- Email: [email protected]
10. Targeted Transgene Integration in Epidermal Stem Cells by Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors
DNA VECTOROLOGY & GENE TARGETING library with specic amino acid positions subjected to saturation mutagenesis, a shufed AAV Cap library (generated from AAV1, 2, 4, 5, 6, 8 & 9), and a library in which the key surface loops of AAV Cap had been substituted between serotypes. These libraries were selected against pooled human serum samples shown to contain AAV2 NAbs, intravenous immunoglobulin (IVIg), or serum samples from individuals excluded from a hemophilia B clinical trial due to high titers of anti-AAV2 NAbs. An initial selection against pooled human serum samples yielded 2 clones (1.45 and γ4.3) that showed the ability to evade neutralization by pooled human serum samples, and a 1.23 and 3.1 fold enhanced resistance to neutralization by IVIg respectively. Subsequently, the γ4.3 variant was used as the basis for the construction of a library in which 6 amino acid residues were subjected to saturation mutagenesis. The resulting library – as well as the shufed and loop substitution libraries – were selected using IVIg and serum from patients excluded from the hemophilia trial. After 2 rounds of evolution (comprising of genetic diversication followed by 3 selection steps against serial dilutions of IVIg or serum samples), library pools emerged with improved in vitro transduction in the presence of IVIg or individual human serum samples. We are currently investigating the composition of individual clones from within these viral pools rescued from the capsid libraries and their transduction in the presence of IVIg or individual patient sera. The identication of clones resistant to individual patient sera or IVIg should enable both the treatment of patients who would otherwise not be eligible for gene therapy, as well as repeated dosing of patients who have developed NAbs against AAV.
DNA Vectorology & Gene Targeting 9. A Simplied Approach for Robust Minicircle DNA Vector Production Using a New Genetically-Modied E. Coli Producer Strain ZYCY10P3S2T
Zhi-Ying Chen,1 Cheng-Yi He,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA. Minicircle DNA vectors are composed of a circular expression cassette devoid of all plasmid backbone DNA. With many features giving them superiority to standard plasmids, minicircles have the potential to become a universal DNA vector system for biomedical research and human gene therapy. The advantages of minicircle DNA vectors include reduction of CpG motifs and reduced-associated toxicity when delivered in lipid-DNA complexes, smaller size facilitating efcient transfection, absence of plasmid backbone DNAs and related transgene expression silencing ensuring high levels of transgene expression in quiescent cells in vitro and in vivo. The minicircles are created in bacterial cells via an intramolecular recombination to eliminate the plasmid DNA backbone sequences. In general the lower yields and increased time for preparation of minicircle DNA vectors compared to routine plasmids have limited their wide appeal to molecular biologists. In addition, the residual contamination of the parental plasmids encoding the minicirclemaking recombination enzymes has limited their appeal for some clinical gene therapeutics. We have now overcome all of these limitations by removing the recombination genes from the parental plasmid and genetically engineering an E. coli to express the minicircle-making enzymes. In the genome of the new bacterial strain ZYCY10P3S2T, we have integrated (1) ten copies of the phage ØC31 gene, which encodes the recombinase that mediates the formation of minicircle via intramolecular recombination, (2) three copies of the ISce1 gene, which encodes the restriction enzyme that destroys the plasmid backbone circle, and (3) araE and LacY mutant, two genes that S4
constitutively express transporters of L-arabinose, which induces the expression of the ØC31 and ISce1 genes under the control of the araC.BAD system.
In concert with this new bacterial producer strain, we have devised a protocol, which can reproducibly produce more than 2 mg/liter of culture of a 2-kb minicircle DNA vector essentially devoid of contaminating parental plasmid. The new protocol is similar to preparation of a routine overnight bacterial culture with only minor modications, including addition of 0.01% of L-arabinose. Thus, our new system greatly simplies minicircle DNA vector production. This, in combination with decreased parental DNA contamination, reduced cost, and a procedure amenable to GMP production, makes minicircles attractive DNA vectors for all non-viral DNA gene transfer studies including routine DNA transfection and human gene therapy. We predict that minicircle DNAs will replace plasmids for any type of laboratory-based gene expression studies in the future.
