Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

Trobridge, Grant D.; Beard, Briar C.; Gooch, Christina; Wohlfahrt, Martin E.; Olsen, Philip; Fletcher, James; Malik, Punam; Kiem, Hans Peter

doi:10.1182/blood-2007-09-115022

Cited by 61 publications

(96 citation statements)

References 32 publications

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“…For in vivo studies, cells were costained with a human/NHPspecific antibody (anti-human CD45 PerCP and anti-primate CD45-BV786 or anti-human CD45 APC Cy7), a mouse CD45-specific antibody (anti-mouse CD45 FITC, eBiosciences clone 30-F11, catalog no. enriched from pigtail macaque BM as previously described (56). Lentivirus vector production.…”

Section: Methodsmentioning

confidence: 99%

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Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

et al. 2015

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Section: Methodsmentioning

confidence: 99%

“…After 12 days, plates were scored for hematopoietic colony phenotypes (CFU-E, CFU-M CFU-GM, CFU-GEMM) under a light microscope. Individual colonies from each experiment were randomly picked for Wright-Giemsa staining of cytospin samples and, in some studies, for analysis of proviral integration by PCR assay (56).…”

Section: Methodsmentioning

confidence: 99%

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

et al. 2015

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“…Hematopoietic recovery and long-term gene marking prior to MGMT P140K -mediated in vivo selection has been previously described for monkeys J02043, J02370, and T04228 (14). Monkey M05189, which received cells transduced with a tricistronic lentivirus vector containing an anti-HIV transgene (C46), MGMT P140K , and GFP, recovered complete blood counts, with normal engraftment kinetics (14) reaching an absolute neutrophil count (ANC) of more than 500 neutrophils per μl by day 17 (24).…”

Section: Efficient Mgmt P140k -Mediated In Vivo Selection and Chemoprmentioning

confidence: 99%

“…Successful HSC gene therapy has been unequivocally demonstrated for patients with X-linked SCID (SCID-X1) (4), adenosine deaminase deficient SCID (5), X-linked chronic granulomatous disease (X-CGD) (6), and adrenoleukodystrophy (ALD) (7). Numerous advances in transduction protocols, including transduction on fibronectin fragment CH-296 (8), the use of multiple cytokines (9,10), and the use of different vector pseudotypes (11)(12)(13), have contributed to gene transfer levels of 20%-30% in clinically relevant nonhuman primates (14). Even with the gene marking levels (defined as the percentage or fraction of genetically modified cells) achieved clinically ranging from approximately 9%-15% in peripheral blood with reduced-intensity busulfan (6) and high-intensity cyclophosphamide and busulfan (7) and levels in the nonhuman primate ranging from 20%-30% in peripheral blood with total body irradiation (TBI) (14), for numerous clinical applications gene marking levels will still likely be below therapeutic thresholds (for review see ref.…”

Section: Introductionmentioning

confidence: 99%

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

Beard¹,

Trobridge²,

Ironside³

et al. 2010

J. Clin. Invest.

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“…Lentiviral vector integrates into the host cell genome, and the foreign gene can be expressed stably and constitutively (24). It can also infect dividing and non-dividing cells, such as nerve cells, hematopoietic stem cells, and liver cells (25). The probability of lentiviral vector gene recombination is higher, the exogenous recombinant is stable, not easy to lose, and easy to be amplified in large amounts (26).…”

Section: Discussionmentioning

confidence: 99%

Construction and characterization of a PDCD5 recombinant lentivirus vector and its expression in tumor cells

et al. 2012

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Abstract. The function and mechanism of the programmed cell death 5 (PDCD5) gene is not completely understood, so it is necessary to build a stable and efficient PDCD5 recombinant lentiviral expression vector. The coding region of the PDCD5 gene was PCR amplified from pCMV-SPORT6. Next, the PDCD5 fragment and the pGC-FU vector were digested with the restriction enzyme AgeI and ligated by in-Fusion technology to build the pGC-FU-PDCD5 plasmid. Competent E. coli DH5α cells were transformed and positive clones were identified by PCR. The PCR products were digested and sequenced. After sequencing, positive clones were selected to grow and propagate. The pGC-FU-PDCD5 plasmid DNA was purified and mixed with the pHelper1.0 and pHelper2.0 packaging plasmids. They were co-transfected into 293T cells. The viral titer was measured by real-time PCR. The expression of GFP and PDCD5 was detected by both Western blotting and fluorescence microscopy. Additionally, the A498, ACHN, HCT116 and LoVo tumor cell lines were transfected with the virus supernatant, and PDCD5 expression was detected in these cells. The constructed pGC-FU-PDCD5 plasmid contained the correct PDCD5 gene sequence. Strong green fluorescence in both the cytoplasm and cell membrane was observed following transfection with purified pGC-FU-PDCD5 into 293T cells. The transfection rate was greater than 95%, and the expression of the PDCD5-GFP fusion protein was confirmed by Western blotting. The titer of the recombinant PDCD5 lentiviral condensed supernatant was measured to be 2x10 9 Tu/ml. The transfection efficiencies of A498, ACHN and HCT116 cells were greater than 90%. However, transfection efficiency of LoVo cells was lower but still greater than 70%. The PDCD5-GFP fusion protein was stable in these transfected tumor cells, as detected by Western blotting. In conclusion, a PDCD5 recombinant lentiviral vector was successfully constructed, and high expression of the plasmid was observed in tumor cells.

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Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

Cited by 61 publications

References 32 publications

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

Construction and characterization of a PDCD5 recombinant lentivirus vector and its expression in tumor cells

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