Expanding genetically engineered HSC: from translation to biology and back

The capacity to maintain and expand hematopoietic stem and progenitor cells (HSPC) in culture represents a fundamental goal in order to broaden the scope of ex vivo gene therapy to new groups of patients and emerging technologies such as gene editing. Using mobilized peripheral blood HSPC from healthy donors and patients affected by genetic diseases, we are applying state-of-the-art genetic engineering protocols, capable of correcting more than 90% of cells, followed by an ex vivo expansion procedure. To this end, we are adopting innovative protocols currently undergoing clinical teesting in the context of allogeneic cord blood transplantation (https://pubmed.ncbi.nlm.nih.gov/31704264/). Using stringent assays, we have shown that genetically-corrected HSPC can be expanded up to 3 times in culture (https://pubmed.ncbi.nlm.nih.gov/28330619/), guaranteeing high diversity (polyclonality) of the product, a desired characteristic when it comes to efficacy and safety of these cells when used for transplant. In order to combinatorially test multiple conditions, in an effort to optimise the expansion protocol, new in vitro readouts able to faithfully assess the stem cell content in the culture are needed. We therefore invested in novel technologies such as single cell RNA sequencing that should allow not only the quantification of the stem cell content, but also to uncover new molecular targets that may be exploited for further improving the degree of HSC expansion (next generation protocols). The current protocol shows promise for the development of an ex vivo gene therapy approach for patients affected by infantile malignant osteopetrosis (https://pubmed.ncbi.nlm.nih.gov/31949009/), a potential candidate for a first-in-man application of ex vivo expanded genetically-engineered grafts. The successive goal is to develop next generation expansion protocols in the context of gene editing protocols.