258 Innate Immune Response Affects AAV Mediated Hepatic Trangene Expression Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S101 IMMUNOLOGIC[.]
IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY I – AAV 185 μg/ml To evaluate the influence of pre-existing NAbs on gene expression, rhesus macaques with NAb titers of 1/20 (n = 3), 1/160 (n = 1) and 1/320 (n = 1) were also injected with 3x1012 GC/kg of the same vector The peak expression in animals with a pre-existing NAb titer of 1/20 was also at day 28 post-administration of vector at a slightly higher level of 580 μg/ml ± 108 μg/ml 201Ig IA The presence of higher levels of pre-existing NAbs did slightly decrease the peak of gene expression as animals with titers of 1/160 and 1/320 had 201Ig IA levels of 71 μg/ml and 117 μg/ml, respectively, at day 28 post-vector injection In conclusion, IM administration of an AAV2/8 vector produced therapeutic systemic levels of an antibody in the presence and absence of pre-existing NAbs The impact of NAbs on skeletal muscle transduction was much less substantial than what we have previously observed with intravenous delivery of vector to target the liver 256 A Comprehensive Study on Immunology and Persistence of AAV8 Vectors with SelfComplementary and Single-Stranded Genome in Mice: Implications for Transient Immune Suppression with AAV8 Vectors Te-Lang Wu,1,2 Hua Li,1 Xiangyang Zhou,1 Katherine A High,3,4 Hildegund C Ertl.1 The Wistar Institute, Philadelphia, PA; 2School of Medicine, University of Pennsylvania, Philadelphia, PA; 3The Children’s Hospital of Philadelphia, PA; 4Howard Hughes Medical Institute, Philadelphia, PA Recent publication describes that self-complementary adenoassociated viral (AAV) vectors of serotype (AAV8) expressing human factor IX (hF.IX) can achieve sustained correction of hemophilia B in human hemophilia B patients High vector doses delivered to the liver caused a definitive transaminitis in one patient and a borderline increase in transaminase in a second patient accompanied by an increase in AAV capsid-specific T cells and a transient decrease in circulating hF.IX levels The clinical course of these patients is reminiscent of the results of a previous trial with single-stranded AAV2 vectors expressing hF.IX where human subjects showed evidence of liver damage accompanied by increases in circulating AAV-specific T cells and a complete loss of the therapeutic protein Nevertheless kinetics of this shared adverse event differed with AAV2 and AAV8 vectors causing rise in transaminases at vs weeks after vector injection, respectively To test if CD8+ T cells to AAV8 vectors, which as fully-gutted vectors fail to encode structural viral proteins, could cause damage at this late time point we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells Our results clearly show that especially within liver CD8+ T cells we can detect such epitopes for at least months indicating that the capsid of AAV8 degrades extremely slowly thus putting AAV8 vector recipients at prolonged risk of immune-mediated elimination of vector transduced cells 257 Characterization of Naturally-Occurring Humoral Immunity to AAV in Sheep Joseph Tellez,1 Kim Van Vliet,2 Yu-shan Tseng,2 Jonathan Finn,3 Nick Tschernia,1 Graỗa Almeida-Porada,4 Valder Arruda,3 Mavis Agbandje-McKenna,2 Christopher Porada.4 Univ of Nevada, Reno; 2Univ of Florida, Gainesville; 3UPenn, Children’s Hospital, Philadelphia; 4Wake Forest Inst for Regen Med., Winston-Salem AAV vectors have received much attention for clinical gene therapy (GT), since they transduce mitotic and quiescent cells and mediate long-term transgene expression Unfortunately, many of the serotypes of AAV commonly employed in GT procedures ubiquitously infect Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy humans, generating pre-existing immunity against the AAV capsid that precludes efficient transduction of the desired target tissue following intravascular administration The absence of similar preexisting immunity in many commonly employed preclinical animals, such as mouse and dog, makes it difficult to predict the likelihood of clinical success of approaches developed in these models Sheep have been used for decades as a clinically predictive model system to study a broad range of diseases Moreover, we have re-established a line of sheep with severe hemophilia A (HA) with a null mutation in the FVIII gene Given the major focus on the development and use of AAV vectors for GT of the hemophilias, we performed the present studies to determine whether sheep possess antibodies to AAV, and to characterize the nature of this pre-existing immunity ELISAs performed on a panel of healthy Merino-Rambouillet sheep revealed naturally occurring antibodies to AAV1, 2, 5, 6, 8, and 9, with the titers against the different serotypes varying from sheep-to-sheep The antibodies present in at least some of these animals were neutralizing against AAV2, 8, and Since our results demonstrated the presence of antibodies recognizing multiple AAV serotypes, epitope mapping was performed to define the capsid peptides recognized