ALBERT

Description: Leukocyte Adhesion Deficiency Type I (LAD-I) is a severe primary immunodeficiency characterized by recurrent and life-threatening bacterial infections. It is caused by mutations in the ITGB2 gene, encoding the integrin β2 common subunit (CD18). These mutations lead to defective or absent expression of β2 integrins on leukocyte surfaces, rendering leukocytes unable to extravasate to infection sites. Severe LAD-I is characterized by less than 2% of normal CD18 neutrophil expression and is fatal during the initial 2 years of life in 60-75% of patients in the absence of allogeneic hematopoietic transplant. As it is the case with other monogenic immunodeficiencies, LAD-I is a disorder that could be corrected by ex vivo gene therapy. To this aim we have developed a lentiviral vector (LV) that has recently obtained the Orphan Drug designation (EU/3/16/1753 and DRU-2016-5430). In this LV the expression of hCD18 is driven by a chimeric promoter with a higher activity in myeloid cells. Comprehensive safety and efficacy preclinical LV-mediated gene therapy studies have been conducted in LAD-I mouse models harboring either hypomorphic or knock-out mutations in the ITGB2 gene. Our studies demonstrate stable engraftment of gene corrected LAD-I mouse hematopoietic stem cells in LAD-I recipients and indicate a phenotypic correction of peripheral blood neutrophils. A complete preclinical safety evaluation of the vector was also carried out demonstrating the absence of hematotoxic and genotoxic effects in treated animals. Further studies have been conducted with GMP-produced LVs in human CD34+ cells aimed at optimization of cell transduction. The use of transduction enhancers (TEs) significantly improved the efficacy of genetic correction of human CD34+ cells transduced with LVs at low MOIs. Additionally, transplants into immunodeficient mice showed no changes in the repopulating ability of CD34+ cells when these cells were transduced in the presence of TEs. As occurred in in vitro cultures, significant increases in the transduction of repopulating cells were also associated with the use of TEs, indicating that optimized TE combinations will enable practical and cost-effective transduction of human HSCs in gene therapy protocols. Taken together, these results demonstrate the efficacy and safety of a gene therapy approach directed towards the therapy of LAD-I patients. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Bueren:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Description: The efficacy of lentiviral gene therapy for the treatment of primary immunodeficiencies (PIDs) has been extensively demonstrated in diseases such as X1-SCID, ADA-SCID, X-CGD and WAS. Leukocyte Adhesion Deficiency type I (LAD-I) has also emerged as a PID that is potentially correctable by gene therapy. To this aim during the recent years we have completed all the pre-clinical work required for the initiation of a gene therapy trial for patients with severe LAD-I. Mutations in the ITGB2 gene (encoding for the β2 subunit of integrins, also known as CD18) impair leukocyte extravasation to inflamed areas. LAD-I patients thus suffer from recurrent and life-threatening bacterial and fungal infections. We have previously demonstrated that the ectopic expression of CD18 driven by a myeloid chimeric promoter in LAD-I leukocytes restores their ability to migrate to inflamed sites. Because the efficacy of hematopoietic gene therapy relies on the stable engraftment of gene-corrected HSCs in treated patients, we have implemented the transduction conditions of human HSCs with this therapeutic lentiviral vector in healthy donor CD34+ cells, either from cord blood or mobilized peripheral blood sources. CD34+ cells were transduced at relatively low multiplicities of infection (MOIs) of 50 i.u./cell in the presence of transduction enhancers. Using an optimized transduction protocol, high transduction efficacies were obtained with minimal loss in hematopoietic progenitors. On average, transduction efficiencies of 70% and mean vector copy numbers of 2-5 copies/cell were identified in hematopoietic progenitor cells. Similar results were obtained when large-scale GMP conditions were used. To assess the transduction efficacy of human HSCs, transduced CD34+ cells were transplanted into immunodeficient NSG mice. In these studies, stable repopulation levels of human cells harboring 1-3 copies of the therapeutic vector per cell were observed up to three months post transplantation. In some cases, secondary transplants were also performed, confirming long-term and stable engraftment of transduced cells. These results have allowed us to obtain the approval for the initiation of the first lentiviral-mediated gene therapy clinical trial for the treatment of LAD-I patients. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Rio:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Law:Rocket Pharmaceuticals: Employment, Equity Ownership. Beard:Rocket Pharmaceuticals: Employment, Equity Ownership. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.