferring these properties have been identified, enabling attempts at re-engineering these properties into more innocuous vectors. This is assisted in the case of AAV-2 by the availability of detailed X-ray crystallographic data. Our strategy for modifying AAV-2 was two-tiered. First, peptides derived from the sequence of a potent NMDA receptor antagonist were incorporated in the capsid. Second, to enhance retrograde transport, we attempted to model a new affinity for the cellular Dynein Motor Complex (DMC) into the virus. Recent studies have identified specific DMC components which associate with particular classes of cargo proteins, including some neurotropic viruses. Indicator genes were next packaged in capsids incorporating either class of mutation alone, both types together, or in the standard capsid. This virus set was then evaluated across a panel of cell types. Much higher gene transfer efficiencies were achieved in several neuronal cell types, including differentiated PC-12 cells and sequestered axons from cultured dorsal root ganglia, by vectors bearing both types of mutation, compared with viruses bearing either alone or the standard capsid. In these cell systems the effects of the mutations were strongly synergistic. This enhanced gene transfer could be competed with excess NMDA antagonist, or with agents that suppressed the DMC. Verification that an inserted peptide conferred binding to the DMC was obtained by coimmunoprecipitation of the predicted DMC peptide together with the mutant capsid protein using an AAV capsid antibody, but not with the standard AAV-2 capsid. These results hold strong promise for the development of practical S334
and convenient methodologies to target gene vectors to specific neuron populations, and provide strong motivation for further work. In addition, the more general strategy of combining distinct mutations affecting unrelated properties in order to generate chimeric vectors with unique new affinities should find applications in targeting other cell types and tissues.
860. Tissue Distribution of Expression Using AAV8-Based Vectors after Intramuscular Injection and Other Routes of Delivery Tsuyoshi Ogura,1 Hiroaki Mizukami,1 YuanYuan Zhang,1 Takashi Okada,1 Akihiro Kume,1 Seiji Madoiwa,2 Jun Mimuro,2 Yoichi Sakata,2 Keiya Ozawa.1 1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan; 2Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan. AAV8-based vectors are shown to be especially efficient in transducing liver and muscle and currently considered as one of the most promising vector system. In this study, we investigated tissue distribution of expression using AAV8-based vectors after intramuscular (IM) injection and other routes of delivery. For this purpose, AAV8-based vectors carrying luciferase gene (Luc) under CAG promoter was administered into tibialis anterior (TA) muscles of C57BL/6 mice. In vivo bioluminescence imaging analysis using IVIS system revealed robust transgene expression not only in lower limbs (around TA) but also in the upper abdominal region (suggesting liver). Analysis by ex vivo bioluminescence imaging after sacrifice confirmed expression in liver as well as TA muscles. To verify whether these findings are unique to AAV8, AAV1- and AAV2based vectors were tested in the same manner. When AAV1- or AAV2-Luc was injected within TA muscles, Luc expression was confined to the injected sites. The level of transgene expression observed in liver after AAV8-Luc injection was comparable to that observed in lower limbs after AAV1-Luc injection. To substantiate ectopic expression after intramuscular injection, vector genomes within the tissues were quantitated at early time points. Vector genome copies were sharply decreased in muscle and increased in liver at 24 hours after injection in the case of AAV8-based vectors. When AAV8-Luc vector was administered intravenously (IV) or intraperitoneally (IP), luciferase expression was confined to liver. A sex-related difference in luciferase expression in liver was observed regardless of the route of administration. In addition to adults, we compared tissue distribution of expression in neonate by the routes of administration of AAV8-Luc vector. Surprisingly, liver specific Luc expression was observed only after IV injection; in IM and IP administration, transgene expression was confined to lower limbs and peritoneum, respectively. Moreover, sex-related difference in transgene expression was not observed in any tissues in neonatal injection. These data suggest remarkable affinity of AAV8-based vectors to liver, and will be useful in designing strategies for gene therapy using AAV8-based vectors in adult and neonate.
861.
AAV Gene Delivery for the Cornea
Jia Liu,1 Sonal S. Tuli,2 David C. Bloom,1 Gregory S. Schultz,3 William W. Hauswirth,1,2 Alfred S. Lewin.1 1 Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL; 2Ophthalmology, University of Florida, College of Medicine, Gainesville, FL; 3Obstetrics and Gynecology, University of Florida, College of Medicine, Gainesville, FL. Purpose: Gene therapy approach has attracted significant interest in curing chronic and potentially recurrent corneal diseases. Adeno-associated Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
AAV VECTORS: VECTOR DEVELOPMENT virus (AAV) is non-pathogenic to the host and has the advantages of long term delivery of genes to both dividing and non dividing cells. The goal of this study includes (1) to compare gene transfer efficiency of different serotypes of AAV vectors in the rabbit cornea, (2) to locate transgene expression in different cell populations of corneal tissue. Methods: An AAV2 vector expressing GFP driven by the chicken- β-actin promoter and CMV enhancer was packaged in 5 different capsid types (1, 2, 5, 7, 8). Both eyes of NZ white rabbits were infected with the same serotype or pseudo type. Equal numbers of AAV particles (1011 vector genomes) were diluted in buffer and applied to the cornea for 10 minutes following excimer ablation of the corneal epithelium to 25 microns. Seven days were allowed for gene expression before corneas were removed. The corneas were fixed in 4% paraformaldehyde before cryosectioning at 10um thickness. One sample was analyzed at every third section. Immunostaining using biotinylated antibody against GFP and an alkaline phosphatase detection as well as a peroxidase detection system were employed. Bright field and fluorescence micrographs were taken for each section using a morphometric microscope, and the level of GFP immunostaining was measured using MCID software. Results: By 7 days all the serotypes of AAV led to expression of the transgene in all layers of the corneal tissue. Epithelial cells were heavily stained compared with stroma and endothelial cells. AAV type 1 was 20 -30% more efficient in gene delivery in comparison with other serotypes, followed by AAV type 8 which exhibited the highest level of penetration to deep layers of the cornea. Conclusions: Among all the serotypes that have been currently tested AAV type 1 and 8 can provide long term and highly efficient transduction. These experiments point the way for delivery of potential therapeutic genes to the cornea ex vivo for the transplant and in vivo. Dr. Hauswirth — AGTC
