Kinetics of lentiviral vector transduction in human CD34+ cells

Abstract

Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34+ cells. We transduced HeLa and CD34+ cells with self-inactivating HIV-1 vector at low and 10-fold higher MOIs, and evaluated vector amounts at various timepoints based upon the rationale that if a given step was not limiting, 10-fold greater vector amounts would be obtained at the 10-fold higher MOI. We observed slower internalization (>60 minutes), a peak of reverse transcription at 24 hours, and completion of integration at 3 days in CD34+ cells. In HeLa cells, vector amounts at high MOI achieved ~10-fold greater values over all timepoints. When compared to HeLa cells, CD34+ cells had a larger difference of vector amounts between high and low MOIs at 2–6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34+ cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts didn’t decrease once detected among CD34+ cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34+ cells in vitro and in xenografted mice. These data suggest that initiation of reverse transcription mainly limits lentiviral transduction for human CD34+ cells. Our findings provide an avenue for optimizing human CD34+ cell transduction.

Restriction of HIV-1-based lentiviral vectors in adult primary marrow-derived and peripheral mobilized human CD34+ hematopoietic stem and progenitor cells occurs prior to viral DNA integration

“…The high levels of total DNA also would not support the idea that the restriction we are observing is due to a limitation at the level of cellular entry from an endocytosis defect as has been described in resting CD4+ T cells [ 46 ]. Total DNA levels were modestly lower at MOIs of both 1 and 10 for both peripheral mobilized and bone marrow derived CD34+ cells, consistent with prior work, but this was not of the same magnitude as the 20–400 fold decrease that we observed in 2-LTR circles compared to total DNA [ 47 ].…”

TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components

“…For the next step, we chose the most promising sgTRAC (sgTRAC3) for application on primary human T cells. Several reports suggest that initial phase of transcription from retroviral genomes might take longer in primary human T cells than in common human T cell lines like Jurkat 46, 47, 48, 49. As a consequence, we decided to stably transduce primary cells with an expression cassette for sgTRAC transcription (with ICLVs) 1 day ahead of TraFo-Cas9 transduction.…”

High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2

“…We performed xenograft mouse transplantation of transduced human CD34 + cells in high-density culture with or without P407 and PGE2 (Figure 5; Figure S6) and demonstrated higher transduction efficiency and similar human cell engraftment in high-density culture (4e6/mL) compared with our standard cell density (1e5/mL). We demonstrated previously that CD34 + cell culture conditions favoring expansion resulted in higher transduction efficiency but lower engrafting abilities in xenograft mouse transplantation 14 . However, high-density CD34 + cell culture enhanced transduction efficiency and reduced cell expansion (Figure 2), suggesting that this low-expansion state might allow CD34 + cells to maintain engrafting ability with efficient transduction.…”

“…We demonstrated that high-density CD34 + cell culture (4e6/mL) alone significantly improves transduction efficiency ∼5-fold compared with our current standard method (1e5/mL) (Figure 1). Usually, improvement in transduction efficiency results from higher cell proliferation because of increased reverse transcription, but these efforts ultimately reduce the engraftment ability of transduced cells 14 . Therefore, we have previously optimized lentiviral transduction in low-density cell culture of CD34 + cells (1e5/mL) with minimal cytokine stimulation (100 ng/mL SCD, FL, and TPO) to balance efficient transduction with high-level engraftment 9, 10, 11.…”