- Email: [email protected]
617. Assessment of Oncogenic Insertional Activation Potential from rAAV Vector
AAV GENOME & HOST INTERACTIONS Differences in AAV transduction in HeLa cells were assessed using a recombinant ssAAV2 vector expressing the firefly Luciferase reporter gene. Analysis of the results obtained from this primary screening identified 1528 genes affecting transduction by AAV vectors by more than 4-fold (184 genes by more than 8-fold). Of these genes, 993 are inhibitors of AAV transduction, whereas 535 are required for efficient transduction by AAV vectors. The effect of these genes on AAV transduction was further confirmed in a secondary screening based on high-content microscopy of cells transduced with a recombinant AAV vector expressing DsRed. Gene ontology analysis of the inhibitor genes revealed a clear overrepresentation of genes related to DNA recombination and repair and cell cycle control, whereas in the subset of genes required for infection an overrepresentation of genes involved in endocytosis, intracellular trafficking and transcription was observed. Consistent with our previous results, among the genes that restrict transduction by AAV vectors, we found members of the MRN complex and other genes involved in cellular DNA damage response, as well as genes involved in the ubiquitin-proteasome system. In addition, a significant number of genes were identified, which have not previously been associated with AAV transduction. Work in progress aims at investigating the mechanisms of action of the identified genes and exploiting this information to develop RNAi or pharmacological strategies to improve AAV transduction in vivo.
615. Enhanced Transgene Expression and SiteSpecific Integration of Adeno-Associated Virus Depends on Methylation Status of the Target Cell Diptiman Chanda,1 Jonathan A. Hensel,1 Jerome T. Higgs,1 Rajat Grover,1 Niroop Kaza,1 Selvarangan Ponnazhagan.1 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL.
Long-term expression of transgenes following vector-mediated delivery is often curtailed due to host defense mechanisms against viral infection. One such event in the eukaryotic cells occurs via DNA methylation and histone modification. Unmethylated CpG regions of microbial DNA are immunogenic and also form targets for DNA methyltransferase activity and recruitment of polycomb and other protein complexes resulting in repression of viral replication as well as transgene expression. Adeno-associated virus (AAV) is a non-pathogenic human parvovirus, which is being tested in many clinical gene therapy applications. Wild-type AAV (wt-AAV) has anti-oncogenic properties mainly in HPV-induced carcinogenesis. There are atleast four CpG islands in the 4.6 kb wtAAV2 genome, which are larger than 400 base-pairs. Moreover, like other gene therapy vectors, recombinant AAV (rAAV) are mostly designed using the cytomegalovirus (CMV) immediate early gene promoter, which also contains a significant number of CpG motifs. Therefore, methylation status of AAV inside the target cell could affect transgene expression. In the present study, we determined the influence of host cell methylation on transgene expression of rAAV and on integration of wt-AAV. When HeLa cells were maintained in a hypomethylating agent, 5-Aza-2’ deoxycytidine (5-Aza) for 72 hours and followed by infection with either recombinant AAV-GFP or wt-AAV showed significantly higher GFP expression and wt-AAV integration respectively when compared to untreated HeLa cells. The enhanced GFP expression was found to be not due to transduction of additional AAV particles following 5-Aza treatment, rather due to the methylation status of the AAV genome. The enhanced GFP expression following 5-Aza treatment was exclusively linked to AAV viral infection and not AAV plasmid transfection, suggesting the role of genomic organization of AAV, flanked by GC-rich terminal repeat hairpin structures. Previous studies have demonstrated that the leading 510 nucleotides at the 5’ end of AAVS1 sequence in the human chromosome 19 are a key to AAV integration and contain homology to the Rep binding site (RBS) and the terminal resolution S236
site (TRS) of AAV. AAVS1 sequence analysis also indicated the 510 nucleotides are a part of a 662 base pairs CpG island starting at 203 nucleotides and ending at 864 nucleotides when the first 1020 base pairs of AAVS1 is analyzed. In order to further characterize the role of methylation on AAV integration, we methylated in vitro a 8.2 kb AAVS1 fragment using CpG methylase and methyl donor, S-adenosyl methionine and transfected this methylated fragment into C18 cells. These cells were infected with wt-AAV after introduction of methylated AAVS1 and when tested failed to show any site-specific integration. Overall, results of the present study showed the influence of host methylation status in AAV-based genetic therapies.
