Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human Hematopoietic Stem and Progenitor Cells

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.

Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells

“…Here, we have shown that CypA is an important cofactor for efficient lentiviral transduction of HSPC. Therefore, we sought a non-immunosuppressive cyclosporine that did not inhibit CypA, identifying CsH as, to our knowledge, the most potent enhancer of HSPC gene transfer (Heffner et al., 2018, Lewis et al., 2018, Wang et al., 2014, Zonari et al., 2017, Petrillo et al., 2015). Our side-by-side comparisons illustrate CsH treatment outperforms even the standard double-hit protocol.…”

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges

“…Importantly, prostaglandin E2 also has a key role in the maintenance of HSCs 91,92 and therefore constitutes a useful agent in the ex vivo engineering of these cells. Lewis et al 93 found that the kinase inhibitor staurosporine boosted lentiviral transduction of HSPCs (possibly by overcoming a barrier to entry). The researchers also found that staurosporine and prostaglandin E2 act through different mechanisms and that a combination of the two had a greater effect on vector transduction than either agent alone.…”

Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

“…No free plasmids were detected 4 days post-transduction by this method, indicating that detection of stable integration in the cell can be measured from that day. Across the literature, analysis of VCN is performed at different timepoints post-transduction form 7 days [ 12 , 93 , 94 ], 9 days [ 52 , 54 ], and up to 14 days [ 9 , 95 , 96 ]. It is important to note that; VCN values may differ when analyzed on different days, and while the differences can be subtle, it can hamper the comparison between trials.…”

“…In the past, to get a sufficient proportion of gene-corrected cells in the therapeutic product, high vector doses (2 transduction hits) and prolonged ex vivo culture (3 days in total) were needed [ 12 , 96 ]. Various TE compounds can be added to the culture media to increase lentiviral transduction efficiency, VCN, and transgene expression; they include Cyclosporin and Rapamycin [ 95 ], Prostaglandin E2 [ 93 ], Staurosporine [ 94 ], or LentiBOOST TM [ 96 , 102 ]. Combinatorial TE application has also been tested, yielding even more potent effects [ 94 , 95 , 96 ].…”

“…Various TE compounds can be added to the culture media to increase lentiviral transduction efficiency, VCN, and transgene expression; they include Cyclosporin and Rapamycin [ 95 ], Prostaglandin E2 [ 93 ], Staurosporine [ 94 ], or LentiBOOST TM [ 96 , 102 ]. Combinatorial TE application has also been tested, yielding even more potent effects [ 94 , 95 , 96 ]. Higher transduction due to TEs was achieved in all HSPCs subpopulation, including the long-term repopulating HSCs, without changing viability, integration sites pattern, global gene expression profiles, in vivo toxicity, or differentiation capacity in vitro (colony-forming assay) and in vivo (NSG mouse model).…”