862. Adipose Tissue Transduction Using AAV8Based Vectors: Inadvertent Gene Transfer into Liver 1
1
2
YuanYuan Zhang, Tsuyoshi Ogura, Jun Mimuro, Takashi Okada,1 Akihiro Kume,1 Yoichi Sakata,2 Keiya Ozawa,1 Hiroaki Mizukami.1 1 Division of Genetic Therapeutics, Jichi Medical School, Minamikawachi, Tochigi, Japan; 2Division of Cell and Molecular Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan. We have shown that intra-adipose tissue injection using AAV1 vectors resulted in tissue-specific transgene expression in db/db mice. To attain a more efficient expression, we tried the same method using AAV8-based vector (AAV8-CMV-Epo). Remarkably higher plasma Epo concentration was observed. When a vector encoding human Factor IX (AAV8-CMV-hFIX) was used at a dose of 2 x 1011 vg/body, more than 10% equivalent of normal human concentration was observed and considered as therapeutic level. In order to confirm tissue specific expression, transduced adipose tissues were surgically removed and plasma Factor IX concentration was pursued one week later. Surprisingly, approximately two-third of plasma concentration remained after total elimination of target tissue. Copy number of hFIX mRNA within the tissue correlated well with the plasma Factor IX concentration, supporting the contribution of both tissues. In vivo imaging analysis by IVIS system suggested transgene expression within liver when AAV8 vector encoding luciferase was used for adipose tissue transduction. Organ specific expression analyzed by ex vivo imaging confirmed robust expression in liver rather than adipose tissue. These data suggest extreme tropism of AAV8-based vectors to liver, and we should be careful about overestimating results when tissues other than liver were targeted. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
863. Directed Evolution of Adeno-Associated Virus (AAV) Vectors for Human Gene Therapy Luca Perabo,1,2 Jan Endell,1 Susan King,1 Daniela Goldnau,1 Kerstin Lux,1 Michael Hallek,1,2,3 Hildegard Buening.1,2 1 Gene Centre, LMU - University of Munich, Munich, Germany; 2 Clinic I for Internal Medicine, University of Cologne, Cologne, Germany; 3GSF - National Center for Research and Environment, Munich, Germany. The success of viral vectors in gene therapy will depend on our ability to solve a number of problems, the most important regarding safety and efficiency of the vector. Overcoming these obstacles requires appropriate molecular engineering of the viral particles. We performed combinatorial manipulation of the viral capsid by error prone PCR to generate mutants with optimized phenotype. To demonstrate the potential of this technology, we randomized the capsid of adeno-associated virus of type 2 (AAV-2), scattering point mutations throughout the capsid gene. The obtained library of viral particles was panned for the selection of viral clones that escape neutralization by human antibodies. We identified several capsid variants that efficiently transduce target cells in vitro in the presence of human serum concentrations that completely neutralize wild type AAV-2. These clones could be used as gene therapy vectors for the treatment of patients with AAV-2 preexisting immunity. The mutants identified in this study unveil undiscovered properties of the viral capsid. This suggests that combinatorial engineering overcomes the limitations of rational design posed by incomplete understanding of the infectious process, offering at the same time a powerful tool to dissect basic viral biology by reverse genetics.
864. Adsorption of Adeno-Associated Virus to Commonly Used Catheter Materials Daisy P. Cross,1 Erica TenBroek,1 Cygni (Howie) Chan,1 Laura Christoferson,1 Orhan Soykan,1 Daniel C. Sigg.2 1 Corporate Science and Technology, Medtronic, Inc., Minneapolis, MN; 2CRM Therapy Delivery, Medtronic, Inc., Minneapolis, MN. Published literature has suggested that various materials that are commonly used in injection catheters (e.g., stainless steel, nitinol) inactivate adenovirus (Adv), a viral vector that is widely used in the field of gene therapy. No such information is available for recombinant adeno-associated virus (rAAV). rAAV has a number of advantages over Adv, most notably, that rAAV is associated with more sustained gene expression. Therefore, we specifically investigated the stability of rAAV when exposed to stainless steel or nitinol catheters. Hollow lumen catheters (stainless steel: 2 catheters, 3 replicates total; nitinol: 2 catheters, 6 replicates total) were filled with rAAV serotype 2 encoding for enhanced green fluorescent protein (rAAV2eGFP) at a concentration of 1 x 1011 genomic particles/mL, then incubated at 37°C. At various time points after filling (0-30 min), virus solution was ejected out of the catheter and applied to cultured cells. Incubation with polypropylene was used as the negative control. EGFP expression was assessed via flow cytometry, and viral titers were assessed via ELISA. rAAV-mediated gene expression appeared to be compromised after contact with various catheter materials in a time-dependent fashion. After 30 min, nitinol reduced rAAV-mediated gene expression to ∼60% of the original activity, and stainless steel to ∼50%. The number of eluted rAAV particles also decreased in a time-dependent fashion. Our data indicates that, unlike findings with Adv, the decreased rAAV-mediated gene expression after material contact is due to adsorption of the rAAV protein capsids to the materials, as opposed to inactivation of the rAAV itself.
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