616. rAAV Hairpin Structure Does Not Contribute to Chromosomal Integration
Marcela P. Cataldi,1,3 Douglas M. McCarty.1,2 1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Dept. of Pediatrics, The Ohio State University, Columbus, OH; 3MCDB Graduate Program, The Ohio State University, Columbus, OH. The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single-stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.
617. Assessment of Oncogenic Insertional Activation Potential from rAAV Vector
Lucia E. Rosas,1 Jessica L. Grieves,1,2 Douglas M. McCarty.1,3 Center for Gene Therapy, The Reseach Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Veterinary Biosciences, Ohio State University College of Veterinary Medicine, Columbus, OH; 3Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH. 1
Although recombinant adeno-associated virus (rAAV) gene delivery vectors have generally proven safe in animal models of human disease, their potential for low frequency integration into host chromosomal DNA raises concerns about genotoxicity and oncogenesis. Interactions between a rAAV vector expressing β-glucuronidase and a host common integration site have been associated with hepatocellular carcinoma (HCC) in MPS VII mice. We Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
AAV GENOME & HOST INTERACTIONS have evaluated the incidence of HCC in tumor-prone C3H/HeJ mice treated with 2 x 1012 vg/kg self-complementary rAAV2-8 vectors at approximately 1 year after IV injection. Two different scAAV vector constructs were compared to saline injected controls. The first was a conventional scAAV vector expressing GFP from a CMV promoter (CMV-GFP). The second was designed to maximize insertional activation of host genes near the integration site to provide a sensitive assay for interactions between integrated rAAV and host genes, and contained the CBA promoter with no protein coding sequences, and no transcription termination signal (CBA-null). The incidence of HCC was significantly increased in C3H/HeJ males treated with either of the scAAV vectors compared to saline controls. Induction of cell cycling at the time of infection by 40% partial hepatectomy led to a further increase in HCC in CBA-null treated animals, but not CMVGFP. Induction of DNA double-strand breaks at the time of infection by treatment with camptothecin (25mg/kg IP) did not increase the incidence of HCC in vector treated animals compared to saline controls. Vector genome (vg) copy numbers were analyzed by qPCR in tumor and normal liver tissue from vector treated animals. Tumors from animals injected with CBA-null vector were more frequently associated with significant amounts of vector DNA than animals receiving CMV-GFP. Vector junctions from several tumors from the CBA-null treated mice were amplified by inverse PCR and sequenced. Two junctions were located in the 5’ non-translated regions of known oncogenes (FGF10 and H-ras1) in a direct orientation relative to the transcription of the CBA promoter. Reverse-transcription-qPCR analysis of RNA levels from the FGF10 associated tumor showed significantly increased levels of FGF10 mRNA compared to normal liver or an unrelated tumor. Characterization of vector integration sites from CMV-GFP treated animals is ongoing. The results suggest that unrestrained promoter activity in a rAAV vector can increase the incidence of HCC by insertional activation in a tumor prone animal. However, HCC incidence was not significantly increased by treatment with this vector in C3H/HeJ females, which are not tumor prone, or in C3H/SCID mice at 6 months post-injection.
618. Investigating the Axonal Transport of AAV Serotype 9 in a Microfluidic System Michael J. Castle,1 Eran Perlson,2 Erika L. F. Holzbaur,1 John H. Wolfe.1 1 University of Pennsylvania, Philadelphia, PA; 2Physiology and Pharmacology, Tel Aviv University, Tel Aviv, Israel.
Though recombinant Adeno-Associated Virus (AAV) vectors are effective tools for long-term gene transfer in the brain, they typically transduce only a small region surrounding the injection site. These vectors thus have limited value for experimental applications that require widespread gene expression, or for clinical treatment of brain disorders requiring global correction. Some AAV serotypes undergo axonal transport to distal brain regions following intraparenchymal injection, although the pattern of distal transduction varies considerably among serotypes, and this transport is not observed along all neuronal pathways. This phenomenon provides a promising way to distribute gene transfer more widely in the brain. Unfortunately, knowledge of the cellular mechanisms underlying the axonal transport of AAV is limited, impeding 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 intraneuronal 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 infection at either the cell bodies or axon endings of cultured E18 rat cortical neurons, facilitating the direct examination of anterograde and retrograde axonal AAV transport. Dye-conjugated AAV9 particles developed for use in this system can be imaged in live cells, revealing the trafficking events following virus entry. Quantitative analysis of Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
this transport indicates that dye-labeled AAV9 moves in both the retrograde and anterograde directions, at average velocities typical of dynein- and kinesin-mediated fast axonal transport. Although both retrograde- and anterograde-directed AAV9 particles pause frequently and for extended durations, these populations are both highly directed, with movement in the reverse direction uncommon. Retrograde-directed AAV9 is composed of multiple compartment populations, including (but likely not limited to) a slow-moving lysosome-colocalized population and a faster-moving late endosome (Rab7)-colocalized population. Anterograde-moving AAV9 particles appear to be a distinct population as well, with anterograde transport observed only after AAV9 has accumulated in the cell body, suggesting that a change of compartment is taking place (e.g., transfer into a synaptic vesicle precursor compartment via the Golgi apparatus). Identification and characterization of this anterograde compartment, as well as of additional compartments composing the population of retrograde-directed particles, is ongoing. By beginning to elucidate the intraneuronal trafficking of AAV9, this work provides valuable insight into the cellular mechanisms underlying AAV transport in vivo, and is a first step toward the development of novel vectors and treatment strategies designed for widespread gene expression.
