- Email: [email protected]
731. Selection of Optimal AAV Serotypes as Clinical Candidates for AAV-Mediated, Liver-Directed Gene Therapy
PRECLINICAL AND CLINICAL APPLICATIONS OF AAV manipulation of epidermal stem cells would allow this established and frequently used approach to be applied to the many genetic diseases that affect the skin, and a gene targeting strategy would avoid problems associated with gene regulation, minimize off-target effects of gene insertion, and allow the treatment of diseases with both dominant and recessive patterns of inheritance. Here we show for the first time that primary human epidermal stem cells cultured from skin biopsies can be efficiently and precisely modified by AAV-vector mediated gene-targeting through recombination of the vector with homologous chromosomal sequence. We designed gene-targeting vectors that allow sorting or growth selection of recombinant clones using a promoter trap strategy. Gene targeting frequencies occur in the range of 0.5% of total cells and approach 100% of sorted cell populations. We show that 10 -20% of the targeted clones exhibit properties consistent with an epidermal stem cell phenotype. As such these fully characterized clonal isolates can be grown long term in culture, stratify to form the various layers of the epidermis, and form an epidermis that functions normally when transplanted to athymic mice. We’ve begun applying this strategy to patient cells containing keratin gene mutations that cause a dominantly inherited, sometimes lethal form of Epidermolyis Bullosa Simplex that presently has no available treatment. These studies outline a feasible therapeutic approach to a genetic disease using a gene targeting strategy, and have implications for the design of gene-targeting approaches to diseases affecting other tissues.
731. Selection of Optimal AAV Serotypes as Clinical Candidates for AAV-Mediated, LiverDirected Gene Therapy
Lili Wang,1 Huan Wang,1,3 Peter Bell,1 Robert J. McCarter,2 Roberto Calcedo,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Genetic Medicine Research, Children’s National Medical Center, Children’s Research Institute, Washington, DC; 3Vaccine Research Institute, Third Affiliated Hospital of Sun Yat-sen University, Guanzhou, China. Adeno-associated virus (AAV)-based vectors are promising vectors for gene therapy of genetic diseases. Besides the vector genome, the capsid plays important roles not only in gene transfer efficiency, but also in host immune response to the capsid and transgene product, toxicity, and vector biodistribution to extrahepatic tissues. The prevalence of neutralizing antibodies in human would also impact the outcome of gene therapy. Thus we developed a strategy to fully characterize the impact of capsid variation on AAV vector biology and identify optimal AAV serotype for liver-directed gene therapy. Such serotypes could serve as clinical candidates (AAVcc) for AAV-mediated, liver-directed gene therapy for genetic disease, such as ornithine transcarbamylase deficiency (OTCD). Starting from a library of 120 novel primate AAV genotypes that were previously isolated from non-human primate or human tissues in our laboratory, we selected 30 AAV capsids that represent the total biodiversity of the expanded family of AAVs. A pilot study was carried out to evaluate the gene transfer efficiency in mouse following intravenous injection of vectors carrying a secreted reporter gene (alpha-1 antitrypsin, A1AT). Based on the pilot study, a subset of six were selected for a more detailed and systematic analysis as potential vectors for liverdirected human clinical trials. The six candidates are AAV8, 6.2, 7, rh64R1, hu37, rh8, and rh32.33, spanning three clades (E, D, and A) as well as two isolates that fall outside the known clades. Vectors expressing GFP from a CMV enhanced chicken β-actin (CB) promoter were packaged with each candidate capsid and injected intravenously into mice. Objective criteria for evaluating and selecting the capsid for AAVcc as well as an algorithm for scoring were established. They include gene transfer efficiency and stability, toxicity, transgene and Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
capsid T cell activation, pre-existing immunity and biodistribution. Statistical analyses were performed on the data and detailed results will be presented. The top candidates that emerged from the mouse study which include AAVs8, rh8, and hu37, are currently being evaluated in nonhuman primates to yield the final AAVcc.
