94 Genetic Correction of Induced Pluripotent Stem Cells from a Wiskott Aldrich Syndrome Patient Normalizes the Immune Defects Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Americ[.]
GENE TARGETING AND GENE CORRECTION I 60% of the cases caused by a mutation in the TCIRG1 gene, which severely affects osteoclasts resorbing activity The resulting increased bone density and most of the other symptoms are recapitulated by oc/oc mice Hematopoietic stem cell (HSC) transplantation is the unique possible treatment, however the chance of cure is limited by the need for a matched donor Therefore, the generation of corrected autologous HSCs may be a novel approach to therapy The aim of our project is to reprogram murine wt and oc/oc fibroblasts into iPSc, to genetically correct the Tcirg1 mutation by homologous recombination, and to generate hematopoietic stem and progenitor cells able to give rise to functional osteoclasts in order to revert the oc/oc phenotype To generate iPSc we employed a third generation polycistronic lentiviral vector carrying the reprogramming genes Oct4, Sox2 and Klf4, subsequently excisable by the Cre recombinase After reprogramming, iPS clones with low vector copy number and normal numerical distribution of chromosomes were treated with Cre and sub-cloned Obtained iPSc showed normal karyotype and their pluripotency was tested by teratoma formation assay, in vitro embryonic germ layers differentiation, and expression of stemness markers by immunocytochemistry and RT-PCR Importantly, iPSc were successfully derived from oc/oc fibroblasts, and then corrected through homologous recombination upon transfection with a BAC containing the wt gene iPSc were guided to differentiate towards the hematopoietic lineage in a 12 days in vitro protocol in the presence of specific cytokines, without additional transgene over-expression All tested iPSc successfully differentiated into hematopoietic cells belonging to different lineages, including Lineage-cKit+Sca1+ phenotypically immature cells Multicolour FACS analysis performed over time revealed differentiation kinetics resembling physiologic fetal hematopoiesis, with CD41+ cells gradually giving rise to CD45+ cells, which comprised mature myeloid cells, as well as high proliferative potential colony-forming cells, including mixed-type colonies Most importantly, we obtained differentiation towards osteoclasts, the relevant cells in our model, which were functional as demonstrated by the dentine resorption assay In vivo transplant into oc/oc recipients is ongoing In conclusion, with our studies we will provide a proof of principle for the future clinical use of a new tool to treat osteopetrosis and potentially other genetic blood disorders vector Chimeric non-intergrating lentivirus (NILV) carrying CCR5ZFN was obtained by co-transfecting 293 T cells with intergrasedefective packaging plasmid and HIV (or VSV-G) envelope plasmid After transductions of different cell lines and primary T cells (activated or resting T cells), genomic DNAs were extracted for characterizing CCR5 disruption by T7 endonuclease assay Thereafter, NILV transduced primary T cells were challenged with HIVBaL to test protection from HIV infection in vitro In addition, CCR5-ZFN NILV transduced resting T cells from healthy donor and HIV patients were adoptively transferred into NOD-scid IL2rγcnull mice (Hu/PBL model) and tested for protection from HIV The results show that NILV/CCR5 ZFN transduced cell lines, as well as activated or resting T cells could successfully disrupt CCR5 gene with various levels of efficiency, and that both activated and resting T cells transduced with CCR5-ZFNs lentiviruses showed resistance to HIV infection in vitro Furthermore, compared to controls, Hu-PBL mice reconstituted with CCR5-ZFN modified resting CD4+ T cells from a healthy donor could control viral replication and maintain their CD4+ T cells count after HIV challenge Likewise, endogenous virus replication was suppressed in NOD-scid IL2rγcnull mice reconstituted with CCR5-ZFN transduced resting T cells from one treatment naïve and two ART-treated HIV seropositive patients Conclusions: Non-intergrating lentivirus provide a useful strategy for delivery of zinc finger nucleases for editing CCR5 and other therapeutically feasible host genes, and HIV envelope pseudotyping of lentivirus provides a feasible strategy for targeted HIV gene therapy to T cells, including resting cells 94 Genetic Correction of Induced Pluripotent Stem Cells from a Wiskott-Aldrich Syndrome Patient Normalizes the Immune Defects 93 Targeting Resting T Cells with NonIntegrating Lentivirus Encoding CCR5-ZFN Suppresses HIV-1 Infection In Vitro and in Humanized Mice Tamara J Laskowski,1 Yasmine Van Caeneghem,2 Rasoul Pourebrahim,1 Xuan Shirley Li,1 Wei Liao,1 Zita Garate,1,3 Ana Crane,1 Zhenya Ni,5 Jose Carlos Segovia,3 Michael Holmes,4 Dan Kaufman,5 Bart Vandekerckhove,2 Brian R Davis.1 Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center, Houston, TX; 2Laboratory for Experimental Immunology, Ghent University, Ghent, Belgium; Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain; 