619. AAV Vector-Mediated Activation of Canonical and Alternative NF-κB Pathways In Vivo: Implications for Innate and Adaptive Immune Responses and Gene Therapy
Giridhara R. Jayandharan,1,2 George V. Aslanidi,1 Ashley T. Martino,1 Stephan C. Jahn,1 George Q. Perrin,1 Roland W. Herzog,1 Arun Srivastava.1 1 Department of Pediatrics, University of Florida Coleege of Medicine, Gainesville, FL; 2Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India. We have reported that infection of HeLa cells with AAV2 vectors in vitro results in activation of the alternative pathway of NF-κB, a central regulator of cellular immune and inflammatory responses. In the present studies, we examined the role of NF-κB in AAV2-mediated gene transfer to liver in mice. In vivo, AAV2-mediated gene transfer results in activation of canonial and alternative NF-κB pathways. AAV2 vectors with wild-type (WT) or tyrosine triple-mutant (TM) capsids activated canonical NF-κB pathway within 2 hrs, resulting in expression of pro-inflammatory cytokines and chemokines (Fig. 1A). This transient process is Toll-like receptor 9 (TLR9)-dependent, and likely reflects the initial sensing of the vector genome by antigenpresenting cells. Western blot analyses (Fig. 1B) of liver homogenates prepared 9 hrs post-vector delivery, showed abundance of nuclear p52 protein component of alternative NF-κB pathway, likely resulting from gene transfer to hepatocytes. Administration of NF-κB inhibitor, Bay11, prior to gene transfer, effectively blocked activation of both pathways. This maneuver prevented pro-inflammatory innate immune responses and also dampened anti-AAV2 capsid antibody formation (Fig. 1C). Importantly, Bay11 did not interfere with long-term transgene expression mediated by both WT and TM AAV2 vectors (Fig. 1D). These studies suggest that transient immuno-suppression with NF-κB inhibitors prior to vector administration eliminates inflammation, and also limits adaptive responses. This strategy could further improve the use of AAV vectors in human gene therapy.
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615. Enhanced Transgene Expression and SiteSpecific Integration of Adeno-Associated Virus Depends on Methylation Status of the Target Cell Diptiman Chanda,1 Jonathan A. Hensel,1 Jerome T. Higgs,1 Rajat Grover,1 Niroop Kaza,1 Selvarangan Ponnazhagan.1 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL.
