1. Objective 1.1 Foamy viral vectors (FV). Gene transfer of a therapeutic gene to HSPCs has been achieved utilizing replication-incompetent integrating retroviruses. Unequivocal clinical benefits were first obtained using -retroviral vectors (GV) in a series of HSPC gene therapy trials for immunodeficiency disorders. However, strong viral enhancers and integration site preferences for these vectors have caused cellular proto-oncogene activation and leukemia. Enhancerless lentiviral vectors (LV) containing weaker cellular promoters for transgene expression were designed to overcome the shortcomings of GV. Despite absence of genotoxic adverse events with these vectors, data are now emerging that inadequate transgene expression from weaker cellular promoters may limit successful correction in various disorders. Vectors based on FV represent an alternative approach for the genetic manipulation of HSPCs. Unlike GV and LV, the FV backbone includes insulator sequences that remarkably reduce genotoxic potential. Hence, we hypothesized that FV are ideal for situations where high transgene expression, necessitating strong promoters, is required for a therapeutic effect. Gene therapy for LAD-1 using FV. Over the past decade, extensive pre-clinical studies have been conducted testing FV in a naturally-occurring canine model of LAD-1. Dogs and patients with LAD-1 suffer from recurrent, life-threatening bacterial and fungal infections. The disease is caused by mutations in the leukocyte integrin CD18 subunit that prevent the formation and surface expression of CD18/CD11 heterodimeric adhesion molecules resulting in an inability of leukocytes to adhere to the endothelium and migrate toward sites of infection. Proof-of-principle of the therapeutic safety and efficacy of HSPC gene therapy with FV containing strong promoters (e.g. MSCV) was provided with the successful long-term (up to 7 years) correction of canine LAD-1 without evidence of leukemogenicity. However, alternative weaker promoters (PGK, EF1a, CD18, and CD11b) directed low levels of expression of CD18 and no clinical benefits were observed in the treated animals. Pre-clinical studies to evaluate the efficacy of FV in human LAD-1 CD34+ cells. In support of our ongoing IND application to the FDA for the clinical use of FV in LAD-1 patients, we have conducted extensive pre-clinical studies to investigate the efficacy of clinical grade FV expressing the human CD18 cDNA (FV-hCD18) in HSPCs collected after G-CSF mobilization and apheresis of subjects with a molecularly confirmed diagnosis of LAD-1. Cells were transduced ex vivo with FV-hCD18 for 16 hours. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 39-42% of cells. Genetic correction of HSPCs from LAD-1 patients restored the chemotactic function of neutrophils differentiated from these progenitor cells in vitro. Transplantation of FV-hCD18-transduced LAD-1 HSPCs into immuno-deficient (NSG) mice resulted in human CD45+ cell engraftment and production of mature human myeloid and lymphoid cells in all mice for up to 5 months. Importantly, high-level, clinically relevant gene marking levels were obtained in vivo. The average percentages of human cells expressing CD18 in the murine BM 5 months after transplantation were 36.0 3.9%. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in most of long-term repopulating HSPCs. Flow cytometry-based lineage analysis of BM from mice transplanted with CD34+ cells transduced with FV-hCD18 revealed human CD18+ cells in both CD13+ myeloid and CD20+ lymphoid compartments. Using next-generation sequencing technology, a total of 101 unique integration sites were recovered in re-populating cells and revealed a polyclonal pattern of integration with no evidence of insertional mutagenesis or tumorigenicity five months after transplantation. First-in-human clinical trial testing safety/efficacy of FV for gene therapy of patients with LAD-1. Based on pre-clinical evidence of safety and efficacy discussed above, significant resources have been allocated during the current funding period to design and submit a first-in-human phase I/II gene therapy clinical trial using FV for the gene therapy of LAD-1. The protocol is under regulatory review with various agencies. A pre-IND type B meeting was held on January 6, 2017 with members of the Center for Biologics Evaluation and Research (CBER) within the FDA. Accrual is expected to begin in 2018. 2. Objective 1.2 Genetic correction by targeted gene addition. The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9), known as CRISPR/Cas9, offers the greatest flexibility for targeted gene therapies. The RNA-guided Cas9 nuclease creates double-stranded breaks at a targeted DNA sequence and subsequent DNA break repair can be exploited to achieve the site-specific addition of new genetic material at the chosen locus via homology-directed repair (HDR). In primary human HSPCs, the low mitotic index presents an impediment to gene editing via HDR, which predominates in the S and G2 phases of the cell cycle, as compared to more efficient non-homologous end joining (NHEJ) resulting in insertions/deletions (indels) in non-cycling cells. With current approaches, only low levels (<1%) of targeted integration via HDR have been reported in human HSPCs. Moreover, targeted gene addition and correction of larger mutations (>50bp) rely on electroporation of bulky exogenous homologous DNA donor template which results in pronounced cytotoxicity to HSPCs. Therefore, new strategies to advance the current state of CRISPR-Cas9-mediated targeted gene delivery are needed. In vivo expansion of genetically edited HSPCs. Recently, McDermott et al. reported that haploinsufficiency associated with monoallelic inactivation of the cxcr4 gene, which encodes a chemokine receptor important for hematopoiesis, may give a proliferative advantage to engrafting HSPCs in a competitive murine transplant model. Thus, monoallelic disruption of cxcr4 by targeted insertion of a therapeutic gene (e.g. CD18) within that locus may enhance in vivo proliferation of rare (<1%) genetically modified HSPCs. In preliminary experiments, we successfully induced a haploinsufficient phenotype by NHEJ-directed indel formation at the cxcr4 locus. When human CD34+ cells were electroporated with Cas9-single guide RNA (sgRNA) ribonucleoprotein complexes (RNPs), we routinely observed >50% occurrence of indel formation via T7 endonuclease I assay and a corresponding knockout of cxcr4 at the protein level. Importantly, in the absence of exogenous homologous DNA template to promote HDR, viability >90% in human CD34+ HSPCs were consistently achieved with this approach. Analysis of clonal isolates indicated that monoallelic indel formation (52%) was favored over either no detectable editing event (31%) or biallelic editing events (17%). Next, we modified this approach with the addition of a plasmid donor template containing a GFP marker gene flanked by 800bp cxcr4 homology arms to favor HDR-driven gene delivery at the cxcr4 locus. Low levels (0.5%) targeted gene insertion and reduced cell viability (20%) were observed, consistent with DNA-induced cytotoxicity in electroporated HSPCs. Experiments investigating the impact of NHEJ- and HDR-driven monoallelic inactivation of cxcr4 on long-term HSPC engraftment in NSG mice are ongoing. Ex vivo expansion of genetically edited HSPCs. Development of a safe and effective ex vivo expansion platform for genetically modified long-term repopulating HSPCs could provide a clinically valuable strategy to increase cell doses and therapeutic efficacy. This is described in a separate reports (Obj.

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