4Sangamo BioSciences, Richmond, CA; 5Dept of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN CCR5 disruption by Zinc finger nuclease (ZFN) is a promising gene therapy approach for generating HIV-resistant T cells However, successful clinical translation of the strategy will depend on the development of a safe and effective method for delivery of ZFNs to relevant cells As permanent disruption of the targeted gene can be achieved with a single ZFN treatment, non-integrating lentivirus is a potentially promising vector for transient ZFN expression to avoid toxicity and off target gene disruption Further, gene delivery to resting T cells would avoid extensive in vitro manipulation and perturbation of the T cell repertoire Here we used a non-intergrating lentivirus with HIV envelope to deliver CCR5-ZFN to human resting T cells from either healthy donor or HIV-1 patients, and investigated the disruption of CCR5 gene and protection of HIV infection in vitro and in humanized mice ZFN constructs targeting both strands of CCR5 locus were connected via a FMDV 2A sequence, and inserted to a lentiviral Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease characterized by thrombocytopenia, recurrent infections and increased autoimmunity This disease is caused by mutations in the WAS gene (WAS) which encodes for the WAS protein (WASp), exclusively expressed in hematopoietic cells and required for proper platelet production and lymphoid cell function Patients with WAS are treated with allogeneic stem cell transplantation or lentiviral hematopoietic stem cell gene therapy We investigated restoration of T- and NK-cell functionality following a virus-free zinc-finger nuclease (ZFN)-mediated genome editing strategy for correction of WAS We generated induced pluripotent stem cells (iPSC) from skin fibroblasts of a WAS patient carrying an insertional frame-shift mutation Subsequently, a WAS-2A-eGFP transgene was targeted at the endogenous chromosomal location by homology-directed repair using ZFN, thereby correcting the gene defect and creating a GFP reporter for WASp expression Hematopoietic progenitor cells were generated from WAS iPSC and gene-corrected iPSC (cWAS) in vitro via spin embryoid bodies Human embryonic stem cell lines WA01 and WA09 were used as controls GFP expression was pronounced in all CD43+ hematopoietic lineages including myeloid, monocytic, Guohua Yi,1 Jang Gi Choi,1 Preeti Bharaj,1 Sojan Abraham,1 Manjunath Swamy,1 Premlata Shankar.1 Center of Excellences-Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, El Paso, TX Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S35 GENE TARGETING AND GENE CORRECTION I lymphoid, erythroid and megakaryocytic lineages Hematopoietic precursors were further cultured on OP9-DL1 to generate T and NK cells NK cells were readily obtained from cWAS and WA01/WA09 progenitors, but to a far more limited extent from WAS progenitors WAS-derived NK cells were unable to generate interferon-g or tumor necrosis factor-α upon stimulation with K562 Cytokine production was restored in cWAS-derived NK cells Interestingly, in T cell generation, although WAS, cWAS, and WA01 lines were able to generate CD5+CD7+ T cell precursors, only low numbers of CD3+ TCRαb and TCRgD cells were obtained with WAS in comparison with WA01 Significantly, T cell generation was restored in cWAS Taken together these results indicate that targeted endogenous integration of the WAS gene in WAS-iPSC results in restoration of the lymphoid defect observed in WAS-iPSC Transplantation of gene-corrected iPSC-derived hematopoietic precursors may offer an alternative to lentiviral gene therapy which carries an inherent risk for insertional oncogenesis 95 Human Artificial Chromosomes or HACBased Gene Delivery Vectors for Biomedicine and Biotechnology Natalay Kouprina,1 Vladimir Larionov.1 National Cancer Institute, NIH, Bethesda Since their description in late nineties, Human Artificial Chromosomes (HACs) carrying a functional kinetochore were considered as a new promising system for gene delivery and expression with a potential to overcome many problems caused by the use of viral-based gene transfer systems Indeed, HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors Moreover, HACs allow therapeutic transgenes to be expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci Recently we constructed an advanced HAC vector carrying a unique gene-loading site and demonstrated its utility for delivery of full-length genes and correction of genetic deficiencies in human cells In addition, we developed a simple HAC-based assay for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells To summarize, the HAC-based vectors have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology 96 Targeted Gene Therapy in the Treatment of X-Linked Hyper-IgM Syndrome Caroline Y Kuo,1 Megan D Hoban,2 Alok V Joglekar,3 Donald B Kohn.4 Division of Allergy & Immunology, University of California, Los Angeles, Los Angeles, CA; 2Department of M.I.M.G., University of California, Los Angeles, Los Angeles, CA; 3Department of Biology, California Institute of Technology, Pasadena, CA; 4Departments