UM171 Enhances Lentiviral Gene Transfer and Recovery of Primitive Human Hematopoietic Cells

Abstract

Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34⁺ and CD34⁺CD45RA^- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the ex vivo production of gene-modified cells and for reducing requirements of virus for a broad range of applications.

Keywords: gene transfer; hematopoietic stem cells; lentivirus.

Figure 1

UM171 Enhances Lentiviral Transduction of Primitive Human Hematopoietic Cells (A) Outline of experimental design. 20,000 CD34⁺ CB cells were prestimulated and transduced with a GFP LV (10⁶ IU/mL, MOI = 5) in the presence of DMSO, SR1, UM171, or UM171+SR1. Cells were then washed and cultured further for 72 hr to assess transduction efficiency by FACS. (B) The proportion of GFP⁺ CD34⁺ cells at the end of 3-day culture. (C) CD34⁺ CB cells were prestimulated and transduced, as outlined in (A), in the presence of DMSO or various concentrations of UM171 (1, 5, 10, 20, 35, 50, and 100 nM). The percentage of GFP⁺ cells (curve) is indicated on the left y axis, and viral copy number per cell (bar graphs) is indicated on the right y axis. (D) Transduction efficiency in CD34⁺ cells exposed to various concentrations of virus ranging from 10⁵ to 10⁹ IU/mL (MOI = 0.5–5000). (E) LV copy number per cell in cells transduced in the presence of 10⁶ or 10⁸ IU/mL virus. (F) Transduction efficiency in various primitive hematopoietic subsets, transduced in the presence of 10⁶ or 10⁸ IU/mL. (G) Absolute yield of total and transduced cells across the range of hematopoietic subsets transduced in the presence of 10⁶ or 10⁸ IU/mL virus. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. The data are shown as mean ± SD.

Figure 2

UM171 Enhances LV Transduction of Transplantable Human HSPCs (A) Outline of experimental design. CD34⁺ CB cells were prestimulated and transduced in the presence or absence of UM171 with a GFP- or a YFP-expressing vector (10⁶ IU/mL, MOI = 1). Immediately after transduction, equal aliquots of YFP-marked and GFP-marked cells were mixed and coinjected into lethally irradiated NOD/SCID-IL2Rγ^−/− (NSG) (n = 8 mice). A small aliquot of cells was cultured for 3 additional days to assess gene transfer into CD34⁺ cells. (B and C) Representative FACS blots showing transduction efficiency in CD34⁺ cells transduced with a GFP-expressing (B) or a YFP-expressing (C) virus. (D) Percentage of GFP or YFP in CD34⁺ cells (n = 3 experiments, each in triplicate). (E) Absolute yield of total and GFP- or YFP-transduced cells (n = 3 experiments, each in triplicate). The bar graphs in (D) and (E) represent mean values ± SD. (F) Percentage of total human engraftment in mouse BM over 30 weeks (blue). Proportion of cells transduced under UM171-stimulated condition is displayed in red, and proportion of cells transduced under control conditions is displayed in black. (G and H) Normal B-lymphoid (G) and myeloid (H) cell production over 30 weeks. Proportion of cells transduced under UM171 stimulated conditions in red and proportion of cells transduced under control conditions in black. The error bars in (F)–(H) are shown as mean values ± SEM. (I and J) FACS blots from a representative mouse BM at 30 weeks post-transplant. (I) CD45 cells. (J) CD33 and CD19 cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

LV Genomic Integration Profile in Cells Transduced with UM171 (A) Clonal distribution of LV in CB CD34⁺ cells transduced in the presence of UM171 versus DMSO. The total number of unique RISs (clones) identified in each sample is listed at the top of each bar (23,170 in the presence of UM171 and 8,054 in the presence of DMSO). Each bar represents the frequency at which each clone in the sample was sequenced from greatest (bottom) to least (top). Clones sequenced at a frequency of ≥1% in the pool are designated by colored boxes. All other clones are grouped into a single gray box at the top of each bar. (B) Genome distribution of identified integration sites. Circos (www.circos.ca/) was used to plot the genomic distribution of integration sites identified in DMSO- and UM171-treated CD34⁺ CB cells as an inverted histogram plot. All integration sites identified are included for each experimental arm (red indicates the presence of UM171; blue indicates the presence of DMSO). The human genome (outer band, version hg19) from chromosome 1 through chromosome Y is shown with each chromosome color coded and corresponding G-banding patterns indicated. The genome was divided into 1-Mb bins. The number of unique integration sites identified in each 1-Mb bin is reflected in the height of the bin histogram. Each histogram gridline represents 25 unique integration sites. The mirror imaging of these two histograms across the genome indicates no significant difference in the global genomic pattern of integration between these two experimental arms. (C) Frequency of LV inserts within and proximal to cancer genes in cells transduced in the presence of DMSO (clear bars) and UM171 (red bars). Bar graph represents the percentage of insertions found within cancer genes or near cancer gene promoters, as determined by analysis using QuickMap. 95% CI for insertions: within cancer genes, 3–24% for DMSO and 3–10% for UM171; <5 kB from cancer genes, 0.1–21% for DMSO and 0.2–8% for UM171; and <50 kB from cancer genes, 3–24% for DMSO and 3–9.7% for UM171. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

UM171 Enhances Transduction of CD34⁺ CB Cells over a Spectrum of Viral Pseudotypes and Viral Types (A) Outline of the experimental design. (B) Proportions of GFP⁺ cells 3 days after transduction with an LV pseudotyped with VSV-G, AMPHO, or RD114/TR envelopes. (C) Proportion of GFP⁺ cells 3 days after transduction with α- and γ-RVs pseudotyped with the VSV-G envelope. (D) Proportion of GFP⁺ cells 1 and 7 days after transduction with nonintegrating viral vectors such as AAV6 (black and red squares) and integrase-defective LV (IDLV; black and red triangles). **p < 0.01, ***p < 0.001. The error bars represent mean values ± SD.

Figure 5

UM171 Enhances Lentiviral Transduction to Adult HSPCs (A) Outline of experimental design. CD34⁺ CB, adult BM, and mPB were prestimulated for 24 hr and transduced for 24 hr at a virus concentration of 10⁷ IU/mL (MOI = 50) in the presence or absence of UM171 (n = 3 experiments, each in triplicate). (B–D) The proportion of GFP⁺ cells at the end of a 3-day culture shown for (B) CB, (C) adult BM, and (D) mPB HSPCs. **p < 0.01, ***p < 0.001, and ****p < 0.0001. The bar graphs represent mean values ± SD.