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. 2014 Aug 7;124(6):913-23.
doi: 10.1182/blood-2013-12-546218. Epub 2014 Jun 9.

Rapamycin Relieves Lentiviral Vector Transduction Resistance in Human and Mouse Hematopoietic Stem Cells

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Free PMC article

Rapamycin Relieves Lentiviral Vector Transduction Resistance in Human and Mouse Hematopoietic Stem Cells

Cathy X Wang et al. Blood. .
Free PMC article

Abstract

Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.

Figures

Figure 1
Figure 1
Rapamycin increases in vitro LV transduction efficiency in human and mouse hematopoietic progenitors. (A) Scheme of human CD34+ and mouse Lin cell transduction and assessment. (B) Human cord blood CD34+ cells were transduced with CG-UbiC-EGFP LV in the presence of rapamycin or DMSO only as a diluent control. Black triangles, DMSO only; red circles, 10 μg/mL rapamycin. Cells were analyzed 10 to 14 days posttransduction. (C) Human cord blood CD34+ cells were transduced with CG-UbiC-EGFP, MOI = 25, in the presence of various concentrations of rapamycin or (D) Torin 1, an active site mTOR inhibitor. In most cases, data are pooled from 4 to 5 independent experiments, each with different cord blood donors and done in duplicate. At lower MOI or rapamycin concentrations, 2 different donors in duplicated experiments were assessed. (E) MFI of EGFP from a representative titration series in panel C. (F) Fold change over DMSO-treated controls in integrated LV copy number per cell, determined by qPCR, in CD34+ cells transduced in the presence of various concentrations of rapamycin and analyzed 10 to 14 days posttransduction. Data are pooled from 2 to 5 independent experiments, each with different cord blood donors. (G) EGFP cell marking and (H) fold change in integrated LV copy number in mouse bone marrow Lin cells transduced with RRL-MND-GFP at various MOIs in the absence (DMSO only) or presence of 1 or 5 μg/mL of rapamycin and analyzed 10 to 12 days posttransduction. Data shown are derived from 4 independent experiments comprising 48 donor animals. LV copy numbers are shown as fold change over the average LV copy number of the DMSO-treated controls. For all panel, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test. BM, bone marrow; hu, human; mu, murine; Rapa, rapamycin.
Figure 2
Figure 2
Increased EGFP marking in mouse hematopoietic lineages arising from rapamycin-treated and LV-transduced Lin cells transplanted into mice. Congenically marked mouse bone marrow Lin cells were transduced for 16 hours with RRL-MND-GFP LV, MOI = 2.5, in the presence of 5 μg/mL rapamycin or DMSO. Cells were washed, and 1 × 106 cells per mouse were injected retro-orbitally into lethally irradiated congenically disparate recipients. Mice were sacrificed 11 (gray triangles or open red circles) or 16 (black triangles or closed red circles) weeks posttransplant. (A) Percent donor cell engraftment in bone marrow and spleen. (B) EGFP marking, assessed by flow cytometry, in total donor bone marrow cells and bone marrow HSCs (LinSca-1+c-Kit+). (C) LV copy number per cell in Lin and Lin+ bone marrow cells as determined by qPCR (described in “Methods”). (D) EGFP marking in splenic subsets analyzed with the following markers: B220+ B cells, B220CD4+ or CD8+ T cells, B220CD4CD8CD11b+GR1lo/neg monocytes, and B220CD4CD8CD11b+GR1hi neutrophils. Each point represents 1 mouse. Cells were pregated on donor-derived cells for all subset assessments. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, and ***P < .001 from a parametric 2-tailed unpaired Student t test.
Figure 3
Figure 3
Increased EGFP marking in human hematopoietic lineages arising from rapamycin-treated and LV-transduced CD34+ cells transplanted into NSG mice. Human cord blood CD34+ cells were prestimulated for 24 hours in HSC-supportive cytokines and then transduced for 12 hours with CG-UbiC-EGFP, MOI = 25, in the presence of DMSO, 10 μg/mL rapamycin, or 20 μg/mL rapamycin. NSG mice each received 3 × 106 CD34+ cells (DMSO control or 10 μg/mL rapamycin treatment) or 2.6 × 106 CD34+ cells (20 μg/mL rapamycin treatment). Mice were sacrificed 19 weeks posttransplant. (A) Total hCD45+ engraftment levels in the bone marrow. (B) EGFP expression and MFI were measured by flow cytometry in live hCD45+ from the BM (left). LV copy number per cell as determined by qPCR (described in “Methods”) (right). (C) EGFP expression in bone marrow and splenic subsets, analyzed with the following human markers: CD34+ for HSCs, CD33+ for myeloid cells, CD19+ for B cells, and CD3+ for T cells. Cells were pregated on hCD45 for subsequent lineage analyses. Each point represents 1 mouse. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test.
Figure 4
Figure 4