732. Micro RNA Regulated Tissue Specific Transduction by rAAV Vector
Jun Xie, Qin Su, Qing Xie, Chris Mueller, Phillip Zamore, Guangping Gao. Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA. Micro RNAs (miRNAs) are small RNA species with approximately 18-24 nucleotides in size. They regulate gene expression by post-transcriptional silencing through pairing to the partially complementary sites located in the 3’ UTRs of transcripts of the target genes. Regulation of transgene expression in a cell type specific manner by endogenous miRNA was first introduced into lentiviral vectors to suppress transgene expression in hematopoietic cells and abolish transgene immunity. Many of the novel AAV vectors that were discovered recently demonstrate strong hepatic tissue tropism while exhibiting unique trandsduction profiles in other tissues. Depending on the applications, intended target tissue and gene of interest to be delivered, inadvertent transduction in liver and other tissues may lead to untoward outcomes which should be avoided. Previously, we have shown that systemic delivery of AAV9 vector can efficiently transduce liver, heart, pancreas, skeletal and diaphragm muscles with an expression cassette driven by an ubiquitous promoter such as chicken β-actin prompter (CB). We also demonstrated that tissue specific expressional targeting in myocardium and islet could be accomplished by introducing tissue/cell type specific promoter into the expression cassette, although levels of transgene expression in the target tissues were somewhat compromised and some off-target expression in liver was detected in the high dose regimen (1012 GC/ mouse). In the present study, we explored use of endogenous miRNAs to regulate tissue specific transduction profiles of rAAV in mouse models. In a proof-of-concept experiment, we tested the potency of miRNA mediated transgene silencing in mouse liver by introducing the binding sites of mi122, the most abundant micro RNA species in the liver, to the TBG promoter (the strongest liver specific promoter in our lab) driven nLacZ expression cassette. The rAAV vectors with and without mi122 binding sites were packaged with AAV9 capsid and delivered at high dose (1x1012 GC/mouse) to mouse liver via IV injections. X-gal histochemical staining of the liver sections at four weeks after gene transfer revealed almost complete suppression of nLacZ transduction in the liver of animals injected with the construct containing mi122 binding sites, suggesting endogenous mi122 RNA mediated transgene silencing in mouse liver is highly effective. The same vector design was then applied to the CBnLacZ construct for a similar comparison of expressional targeting for mouse liver, heart and some other tissues. The mi122 binding site bearing construct led to a 100% suppression of liver transduction without any impact on the heart and pancreas transduction. Other manipulations in endogenous microRNA mediated regulation of transgene expression have also been investigated. These include the dose response of gene silencing to the numbers of miRNA binding sites, simultaneous transgene suppression in multiple tissues and cell types through different miRNA species, and rescue of transgene expression from miRNA mediated gene silencing by miRNA sponge, etc.
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731. Selection of Optimal AAV Serotypes as Clinical Candidates for AAV-Mediated, LiverDirected Gene Therapy
Lili Wang,1 Huan Wang,1,3 Peter Bell,1 Robert J. McCarter,2 Roberto Calcedo,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Genetic Medicine Research, Children’s National Medical Center, Children’s Research Institute, Washington, DC; 3Vaccine Research Institute, Third Affiliated Hospital of Sun Yat-sen University, Guanzhou, China. Adeno-associated virus (AAV)-based vectors are promising vectors for gene therapy of genetic diseases. Besides the vector genome, the capsid plays important roles not only in gene transfer efficiency, but also in host immune response to the capsid and transgene product, toxicity, and vector biodistribution to extrahepatic tissues. The prevalence of neutralizing antibodies in human would also impact the outcome of gene therapy. Thus we developed a strategy to fully characterize the impact of capsid variation on AAV vector biology and identify optimal AAV serotype for liver-directed gene therapy. Such serotypes could serve as clinical candidates (AAVcc) for AAV-mediated, liver-directed gene therapy for genetic disease, such as ornithine transcarbamylase deficiency (OTCD). Starting from a library of 120 novel primate AAV genotypes that were previously isolated from non-human primate or human tissues in our laboratory, we selected 30 AAV capsids that represent the total biodiversity of the expanded family of AAVs. A pilot study was carried out to evaluate the gene transfer efficiency in mouse following intravenous injection of vectors carrying a secreted reporter gene (alpha-1 antitrypsin, A1AT). Based on the pilot study, a subset of six were selected for a more detailed and systematic analysis as potential vectors for liverdirected human clinical trials. The six candidates are AAV8, 6.2, 7, rh64R1, hu37, rh8, and rh32.33, spanning three clades (E, D, and A) as well as two isolates that fall outside the known clades. Vectors expressing GFP from a CMV enhanced chicken β-actin (CB) promoter were packaged with each candidate capsid and injected intravenously into mice. Objective criteria for evaluating and selecting the capsid for AAVcc as well as an algorithm for scoring were established. They include gene transfer efficiency and stability, toxicity, transgene and Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
capsid T cell activation, pre-existing immunity and biodistribution. Statistical analyses were performed on the data and detailed results will be presented. The top candidates that emerged from the mouse study which include AAVs8, rh8, and hu37, are currently being evaluated in nonhuman primates to yield the final AAVcc.