of Pediatrics and M.I.M.G., University of California, Los Angeles, Los Angeles, CA X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency with absent IgG, IgA, IgE and normal/elevated IgM due to defects in the CD40 ligand (CD40L) gene CD40L is expressed on T-lymphocytes and is essential in the interaction between B and T cells that induces class switch recombination To date, hematopoietic stem cell transplantation (HSCT) is the only curative modality, but it carries significant risks and is generally not considered until there is uncontrolled disease, suggesting that alternative methods of treatment should be addressed Previous S36 studies using transplantation of CD40L-/- bone marrow corrected by retroviral-vector transfer of CD40L cDNA for XHIM in mouse models resulted in abnormal lymphoproliferation due to unregulated expression of the gene An alternative to ectopic viral-mediated gene addition may be the use of targeted gene repair Our central hypothesis is that custom TAL effector nucleases (TALENs), combined with the effective delivery of a homologous donor sequence containing CD40L cDNA, will allow for targeted integration and provide physiologic expression of the endogenous CD40L gene to safely provide longterm immune reconstitution Custom TALENs targeting the 5’ untranslated region of the CD40L gene were created and their function validated using a surveyor endonuclease assay Allelic disruption levels of up to 31% at the target CD40L locus in K562 cells were achieved K562 cells were then electroporated with the TALEN pair and a donor template homologous to the CD40L gene but containing a single base pair change that introduces a unique EcoRV restriction enzyme site PCR amplification of genomic DNA and digestion analysis demonstrated integration of the EcoRV restriction site, representing site-specific gene modification at CD40L In order to evaluate the capacity for targeted integration of a cassette at the cut site, K562 and Jurkat cells were electroporated with the TALEN pair and a donor molecule with a promoterless GFP reporter gene flanked by homology arms that parallel the cut site In/Out PCR using a forward primer within the GFP region and a reverse primer in the genomic DNA outside the donor molecule demonstrated proper integration of the cassette into K562 cells Targeted insertion of the GFP reporter into the CD40L locus should also provide a measure of physiologic induction of CD40L expression Thus, expression of the targeted GFP reporter was evaluated in Jurkat cells (which naturally express ˜60% CD40L), with levels of up to 10% detected by FACs and increasing to 22% upon activation with PHA In all, these results demonstrate that site-specific gene insertion using TALENs at CD40L is achievable and sets the stage for further work to demonstrate that physiologic expression of the endogenous CD40L gene, rather than constitutive expression from ectopically inserted CD40L transgenes, could provide a viable therapy for immune reconstitution in XHIM 97 Targeted Lentiviral Vector Integration at “Safe Harbor” Genomic loci Pingjuan Li,1 Michael Marino,1 Jakob Reiser.1 Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD Lentiviral vectors provide powerful tools for therapeutic gene delivery in vitro, ex vivo and in vivo because of their ability to mediate long-term transgene expression However, there are safety concerns since these vectors integrate nonspecifically into actively transcribed genes Our goal is to engineer lentiviral vectors so that their integration preference is shifted to genomic “safe harbor” sites The AAVS1 locus on chromosome 19 constitutes such a genomic “safe harbor” We generated integrase-defective lentiviral vectors (IDLVs) capable of integrating at the AAVS1 locus through homologous recombination (HR) We are now in the process of improving the efficiency of this approach using site-specific nucleases, including zinc finger nucleases (ZFNs) and zinc finer nickases (ZF nickases) ZF nickases have recently been reported to greatly reduce off-target effects in gene repair To assess the efficiency of HR at the endogenous AAVS1 locus in HEK 293 cells, we co-delivered, by IDLVs, the coding regions for the AAVS1 site-specific ZFNs and ZF nickases, and a donor vector bearing a puromycin resistance gene, flanked by homology arms corresponding to the AAVS1 site Puromycin-resistant cell clones were counted Our data indicate that the AAVS1 specific ZFN and ZF nickase can significantly increase the efficiency of HR at the AAVS1 Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... of California, Los Angeles, Los Angeles, CA; 3Department of Biology, California Institute of Technology, Pasadena, CA; 4Departments of Pediatrics and M.I.M.G., University of California, Los Angeles,... that the AAVS1 specific ZFN and ZF nickase can significantly increase the efficiency of HR at the AAVS1 Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of. .. Custom TALENs targeting the 5’ untranslated region of the CD40L gene were created and their function validated using a surveyor endonuclease assay Allelic disruption levels of up to 31% at the target