Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically RelevantEx VivoGene Therapy Settings

Abstract

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow–derived CD34+ cells while better preserving their engraftment capacity in vivo. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.

Innovative Cell-Based Therapies and Conditioning to Cure RAG Deficiency

“…Preclinical GT studies are now in progress to implement transgene expression. To this end, the recent description of the effect of immunomodulatory compounds is particularly relevant, leading to increased transduction levels in long-term HSC while preserving engraftment potential (81)(82)(83). Cyclosporines A and H have been recently demonstrated improve transduction without altering HSCP subpopulation composition nor the cell-cycle status (81,82).…”

Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering

“…CsH yields a 5–10-fold increase in transduction efficiency in long-term repopulating HSPC in vivo, rendering it the most potent enhancer of HSPC gene transfer described thus far [ 11 , 93 ]. Importantly, CsH does not impact viability nor engraftment capacity of human HSPC, conversely to other enhancers such as Rapamycin (Rapa) or Cyclosporine A (CsA) that have shown some degree of toxicity, in particular in the clinically relevant mPB-derived HSPC [ 121 ]. Indeed, cell-source dependent effects of enhancers should be carefully evaluated as significant differences may exist with cord-blood and BM-derived HSPC being usually less sensitive as compared to mPB-derived CD34 + cells [ 121 ].…”

“…Importantly, CsH does not impact viability nor engraftment capacity of human HSPC, conversely to other enhancers such as Rapamycin (Rapa) or Cyclosporine A (CsA) that have shown some degree of toxicity, in particular in the clinically relevant mPB-derived HSPC [ 121 ]. Indeed, cell-source dependent effects of enhancers should be carefully evaluated as significant differences may exist with cord-blood and BM-derived HSPC being usually less sensitive as compared to mPB-derived CD34 + cells [ 121 ]. Of note, CsH significantly enhances also non-integrating LV-based gene editing as it increases the donor DNA template availability in HPSC [ 121 ].…”

“…Indeed, cell-source dependent effects of enhancers should be carefully evaluated as significant differences may exist with cord-blood and BM-derived HSPC being usually less sensitive as compared to mPB-derived CD34 + cells [ 121 ]. Of note, CsH significantly enhances also non-integrating LV-based gene editing as it increases the donor DNA template availability in HPSC [ 121 ]. This is of particular interest in settings in which larger donor DNA cassettes that AAV is unable to accommodate are required.…”

Successfully transfected primary peripherally mobilized human CD34+ hematopoietic stem and progenitor cells (HSPCs) demonstrate increased susceptibility to retroviral infection

“…A number of different approaches have been investigated to understand the basis of the observed limitations to transduction of these cells and improve efficiency. Investigators have exposed CD34+ HSPCs to various chemicals such as Cyclosporin A, Cyclosporin H, Rapamycin, stem cell factor, thrombopoietin, Flt3 ligand, interleukins, proteasome inhibitors and other agents before transduction with various levels of success [4][5][6][7][8][9][10][11][12][13][14].…”