732. Micro RNA Regulated Tissue Specific Transduction by rAAV Vector
Jun Xie, Qin Su, Qing Xie, Chris Mueller, Phillip Zamore, Guangping Gao. Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA. Micro RNAs (miRNAs) are small RNA species with approximately 18-24 nucleotides in size. They regulate gene expression by post-transcriptional silencing through pairing to the partially complementary sites located in the 3’ UTRs of transcripts of the target genes. Regulation of transgene expression in a cell type specific manner by endogenous miRNA was first introduced into lentiviral vectors to suppress transgene expression in hematopoietic cells and abolish transgene immunity. Many of the novel AAV vectors that were discovered recently demonstrate strong hepatic tissue tropism while exhibiting unique trandsduction profiles in other tissues. Depending on the applications, intended target tissue and gene of interest to be delivered, inadvertent transduction in liver and other tissues may lead to untoward outcomes which should be avoided. Previously, we have shown that systemic delivery of AAV9 vector can efficiently transduce liver, heart, pancreas, skeletal and diaphragm muscles with an expression cassette driven by an ubiquitous promoter such as chicken β-actin prompter (CB). We also demonstrated that tissue specific expressional targeting in myocardium and islet could be accomplished by introducing tissue/cell type specific promoter into the expression cassette, although levels of transgene expression in the target tissues were somewhat compromised and some off-target expression in liver was detected in the high dose regimen (1012 GC/ mouse). In the present study, we explored use of endogenous miRNAs to regulate tissue specific transduction profiles of rAAV in mouse models. In a proof-of-concept experiment, we tested the potency of miRNA mediated transgene silencing in mouse liver by introducing the binding sites of mi122, the most abundant micro RNA species in the liver, to the TBG promoter (the strongest liver specific promoter in our lab) driven nLacZ expression cassette. The rAAV vectors with and without mi122 binding sites were packaged with AAV9 capsid and delivered at high dose (1x1012 GC/mouse) to mouse liver via IV injections. X-gal histochemical staining of the liver sections at four weeks after gene transfer revealed almost complete suppression of nLacZ transduction in the liver of animals injected with the construct containing mi122 binding sites, suggesting endogenous mi122 RNA mediated transgene silencing in mouse liver is highly effective. The same vector design was then applied to the CBnLacZ construct for a similar comparison of expressional targeting for mouse liver, heart and some other tissues. The mi122 binding site bearing construct led to a 100% suppression of liver transduction without any impact on the heart and pancreas transduction. Other manipulations in endogenous microRNA mediated regulation of transgene expression have also been investigated. These include the dose response of gene silencing to the numbers of miRNA binding sites, simultaneous transgene suppression in multiple tissues and cell types through different miRNA species, and rescue of transgene expression from miRNA mediated gene silencing by miRNA sponge, etc.
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