Rapamycin enhances LV transduction via an mTOR-dependent, nonautophagy mechanism. (A) Human cord blood CD34+ cells were transduced with CG-UbiC-EGFP, MOI = 25, in the absence or presence of the autophagy-stimulating peptide Tat-beclin 1. Black triangles, Tat-beclin 1 scrambled peptide control; blue circles, Tat-beclin 1. Data are pooled from 2 independent experiments, each with different cord blood donors and done in duplicate. (B) Bone marrow Lin cells from wild-type or beclin 1+/− mice were transduced with RRL-MND-GFP, MOI = 0.5, in the presence of 5 μg/mL rapamycin or DMSO. EGFP marking was assessed by flow cytometry 10 days posttransduction. Data points represent cells from individual donor mice. Black triangles, wild-type cells; blue circles, beclin 1+/− cells. (C) Human cord blood CD34+ cells were transduced with CG-UbiC-EGFP, MOI = 25, in the presence of combinations of either rapamycin or (D) Torin 1 with FK506, an FKBP12-binding compound. Black triangles, DMSO only; green squares, 10 μg/mL FK506. Data are duplicate transductions from 2 representative experiments with different cord blood donors. For all panels, lines represent group mean and error bars represent standard deviation. ****P < .0001 from a parametric 2-tailed unpaired Student t test.
Figure 5
Figure 5
Rapamycin does not affect LV binding but enhances LV uptake into the cytoplasm. (A) Human cord blood CD34+ cells were treated with CG-UbiC-EGFP, MOI = 25, at 4°C (to prevent endocytosis and limit binding to the cell surface) for 2 hours, in the presence of 10 μg/mL rapamycin or DMSO. After transduction 60 000 to 80 000 cells were lysed and p24 protein content was determined to assess LV binding (left), and the remaining ∼10 000 cells were cultured at 37° for 10 days and evaluated for EGFP marking to determine transduction efficacy (right). Data are shown for 2 independent experiments with different cord blood donors, each done in triplicate. (B) Human cord blood CD34+ cells were prestimulated and treated with rapamycin or DMSO for 12 hours, and cell-surface LDL receptor (LDLR) and α-dystroglycan (α-DG) levels were determined by flow cytometry. (C) Human cord blood CD34+ cells were treated with DMSO (black triangles) or 10 μg/mL rapamycin (red circles) after allowing LVs to either prebind to the cell surface for 2 hours at 4°C or (D) preinternalize for 2 hours at 37°C followed by cleaving off external LVs with 0.05% trypsin (to remove bound LVs that have not internalized). Control represents same cells transduced under standard conditions outlined in Figure 1A. Data are duplicate or triplicate transductions from 1 experiment representative of 3 experiments with different cord blood donors. (E) Human cord blood CD34+ cells were transduced in the presence or absence of indicated concentrations of rapamycin, with CG-UbiC-mCherry, MO I = 25, carrying the BLAM-Vpr protein. After a 6-hour transduction, cells were loaded with the BLAM substrate CCF4-AM, and LV entry was quantified by flow cytometric detection of cells exhibiting cleaved CCF4. Data are pooled from 4 independent experiments, each with different cord blood donors. (F) Human cord blood CD34+ cells were transduced with CG-UbiC-EGFP, MOI = 25, and the following HIV-1 reverse-transcription products were quantified by qPCR: strong-stop DNA (Early RT), full-length DNA (Late RT), and 2-long terminal repeat circles. (G) Ratios between each pair of adjacent reverse-transcription products from panel F were calculated. Black, DMSO only; red, 10 μg/mL rapamycin. Data are pooled from 4 independent experiments, each with different cord blood donors. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test (A,C,D-E) or paired Student t test (F-G). LTR, long terminal repeat.
Figure 6
Figure 6
In vivo LV genomic integration profile in rapamycin-treated mouse bone marrow Lin cells. (A) Total number of unique LV integration sites (RIS) identified in BM Lin cells from primary transplant recipient mice described in Figure 2. *P < .05 from a parametric 2-tailed unpaired Student t test. (B) Percentage of the total number of RIS mapped to each chromosome. White, DMSO only; red, 5 μg/mL rapamycin. Asterisk (*) denotes borderline significant differences between the frequency of integration in chromosomes 7 and 13 (P = .077 and .076, respectively). (C) Genome distribution of identified integration sites. The mouse genome is represented by chromosomes 1 to 19, x and y clockwise from top. Inner circles represent all mice from a given treatment group: rapamycin-treated mice (first inner circle, warm colors) and control mice (second inner circle, cool colors). Each colored dot represents an individual integration event and is color-coded by the mouse in which it was identified within the specified treatment group. Integrations mapped to chromosomes 7 and 13 are magnified (center). For all panels, lines represent group mean and error bars represent standard deviation.
Figure 7
Figure 7
Rapamycin enhances in situ transduction of mouse bone marrow cells following IO infusion. C57BL/6 mice were treated with IO infusions of RRL-MND-GFP, 3.3 × 109 TU/mouse, in the absence or presence of 250 μg/kg rapamycin. Bone marrow cells were isolated 62 days after injection, and EGFP expression and MFI in bone marrow LinSca-1+c-Kit+ cells were analyzed by flow cytometry. LV copy number per cell was determined by qPCR. Each point represents 1 mouse. Lines represent group mean, and error bars represent standard deviation. ***P < .001 from a parametric 2-tailed unpaired Student t test.

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