295. Hematopoietic Stem Cell Gene Therapy (2.0) Based on Purified CD34+CD38- Cells
Authors: Erika Zonari, Francesco Boccalatte, Tiziana Plati, Alessandro Aiuti, Giuliana Ferrari, Eugenio Montini, Luigi Naldini, Bernhard Gentner
Journal: Molecular Therapy (2015)

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety. First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells. In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by gene-modified CD34+CD38- cells was slower (15wks) and incomplete (<60% at 24wks) in this setting. Additional, non-competitive transplants confirmed that freshly isolated CD34+CD38int cells possess long-term reconstituting capacity, which is lost after 2 days of standard culture. To improve the engraftment of the gene-modified CD34+CD38- cells when administered with uncultured CD38int/+ supporter cells, we optimized culture conditions of the CD38- and cell doses of the CD38int/+ population. We achieved accelerated engraftment kinetics of gene-modified CD38- cells by >5 wks and re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells. Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC. In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.