1. Objective 1: Develop approaches for the genetic correction of human HSCs Development of a clinical trial for LAD-1. A clinical trial for gene therapy of LAD-1 using first-in-human foamy viral vectors is in preparation. Documents are expected to be submitted to the Food and Drug Administration (FDA) in the fall 2015. In preparation for this trial, we have performed process-development and scale-up of FVV production (using the ΔΦMSCV-GFP and ΔΦMSCV-hCD18 FVV) in collaboration with investigators at CCHMC Vector Production Facility (VPF) (Dr. Johannes C.M. van der Loo, Dr. Carolyn Lutzko and Dr. Punam Malik) and Dr. David Russell (University of Washington). A considerable effort was needed to develop processes for large-scale concentration and purification of this extremely serum dependent virus. Since FV is a non-pathogenic virus, its isolation and purification techniques had to be developed. Assays required for assuring safety and potency of ΔΦMSCV-hCD18 FVV were developed, and some of the final safety assays still need further development to a cGMP-grade assay. The protocol for production of clinical grade FVV stocks for gene therapy of LAD-1 has been optimized. Optimization of the CRISPR/Cas9 genome editing technology in human HSCs. We have investigated multiple approaches for transfection of human CD34+ cells that have resulted in low efficiency of delivery or unacceptable cell toxicity, including cationic lipids, nucleofection, DMRIE-C, PEI, CellfectinII, 293fectin, Superfect, Effectine, Endofectin-Plus, Glycofect, Escort V, and Lipofectamine 3000. We are currently focusing on alternative approaches, including large-scale production of non-integrating retroviral vectors carrying Cas9 and gRNA cassettes, and induced transduction by osmocytosis and propanebetaine (iTOP, Cell 2015). A recent publication has indicated that HSCs with CXCR4 haploinsufficiency have a competitive repopulation advantage, perhaps due to enhanced proliferation. We have developed and amplified the Cas9/gRNA plasmids necessary for targeted introduction of a gene of interest (GFP and CD18) in the CXCR4 locus via homologous recombination (HR) DNA repair pathways. Activation of HR pathways requires active cell division; we have therefore tested the ability of a recently described small molecule (UM729) to increase HSC proliferation/self-renewal. Preliminary data indicate successful amplification of CD34+ cells, setting the stage for testing Cas9/gRNA for targeting the CSCR4 locus once approaches for delivering macromolecules in human CD34+ cells (step 1 above) have been optimized. In FY2016, we plan to assess the impact of CXCR4 haploinsufficiency on human HSC engraftment and proliferation using the gold-standard immuno-deficient (NSG) murine model. 2. Objective 2: Develop approaches for expansion of genetically corrected human HSCs Investigation of pathways regulating HSC self-renewal and differentiation We have determined that G-CSF mobilized human CD34+ cells cultured ex vivo for 21 days in the presence of immobilized Notch (Delta-1ext-IgG) ligand results in 5-fold expansion of long-term repopulating HSCs when cultured under hypoxic conditions (1.5% O2) compared to cultures performed under normoxia (21% O2) where maintenance but no expansion of HSCs was observed. These data suggest synergy between hypoxia and Notch pathways. We have also initiated investigations testing whether transient downregulation of DNMT3a expression using SiRNA in human CD34+ cells may prevent differentiation of HSCs and favor their self-renewal during short-term culture. Conditions for optimal detection of DNMT3A expression by Q-PCR and Western blots have been developed. A first attempt at transiently downregulating DNMT3A expression has failed using commercially available SiRNA. We are testing an alternative SiRNA (Silencer Select, Life Technologies). By exploiting the extensive HSC amplification after transplantation, we aim to identify novel factors/pathways involved in HSC self-renewal. We have performed preliminary experiments to identify conditions for optimal HSC amplification after transplantation, including cell dose, timing of cell collection after transplantation, and the impact of serial transplantations. We found that lower cell doses (e.g. 1x106 cells) result in superior HSC expansion in vivo compared to larger cell doses (e.g. 1x107 cells). The optimal timing for cell collection was 1 week; longer times in vivo (e.g. 2, 3, or 4 weeks) resulted in progressive differentiation to more mature progeny. Serial transplantations have led to engraftment levels too low to allow reliable detection and selection of HSCs. In FY16, we will further optimize conditions to detect sufficient number of HSCs after transplantation, namely transplanting larger cell doses and shortening time of cell collection to 3 days in serial transplant experiments. Pending optimization of these conditions, we will compare gene expression and methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, and CHIP-Seq approaches. Development of a programmable 3-dimensional bone marrow niche for expansion of human HSCs We have recently started to adapt a programmable 3D silk scaffold system developed by Dr. Kaplan at Tufts University for expansion of HSCs ex vivo. IN FY15, we have optimized most components of this system, including: 1- The basic silk scaffold to mimic the 3D bone marrow microenvironment; 2- The tube structures to mimic bone marrow vasculature; 3- The various cellular and extra-cellular matrix components (e.g. megakaryocytes, endothelial cells, fibronectin); 4- The biophysical properties of the system (e.g. bioreactor flow rate, oxygen tension, etc). 3. Objective 3: Develop approaches for efficient differentiation of human iPSCs into functional HSCs Development of a culture system for hematopoietic differentiation of normal human iPSCs Generation of normal human iPSC lines. We have established several iPSC lines derived from G-CSF mobilized peripheral blood (MPB) CD34+CD38- and CD34+CD38+ of normal control individuals using a non-integrating Sendai virus delivery system expressing the transcription factors Sox2, Klf4, c-Myc and Oct2. Differentiation of normal human iPSCs into hematopoietic cells. We have established a novel system for de novo generation of easily accessible suspension human hematopoietic cells (CD45+CD34+) from iPSCs. A patent application (# PCT/US2014/058583) describing this protocol has been approved and published on April 9, 2015. Characterization of normal human iPSC-derived hematopoietic cells. Up to 60% of iPSC-differentiated cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to primary CD34+ cells. These cells, however, failed to home to the bone marrow of immuno-deficient (NSG) animals and did not result in long-term engraftment after transplantation. Differentiation of genetically corrected iPSCs derived from patients with inherited bone marrow failure syndromes into transplantable HSCs We have obtained original and genetically corrected iPSC lines derived from individuals with inherited bone marrow failure syndromes (Fanconi Anemia and Diamond-Blackfan Anemia) from the laboratories of Dr. Juan Carlos Izpisua-Belmonte (Salk Institute) and Dr. MJ Weiss (St. Jude Childrens Research Hospital), respectively. Culture conditions for optimal growth of these lines are being optimized. In FY16, the differentiation protocol developed for normal iPSCs will be evaluated for hematopoietic differentiation of patient-derived iPSCs.

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