by these antibodies A library of 15mer peptides overlapping by 10 amino acids was used to cover the entire VP1 sequence from the capsids of AAV2, 5, 8, and These analyses revealed that, although many of the recognized epitopes were common to the capsids of all AAV serotypes tested, each animal also harbored antibodies to epitopes that were unique to each specific capsid Using the AAV capsid structures determined by X-ray crystallography, these peptides were mapped on the capsid Although some of these antibodies recognized epitopes on the surface of the capsid, surprisingly, many of the epitopes were located within regions of the capsid that are internal or buried in the capsid structure These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid, as well as capsid epitopes that are presented following proteolysis during the course of infection These antibodies recognize many of the serotypes of AAV commonly employed as GT vectors The close parallels between human and sheep physiology, coupled with our re-establishment of sheep with severe HA and the presence of these antibodies, suggest that sheep may represent an ideal large animal model in which to study GT for HA, and other diseases, in the context of pre-existing immunity to AAV, and allow the development of novel strategies for circumventing this clinically important immunologic barrier 258 Innate Immune Response Affects AAVMediated Hepatic Trangene Expression Irene Gil,1 Marianna Di Scala,1 Lucia Vanrell,1 Africa Vales,1 Cristina Olague,1 Jesus Prieto,1,2 Kathy High,3 Federico Mingozzi,3 Gloria Gonzalez-Aseguinolaza.1 Gene Therapy and Hepatology, CIMA, Pamplona, Spain; Ciberehd, Pamplona, Spain; 3University of Pennsylvania School of Medicine, Philadelphia This study evaluates the effect of IL-12 over AAV serotype mediated gene expression and over the induction of specific immune responses against AAV8 capsid proteins in mice Our results indicate that IL-12 expression downregulates AAV8 mediated gene expression through the induction of IFN-γ The effect of IFN-γ could be detected shortly after viral injection suggesting the involvement of the innate immune system In fact the results indicate a major role for NK cells activation and a partial role of T cells on gene expression inhibition The downregulation of gene expression occurs with no variation in the number of viral genomes indicating an epigenetic mechanism of gene silencing at early time points However, sustained expression of low IL-12 levels induce hepatocyte death, loss of viral genomes, and concomitant decrease of gene expression at a later stage Splenocytes were obtained from mice treated with an AAV8 that express IL-12 in an inducible manner produced IFN-γ after stimulation AAV8 capsid S101 OLIGONUCLEOTIDE & RNAI THERAPEUTICS I derived peptides AAV expressing IL-12 induces a weaker immune response against AAV capsid than that induced by an adenovirus expressing capsid protein and different antigenic determinants are presented Furthermore, the immune response induced by AAV8 expressing IL-12 failed to eliminate AAV8 transduced hepatocytes In conclusion, IFN-γ secretion induced by IL-12 downregulates but not eliminates AAV mediated gene expression Sustained, cytokineinduced activation of liver lymphocytes eliminates AAV transduced hepatocytes in a non-specific manner resulting in the elimination of transgene expression, a situation that resembles a chronic infection 259 Prevalence and Correlation of Neutralizing Antibodies Against AAV2, 7, 8, and RH32.33 in the Human Sera from Southern China Jianxi Lu,1 Mingzhu Mei,1 Lan Sun,4 Qiang Wang,3 You Lu,4 Gang Li,1 Guangping Gao.2 Vaccine Research Institute, The 3rd Affiliated Hospital of Sun Yatsen University, Guangzhou, Guangdong, China; 2Microbiology & Physiological Systems, University of Massachusetts Medical School, Worcester, MA; 3Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perlman School of Medicine, University of Pennsylvania, Philadelphia, PA; 4Thoracic Cancer, Cancer Center, West China School of Clinical Medicine, Sichuan University, Chengdu, China Recent progress in clinical gene therapy of hemophilia B highlighted the potential of recombinant aedno-associated viruses (rAAVs) in human applications However, discovery of a large family of naturally existing primate AAVs suggested that the pre-existing immunity against different AAVs resulted from natural infections could become one of the potential obstacles to rAAV-mediated gene transfer to patients Therefore, assessment of prevalence of pre-existing neutralizing antibodies (PENABs) against different AAV serotypes that hold promise for therapeutic gene transfer as well as possible cross-reactivity between different PENABs and AAV serotypes are becoming a critically important step towards further clinical development of rAAV gene therapy To this end, we anonymously collected serum samples from 113 healthy subjects (24 men, 89 women; age 18-81) from Guangzhou, South China after local IBC approval We initially screened the samples for Nabs against AAV2, 7, 8, and rh32.33 by transduction inhibitionbased in