Long-term expression of transgenes following vector-mediated delivery is often curtailed due to host defense mechanisms against viral infection. One such event in the eukaryotic cells occurs via DNA methylation and histone modification. Unmethylated CpG regions of microbial DNA are immunogenic and also form targets for DNA methyltransferase activity and recruitment of polycomb and other protein complexes resulting in repression of viral replication as well as transgene expression. Adeno-associated virus (AAV) is a non-pathogenic human parvovirus, which is being tested in many clinical gene therapy applications. Wild-type AAV (wt-AAV) has anti-oncogenic properties mainly in HPV-induced carcinogenesis. There are atleast four CpG islands in the 4.6 kb wtAAV2 genome, which are larger than 400 base-pairs. Moreover, like other gene therapy vectors, recombinant AAV (rAAV) are mostly designed using the cytomegalovirus (CMV) immediate early gene promoter, which also contains a significant number of CpG motifs. Therefore, methylation status of AAV inside the target cell could affect transgene expression. In the present study, we determined the influence of host cell methylation on transgene expression of rAAV and on integration of wt-AAV. When HeLa cells were maintained in a hypomethylating agent, 5-Aza-2’ deoxycytidine (5-Aza) for 72 hours and followed by infection with either recombinant AAV-GFP or wt-AAV showed significantly higher GFP expression and wt-AAV integration respectively when compared to untreated HeLa cells. The enhanced GFP expression was found to be not due to transduction of additional AAV particles following 5-Aza treatment, rather due to the methylation status of the AAV genome. The enhanced GFP expression following 5-Aza treatment was exclusively linked to AAV viral infection and not AAV plasmid transfection, suggesting the role of genomic organization of AAV, flanked by GC-rich terminal repeat hairpin structures. Previous studies have demonstrated that the leading 510 nucleotides at the 5’ end of AAVS1 sequence in the human chromosome 19 are a key to AAV integration and contain homology to the Rep binding site (RBS) and the terminal resolution S236
site (TRS) of AAV. AAVS1 sequence analysis also indicated the 510 nucleotides are a part of a 662 base pairs CpG island starting at 203 nucleotides and ending at 864 nucleotides when the first 1020 base pairs of AAVS1 is analyzed. In order to further characterize the role of methylation on AAV integration, we methylated in vitro a 8.2 kb AAVS1 fragment using CpG methylase and methyl donor, S-adenosyl methionine and transfected this methylated fragment into C18 cells. These cells were infected with wt-AAV after introduction of methylated AAVS1 and when tested failed to show any site-specific integration. Overall, results of the present study showed the influence of host methylation status in AAV-based genetic therapies.
616. rAAV Hairpin Structure Does Not Contribute to Chromosomal Integration
Marcela P. Cataldi,1,3 Douglas M. McCarty.1,2 1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Dept. of Pediatrics, The Ohio State University, Columbus, OH; 3MCDB Graduate Program, The Ohio State University, Columbus, OH. The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single-stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.
617. Assessment of Oncogenic Insertional Activation Potential from rAAV Vector
Lucia E. Rosas,1 Jessica L. Grieves,1,2 Douglas M. McCarty.1,3 Center for Gene Therapy, The Reseach Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Veterinary Biosciences, Ohio State University College of Veterinary Medicine, Columbus, OH; 3Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH. 1
Although recombinant adeno-associated virus (rAAV) gene delivery vectors have generally proven safe in animal models of human disease, their potential for low frequency integration into host chromosomal DNA raises concerns about genotoxicity and oncogenesis. Interactions between a rAAV vector expressing β-glucuronidase and a host common integration site have been associated with hepatocellular carcinoma (HCC) in MPS VII mice. We Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
AAV GENOME & HOST INTERACTIONS have evaluated the incidence of HCC in tumor-prone C3H/HeJ mice treated with 2 x 1012 vg/kg self-complementary rAAV2-8 vectors at approximately 1 year after IV injection. Two different scAAV vector constructs were compared to saline injected controls. The first was a conventional scAAV vector expressing GFP from a CMV promoter (CMV-GFP). The second was designed to maximize insertional activation of host genes near the integration site to provide a sensitive assay for interactions between integrated rAAV and host genes, and contained the CBA promoter with no protein coding sequences, and no transcription termination signal (CBA-null). The incidence of HCC was significantly increased in C3H/HeJ males treated with either of the scAAV vectors compared to saline controls. Induction of cell cycling at the time of infection by 40% partial hepatectomy led to a further increase in HCC in CBA-null treated animals, but not CMVGFP. Induction of DNA double-strand breaks at the time of infection by treatment with camptothecin (25mg/kg IP) did not increase the incidence of HCC in vector treated animals compared to saline controls. Vector genome (vg) copy numbers were analyzed by qPCR in tumor and normal liver tissue from vector treated animals. Tumors from animals injected with CBA-null vector were more frequently associated with significant amounts of vector DNA than animals receiving CMV-GFP. Vector junctions from several tumors from the CBA-null treated mice were amplified by inverse PCR and sequenced. Two junctions were located in the 5’ non-translated regions of known oncogenes (FGF10 and H-ras1) in a direct orientation relative to the transcription of the CBA promoter. Reverse-transcription-qPCR analysis of RNA levels from the FGF10 associated tumor showed significantly increased levels of FGF10 mRNA compared to normal liver or an unrelated tumor. Characterization of vector integration sites from CMV-GFP treated animals is ongoing. The results suggest that unrestrained promoter activity in a rAAV vector can increase the incidence of HCC by insertional activation in a tumor prone animal. However, HCC incidence was not significantly increased by treatment with this vector in C3H/HeJ females, which are not tumor prone, or in C3H/SCID mice at 6 months post-injection.
