Development of an alternative, potentially curative, treatment strategy by performing gene?editing by homologous recombination (HR) to directly correct the E6V mutation in the HBB gene in autologous hematopoietic stem and progenitor cells (HSPCs) from sickle cell patients, and then reinfusing these modified cells post-myeloablation (autologous HSCT (auto-HSCT) (product ?gcHBBSCD?). This obviates the need to find donors for transplantation and decreases the risk of immune related complications due to Human Leucocyte Antigen (HLA) ?mismatch between donor and recipient (e.g. rejection, graft-versus host disease (GVHD), poor immune reconstitution). These advantages suggest the gene editing strategy might effectively be applicable to a broader range of patients with SCD than matched-sibling donor allo-HSCT and may one day also be safer and more effective treatment for patients with SCD. HBB gene correction offers numerous advantages over current lentiviral (LV) gene therapy for the treatment of severe SCD. 1) Early results with LV gene therapy for SCD show low transduction efficiencies in HSPCs; 2) Our gcHBB-SCD offers precise editing at the endogenous HBB loci, in contrast to LV vectorbased gene transfer/addition methods, thus maintaining endogenous regulatory elements preserving physiologic regulation of gene expression; 3) LVs integrate semi-randomly within the genome, thus the possibility always remains of insertional mutagenesis near oncogenes and/or tumor suppressor genes; and 4) By directly correcting the endogenous gene, one also removes expression of pathologic HgbS from the cell thus avoiding the problem of competing with the highly expressed pathologic protein. In addition, this semi-random lentiviral integration leads to heterogeneity in consequent expression such that some cells will not be fully protected from sickling. These reasons provide strong rationale for initiation of a Phase I/II trial using gcHBB-SCD for the treatment of SCD. This approach will be a first-in-human gene correction clinical trial for any disease, including sickle cell disease. While the primary endpoint will be safety and feasibility as a Phase I/II clinical trial, the ultimate aim is to hematologically cure patients with SCD. We have partnered with the California Institute of Regenerative Medicine (CIRM) to fund the INDenabling safety and efficacy studies over 18 months in order to gain FDA approval to initiate a Phase I/II clinical evaluation of a cell therapy product derived from autologous HSPCs from sickle cell patients (gcHBB-SCD). In this approach, the mutated HBB gene is corrected ex vivo using the CRISPR/Cas9 system to create a double strand break (DSB), which stimulates homologous recombination in the presence of a recombinant adeno-associated virus serotype 6 (gene corrected sickle cell disease-AAV6 donor vector: ?gcSCD-AAV6?). When combining a high-fidelity Cas9 version with gcSCD-AAV6, we achieve high levels of sickle cell correction in SCD patient-derived CD34+ HSPCs that also heavily reduced off-target INDELs when compared with ?WT? Cas9 protein. Thus, the overall system involves using Cas9 as part of an RNP to make the targeted break in the HBB gene, combined with rAAV6 to deliver the gene correcting DNA (RNP/AAV6 system). We have demonstrated that high frequencies (>50%) of HBB gene corrected CD34+ HSPCs are maintained after transplantation into NSG mice, supporting the therapeutic potential of this approach. As our trial will use plerixafor to mobilize peripheral blood CD34+ HSPCs from sickle cell disease patients as the starting blood stem cell source for ex vivo gene correction, we performed process development on this HSPC source. In 14 independent experiments we have obtained an average of 80% cell viability, 94% CD34+ purity and 67% gene correction frequency. We have also shown the feasibility of manufacturing plerixafor mobilized-HSPCs at near clinical scale. In 6 manufacturing runs, 492 million HBB gene corrected cells were generated. These cells were then parlayed into the necessary long-term safety and tumorigenicity study that is being performed by a CRO to show that HBB modified HSPCs can engraft and differentiate into human lineages in immunodeficient mice. These results will be delivered by May 2019 (attached as a PDF appendix). The primary goal of this Phase I/II clinical trial is to determine the feasibility and safety of the treatment strategy. The secondary goal is to determine the efficacy by analyzing the fraction of treated patients who achieve a Hemoglobin S (HgbS) level of <50% and/or elimination of symptoms and sequelae associated with SCD. We plan on submitting our IND to the FDA by July 5th , 2019 (this date remains on our timeline). To enable manufacturing of the autologous investigational cell product, we are in need of clinical-grade AAV6. Currently, through a CIRM CLIN-1 grant, we have a funded a path forward for all other GMP-compatible genome editing reagents, including sgRNA (Agilent) and HiFi Cas9 protein (Aldevron). Also, we have been able to initiate process development on AAV6 large-scale production and established a contract with a CMO (Vigene) to manufacture the vector for this clinical trial. Here we are requesting funding through the NIH initiative to cure sickle cell disease to support the critical manufacture of AAV6 that is required for initiation of our clinical trial in 2019. The total amount requested is $1,854,255 over a 6-month period to support AAV6 vector manufacturing (which could be distributed as milestone payments). We note that the NHLBI supports lentiviral production centers and cell manufacturing centers for cell and gene therapy programs but does not currently support an AAV6 manufacturing center. Thus, we established a contract with a CMO (Vigene) for GMP vector manufacturing and have utilized Fraser Wright (biosketch attached and world leader in AAV manufacturing) as a consultant. As part of our contract our team (including Fraser Wright) has performed a site visit of the CMO auditing manufacturing capacity, batch records, and past performance. We chose this CMO after evaluation of several alternative CMO?s and chose this one because it had could manufacture a clinical lot of AAV6 consistent with our timeline and because the cost was 50% cheaper than others (1.7 million as compated to >3.5 million from the most competitive CMO). Our team (including Fraser Wright) then audited the facility to determine its manufacturing capacity, its quality control processes, and its batch records (among other things). The facility and team passed our internal audit and specifications and thus we have finalized the contract. We now have bi-weekly phone calls to closely monitor the progress of manufacture of the clinical lot of AAV6 for our gene correction program. We will perform an additional audit in June, 2019 to assure that the CMO continues to perform at the level that our specifications require. This proposal is to specifically request the funds to cover the manufacture of an essential reagent for our gene correction for sickle cell disease program?a reagent that currently can not be provided by other NHLBI or NIH funded sources.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Special Emphasis Panel (ZHL1)
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Mondoro, Traci
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Stanford University
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United States
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