vitro Nab assay using LacZ reporter gene and quantitative chemiluminescent measurement as described by Wang et al, 2010 Our data indicated that the Nab prevalence (Nab titer ≥1:20) for AAV2, 7, 8, and rh32.33 is 80.7%,78.9%,74.6%,68.4% and 13.2% in the study group, respectively There is no difference between men and women in the prevalence of Nabs to all the AAV serotypes tested Of note, for AAV2, 7, 8, and 9, all serum samples from the subjects at the ages ranging from 40 to 59 years old (n=16) had Nab titers ≥1:5 (among the 16, the number of subjects who have a Nab titer≥ 1:20 AAV2: 15, AAV7: 15, AAV8: 14, AAV9: 14), suggesting that AAV sero-positivity in this age group is more prevalent Among the 113 subjects, there is a relatively strong correlation of simultaneous detection of NAbs to AAV8 and AAV9 (R square = 0.82) To verify our data independently, based on the limited availability of serum samples, we selected a group of samples for a different in vitro Nab assay that used EGFP as a reporter gene and microscopic counts of EGFP positive cells to assess transduction inhibition The data generated by this assay are in general agreement with those from LacZ transduction-based assay In addition, several samples with known in vitro NAB titers to AAV2 were verified in vivo NAB assays as described by Wang et al, 2010 Taken together, our study should be informative and helpful to the design of future clinical trials of rAAV gene therapy S102 260 Epirubicin Potentiates rAAV2/5-Mediated TRAIL Expression in Fibroblast-Like Synoviocytes and Augments the Anti-Arthritis Effects of rAAV2/5-TRAIL Juan Shi,1 Xin You,2 Dexian Zheng.1 Biochem & Mol Biol, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China; 2Rheumatol & Clin Immunol, PUMC Hospital, Beijing, China Objective Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor in fibroblast-like synoviocytes (FLSs) This study was undertaken to investigate the functions of recombinant adeno-associated virus (rAAV2/5)TRAIL in FLSs and arthritic mice Methods Primary human FLSs were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin (EPB) The transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry The disease-modulating activity of rAAV2/5-TRAIL plus EPB was investigated in mice with collagen-induced arthritis (CIA) Results Subtoxic doses of EPB potentiated rAAV2/5-mediated TRAIL expression in FLSs and simultaneously enhanced the sensitivity of FLSs to TRAIL EPB upregulated DR4 and DR5 expression and downregulated FLIP expression, thereby enhancing the activation of procaspase-3, procaspase-8 and procaspase-9 An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and EPB chemotherapy provided augmented anti-arthritis effects in a CIA mouse model The intra-articular injection of rAAV2/5-TRAIL combined with EPB treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals Histological evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the synthetic treatment mice A viral genome copy number assay indicated that EPB dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution Conclusion These results suggest that EPB enhances the anti-arthritis effect of rAAV2/5-TRAIL, and combination treatment might be an important therapeutic alternative with practical significance for rheumatoid arthritis Oligonucleotide & RNAi Therapeutics I 261 Nanoparticles as Delivery Systems for Antisense Oligoribonucleotides Aimed at Exon Skipping: Results in the Dystrophinopathic mdx Animal Model by Intraperitoneal Ad Oral Administration Routes Alessandra Ferlini,1 Paola Rimessi,1 Elena Bassi,1 Maria Sofia Falzarano,1 Marina Fabris,1 Patrizia Sabatelli,2 Nadir M Maraldi,2 Katia Sparnacci,3 Michele Laus,3 Paolo Bonaldo,4 Paola Braghetta.4 Medical Genetics, Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy; 2CNR IOR Bologna, University and CNR Bologna, Italy; 3Environmental and Life Sciences INSTM, University of Eastern Piedmont, Alessandria, Italy; 4Histology, Microbiology, and Medical Biotechnology, Univerisity of Padua, Italy We have tested different types of polymeric cationic core-shell nanoparticles (NPs) for delivering 2-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs), in mdx mice Both T1 and ZM2 NP bind and convey AONs: intraperitoneal (IP) injections of low doses (52.5mg/kg) of NP-AON complex restored dystrophin protein synthesis in skeletal and cardiac muscles, allowing protein localization in up to 40% of muscle fibers with skipping level up to 20% A group Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... the immune response induced by AAV8 expressing IL-12 failed to eliminate AAV8 transduced hepatocytes In conclusion, IFN-γ secretion induced by IL-12 downregulates but not eliminates AAV mediated. .. ≥1:5 (among the 16, the number of subjects who have a Nab titer≥ 1:20 AAV2 : 15, AAV7 : 15, AAV8 : 14, AAV9 : 14), suggesting that AAV sero-positivity in this age group is more prevalent Among the 113...OLIGONUCLEOTIDE & RNAI THERAPEUTICS I derived peptides AAV expressing IL-12 induces a weaker immune response against AAV capsid than that induced by an adenovirus expressing capsid protein