618. Investigating the Axonal Transport of AAV Serotype 9 in a Microfluidic System Michael J. Castle,1 Eran Perlson,2 Erika L. F. Holzbaur,1 John H. Wolfe.1 1 University of Pennsylvania, Philadelphia, PA; 2Physiology and Pharmacology, Tel Aviv University, Tel Aviv, Israel.
Though recombinant Adeno-Associated Virus (AAV) vectors are effective tools for long-term gene transfer in the brain, they typically transduce only a small region surrounding the injection site. These vectors thus have limited value for experimental applications that require widespread gene expression, or for clinical treatment of brain disorders requiring global correction. Some AAV serotypes undergo axonal transport to distal brain regions following intraparenchymal injection, although the pattern of distal transduction varies considerably among serotypes, and this transport is not observed along all neuronal pathways. This phenomenon provides a promising way to distribute gene transfer more widely in the brain. Unfortunately, knowledge of the cellular mechanisms underlying the axonal transport of AAV is limited, impeding 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 intraneuronal 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 infection at either the cell bodies or axon endings of cultured E18 rat cortical neurons, facilitating the direct examination of anterograde and retrograde axonal AAV transport. Dye-conjugated AAV9 particles developed for use in this system can be imaged in live cells, revealing the trafficking events following virus entry. Quantitative analysis of Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
this transport indicates that dye-labeled AAV9 moves in both the retrograde and anterograde directions, at average velocities typical of dynein- and kinesin-mediated fast axonal transport. Although both retrograde- and anterograde-directed AAV9 particles pause frequently and for extended durations, these populations are both highly directed, with movement in the reverse direction uncommon. Retrograde-directed AAV9 is composed of multiple compartment populations, including (but likely not limited to) a slow-moving lysosome-colocalized population and a faster-moving late endosome (Rab7)-colocalized population. Anterograde-moving AAV9 particles appear to be a distinct population as well, with anterograde transport observed only after AAV9 has accumulated in the cell body, suggesting that a change of compartment is taking place (e.g., transfer into a synaptic vesicle precursor compartment via the Golgi apparatus). Identification and characterization of this anterograde compartment, as well as of additional compartments composing the population of retrograde-directed particles, is ongoing. By beginning to elucidate the intraneuronal trafficking of AAV9, this work provides valuable insight into the cellular mechanisms underlying AAV transport in vivo, and is a first step toward the development of novel vectors and treatment strategies designed for widespread gene expression.
619. AAV Vector-Mediated Activation of Canonical and Alternative NF-κB Pathways In Vivo: Implications for Innate and Adaptive Immune Responses and Gene Therapy
Giridhara R. Jayandharan,1,2 George V. Aslanidi,1 Ashley T. Martino,1 Stephan C. Jahn,1 George Q. Perrin,1 Roland W. Herzog,1 Arun Srivastava.1 1 Department of Pediatrics, University of Florida Coleege of Medicine, Gainesville, FL; 2Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India. We have reported that infection of HeLa cells with AAV2 vectors in vitro results in activation of the alternative pathway of NF-κB, a central regulator of cellular immune and inflammatory responses. In the present studies, we examined the role of NF-κB in AAV2-mediated gene transfer to liver in mice. In vivo, AAV2-mediated gene transfer results in activation of canonial and alternative NF-κB pathways. AAV2 vectors with wild-type (WT) or tyrosine triple-mutant (TM) capsids activated canonical NF-κB pathway within 2 hrs, resulting in expression of pro-inflammatory cytokines and chemokines (Fig. 1A). This transient process is Toll-like receptor 9 (TLR9)-dependent, and likely reflects the initial sensing of the vector genome by antigenpresenting cells. Western blot analyses (Fig. 1B) of liver homogenates prepared 9 hrs post-vector delivery, showed abundance of nuclear p52 protein component of alternative NF-κB pathway, likely resulting from gene transfer to hepatocytes. Administration of NF-κB inhibitor, Bay11, prior to gene transfer, effectively blocked activation of both pathways. This maneuver prevented pro-inflammatory innate immune responses and also dampened anti-AAV2 capsid antibody formation (Fig. 1C). Importantly, Bay11 did not interfere with long-term transgene expression mediated by both WT and TM AAV2 vectors (Fig. 1D). These studies suggest that transient immuno-suppression with NF-κB inhibitors prior to vector administration eliminates inflammation, and also limits adaptive responses. This strategy could further improve the use of AAV vectors in human gene therapy.
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