Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2–4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.
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We dedicate this manuscript to the late Dr Brian Sorrentino whose work on LV-XSCID gene therapy provides new hope for affected patients and their families. We also thank Dr Mitchell Weiss for helpful advice on this manuscript. This work was supported by The National Heart, Lung, and Blood Institute grant P01 HL053749, the Assisi Foundation of Memphis grant #94-000 R18 “Cell and Gene Therapy” and St. Jude Children’s Research Hospital Research Consortium “Novel Gene Therapies for Sickle Cell Disease.” LentiBOOST was acquired from Sirion Biotech GmbH. DJR received support from the Seattle Children’s Program for Cell and Gene Therapy.
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Jang, Y., Kim, YS., Wielgosz, M.M. et al. Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy. Gene Ther 27, 545–556 (2020). https://doi.org/10.1038/s41434-020-0150-z
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