10. Targeted Transgene Integration in Epidermal Stem Cells by Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors
Giulietta Maruggi,1 Angelo Lombardo,2 Francesca Miselli,1 Pietro Genovese,2 Michael C. Holmes,3 Philip D. Gregory,3 Alessandra Recchia,1 Fulvio Mavilio,1 Luigi Naldini.2 1 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 2San Raffaele-Telethon Institute for Gene Therapy, Vita-Salute San Raffaele University, Milan, Italy; 3 Sangamo Biosciences Inc., Richmond, CA. Transplantation of autologous, genetically corrected epidermal stem cells has been successfully used to treat junctional epidermolysis bullosa, a genetic skin adhesion disorder. The therapeutic value of this approach has been already tested in the clinic, although safety concerns exist about the use of gamma-retroviral vectors in human stem cells. Thus, the development of alternative approaches is needed. We previously developed a gene-targeting platform based on Zinc Finger Nucleases (ZFN) and Integrase-Defective Lentiviral Vectors (IDLV) to insert transgenes into pre-determined sites of the genome by means of Homologous Recombination (HR). Here, we assessed the feasibility of this approach in human keratinocytes by using two different sets of ZFN, one designed to target the CCR5 gene and the other to target the AAVS1 locus on chromosome 19. To identify which of the two platforms performs better in human keratinocytes, we rst compared their targeting efciency in the keratinocyte cell line HaCat by co-introducing the ZFN with homolgous donor templates that drive insertion of a GFP-expression cassette at the site of cleavage. By IDLV-mediated delivery of the ZFN pair and cognate donor constructs for each target site, we achieved high levels of targeted insertion (up to 20% of cells) of the GFP cassette into the intended locus. We then tested the CCR5 targeting platform in human primary keratinocytes and found up to 4% GFP-positive cells with evidence of site-specic transgene insertion. The efciency of the AAVS1 platform is currently being tested in vitro and in vivo, upon xenotransplantation of human skin implants on immunodecient mice. Overall, these experiments provide a proof-of-principle study on the feasibility of ZFN/IDLV driven targeted integration in keratinocytes and epidermal stem cells.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
DNA Vectorology & Gene Targeting 9. A Simplied Approach for Robust Minicircle DNA Vector Production Using a New Genetically-Modied E. Coli Producer Strain ZYCY10P3S2T
Zhi-Ying Chen,1 Cheng-Yi He,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA. Minicircle DNA vectors are composed of a circular expression cassette devoid of all plasmid backbone DNA. With many features giving them superiority to standard plasmids, minicircles have the potential to become a universal DNA vector system for biomedical research and human gene therapy. The advantages of minicircle DNA vectors include reduction of CpG motifs and reduced-associated toxicity when delivered in lipid-DNA complexes, smaller size facilitating efcient transfection, absence of plasmid backbone DNAs and related transgene expression silencing ensuring high levels of transgene expression in quiescent cells in vitro and in vivo. The minicircles are created in bacterial cells via an intramolecular recombination to eliminate the plasmid DNA backbone sequences. In general the lower yields and increased time for preparation of minicircle DNA vectors compared to routine plasmids have limited their wide appeal to molecular biologists. In addition, the residual contamination of the parental plasmids encoding the minicirclemaking recombination enzymes has limited their appeal for some clinical gene therapeutics. We have now overcome all of these limitations by removing the recombination genes from the parental plasmid and genetically engineering an E. coli to express the minicircle-making enzymes. In the genome of the new bacterial strain ZYCY10P3S2T, we have integrated (1) ten copies of the phage ØC31 gene, which encodes the recombinase that mediates the formation of minicircle via intramolecular recombination, (2) three copies of the ISce1 gene, which encodes the restriction enzyme that destroys the plasmid backbone circle, and (3) araE and LacY mutant, two genes that S4
constitutively express transporters of L-arabinose, which induces the expression of the ØC31 and ISce1 genes under the control of the araC.BAD system.
In concert with this new bacterial producer strain, we have devised a protocol, which can reproducibly produce more than 2 mg/liter of culture of a 2-kb minicircle DNA vector essentially devoid of contaminating parental plasmid. The new protocol is similar to preparation of a routine overnight bacterial culture with only minor modications, including addition of 0.01% of L-arabinose. Thus, our new system greatly simplies minicircle DNA vector production. This, in combination with decreased parental DNA contamination, reduced cost, and a procedure amenable to GMP production, makes minicircles attractive DNA vectors for all non-viral DNA gene transfer studies including routine DNA transfection and human gene therapy. We predict that minicircle DNAs will replace plasmids for any type of laboratory-based gene expression studies in the future.
10. Targeted Transgene Integration in Epidermal Stem Cells by Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors
Giulietta Maruggi,1 Angelo Lombardo,2 Francesca Miselli,1 Pietro Genovese,2 Michael C. Holmes,3 Philip D. Gregory,3 Alessandra Recchia,1 Fulvio Mavilio,1 Luigi Naldini.2 1 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 2San Raffaele-Telethon Institute for Gene Therapy, Vita-Salute San Raffaele University, Milan, Italy; 3 Sangamo Biosciences Inc., Richmond, CA. Transplantation of autologous, genetically corrected epidermal stem cells has been successfully used to treat junctional epidermolysis bullosa, a genetic skin adhesion disorder. The therapeutic value of this approach has been already tested in the clinic, although safety concerns exist about the use of gamma-retroviral vectors in human stem cells. Thus, the development of alternative approaches is needed. We previously developed a gene-targeting platform based on Zinc Finger Nucleases (ZFN) and Integrase-Defective Lentiviral Vectors (IDLV) to insert transgenes into pre-determined sites of the genome by means of Homologous Recombination (HR). Here, we assessed the feasibility of this approach in human keratinocytes by using two different sets of ZFN, one designed to target the CCR5 gene and the other to target the AAVS1 locus on chromosome 19. To identify which of the two platforms performs better in human keratinocytes, we rst compared their targeting efciency in the keratinocyte cell line HaCat by co-introducing the ZFN with homolgous donor templates that drive insertion of a GFP-expression cassette at the site of cleavage. By IDLV-mediated delivery of the ZFN pair and cognate donor constructs for each target site, we achieved high levels of targeted insertion (up to 20% of cells) of the GFP cassette into the intended locus. We then tested the CCR5 targeting platform in human primary keratinocytes and found up to 4% GFP-positive cells with evidence of site-specic transgene insertion. The efciency of the AAVS1 platform is currently being tested in vitro and in vivo, upon xenotransplantation of human skin implants on immunodecient mice. Overall, these experiments provide a proof-of-principle study on the feasibility of ZFN/IDLV driven targeted integration in keratinocytes and epidermal stem cells.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy