Abstract
Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products.
Highlights
Encouraging ex vivo gene therapy utilizing lentiviral vector (LVV) transduced human CD34+ hematopoietic stem and progenitor cells (HSPCs) has been documented in multiple diseases.[1,2,3,4,5,6] Even with numerous examples of promising clinical outcomes, transduction of long-term HSPCs (LT-HSPCs) remains challenging
Staurosporine Treatment Increases Transduction of Human CD34+ Cells Consistently achieving both high average vector copy numbers (VCNs) and a high proportion of transduced cells (%LVV+) in HSPCs is a challenge in the gene therapy field
Even in cell lots capable of achieving high levels of transduction, less than 50% of the cells are BlaM+, indicating there is a barrier to LVV entry
Summary
Encouraging ex vivo gene therapy utilizing lentiviral vector (LVV) transduced human CD34+ hematopoietic stem and progenitor cells (HSPCs) has been documented in multiple diseases.[1,2,3,4,5,6] Even with numerous examples of promising clinical outcomes, transduction of long-term HSPCs (LT-HSPCs) remains challenging. Overcoming transduction barriers in LT-HSPCs, especially in indications where a high proportion of genetically modified cells is necessary for therapeutic benefit, is a significant focus of the field.[7,8,9,10,11] To facilitate transgene delivery into LT-HSPCs, we and others have employed small molecules or peptides that can be added to the transduction process to overcome barriers preventing LVV transduction and to increase the proportion of transduced LT-HSPCs. Similar efforts have been undertaken by others and have led to the identification of rapamycin, cyclosporin, vectofusin, and prostaglandin E2 (PGE2) to increase LVV transduction efficiency in cells.[7,8,9,10,11]. HIV infection and LVV transduction utilize different mechanisms to overcome the cellular membrane barrier, it has been shown that other methods used to increase transduction efficiency of LVV in HSPCs, such as spinoculation, which has been utilized to transduce HSPCs with both gammaretroviral vector and LVV, cause a similar activation of cortical actin dynamics, suggesting that this cellular membrane entry barrier might be a common restriction point for both HIV infection and LVV transduction.[15,16,17,18]
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