Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application

Abstract

Ex vivo retroviral gene transfer into CD34 + hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34 + HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34 + CD90 + HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy.

Materials promoting viral gene delivery

“…In combination with polybrene the TE was further elevated, which was explained by the distinct modes of each adjuvant, polybrene compensating electrostatic repulsion and poloxamer 338 fluidization the membrane. 240 More recently, poloxamer 338 was commercialized as LentiBOOST™, and applied for LV delivery to CD34+ stem cells, [241][242][243] CD4+ and CD8+ stem cells, 244 and T-cells. 245 Enhanced TE, non-toxicity and clinical relevance were emphasized in all of these studies.…”

“…356,357 In comparative transduction studies of LV delivery to CD34+ stem cells, protamine sulfate showed a TE similar to Vectofusin-1 and higher than that of Retronectin. 243 TE could be enhanced even further by combining protamine sulfate with chondroitin sulfate. 358 Retronectin is a 574 amino acid long recombinant polypeptide based on the fibronectin fragment CH296.…”

Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity

“…Genomic DNA extraction was performed using the QIAamp DNA Blood Mini Kit (QIAGEN). LV DNA was measured using specific probes for the vector packaging sequence (c) and for the cellular albumin gene (Table S1), 51 and values were compared to a standard curve to determine the number of transducing units per mL of vector.…”

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.…”

“…This is of high importance in multi-center studies where there might be a need to agree on a release VCN value across countries and different regulatory agencies. Furthermore, it would be interesting to introduce new techniques for further characterization of the therapeutic gene product such as the abundant heterogeneity regarding stem cell subpopulations and the actual percentage of transduced cells (by flow cytometry or colony-forming assays); these are key features for the success of the gene therapy outcome that is currently being assessed differently in different trials [ 9 , 12 , 37 , 54 , 96 ].…”