与MGMT和普遍存在的或红细胞特异性GFP慢病毒的协同转导允许在体内富集双阳性造血祖细胞。

ISRN Hematology Pub Date : 2012-01-01 Epub Date: 2012-07-19 DOI:10.5402/2012/212586
Justin C Roth, Mourad Ismail, Jane S Reese, Karen T Lingas, Giuliana Ferrari, Stanton L Gerson
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引用次数: 6

摘要

MGMT的P140K点突变体允许在体内大量富集造血干细胞(HSC)。因此,结合MGMT和治疗性基因表达的双基因载体可以在动物模型中富集基因校正的造血干细胞。然而,双基因载体的表达水平往往降低一个或两个基因。此外,在细胞分化的不同阶段表达选择和治疗基因可能是可取的。在这方面,我们评估了两种单独的单基因慢病毒是否可以有效地共转导造血细胞,包括用于双阳性细胞富集的MGMT。使用一系列MGMT: GFP病毒比率、moi和体外选择严格度来评估共转导效率。当每种病毒的比例相等时,共转导效果最佳,但选择后,低MGMT: GFP病毒比例导致双阳性细胞比例最高。该策略随后在小鼠模型中进行了评估,用于体内选择与普遍存在的MGMT表达载体和红细胞特异性GFP载体共转导的造血干细胞。虽然在移植受体中MGMT和GFP的表达百分比不同,但药物选择使MGMT阳性白细胞和GFP阳性红细胞群富集。这些数据表明,共转导是体内快速富集和评估治疗性慢载体的手段。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo.

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo.

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo.

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo.

The P140K point mutant of MGMT allows robust hematopoietic stem cell (HSC) enrichment in vivo. Thus, dual-gene vectors that couple MGMT and therapeutic gene expression have allowed enrichment of gene-corrected HSCs in animal models. However, expression levels from dual-gene vectors are often reduced for one or both genes. Further, it may be desirable to express selection and therapeutic genes at distinct stages of cell differentiation. In this regard, we evaluated whether hematopoietic cells could be efficiently cotransduced using low MOIs of two separate single-gene lentiviruses, including MGMT for dual-positive cell enrichment. Cotransduction efficiencies were evaluated using a range of MGMT : GFP virus ratios, MOIs, and selection stringencies in vitro. Cotransduction was optimal when equal proportions of each virus were used, but low MGMT : GFP virus ratios resulted in the highest proportion of dual-positive cells after selection. This strategy was then evaluated in murine models for in vivo selection of HSCs cotransduced with a ubiquitous MGMT expression vector and an erythroid-specific GFP vector. Although the MGMT and GFP expression percentages were variable among engrafted recipients, drug selection enriched MGMT-positive leukocyte and GFP-positive erythroid cell populations. These data demonstrate cotransduction as a mean to rapidly enrich and evaluate therapeutic lentivectors in vivo.

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