Cai Shanbao, Ernstberger Aaron, Wang Haiyan, Bailey Barbara J, Hartwell Jennifer R, Sinn Anthony L, Eckermann Olaf, Linka Yvonne, Goebel W Scott, Hanenberg Helmut, Pollok Karen E
Department of Pediatrics, James Whitcomb Riley Hospital for Children and Indiana University School of Medicine, Indianapolis, IN 46202-5525, USA.
Exp Hematol. 2008 Mar;36(3):283-92. doi: 10.1016/j.exphem.2007.11.009.
Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy.
Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.
Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months.
In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.
采用临床相关的转导策略，我们研究了谱系阴性骨髓（Lin(neg) BM）中的造血干细胞能够在多大程度上被一种表达DNA修复蛋白O(6)-甲基鸟嘌呤DNA甲基转移酶（MGMT(P140K)）的泡沫病毒（FV）载体进行基因改造，并通过亚髓细胞消融或髓细胞消融烷化剂疗法在体内进行选择。
用FV载体MD9-P140K以低感染复数转导Lin(neg) BM，该载体共表达MGMT(P140K)和增强型绿色荧光蛋白，将其移植到C57BL/6小鼠体内，并用亚髓细胞消融或髓细胞消融烷化剂疗法对小鼠进行治疗。在移植后6个月分析骨髓中体内选择的MD9-P140K转导细胞的存在情况，随后将其移植到二级受体动物体内。
亚髓细胞消融治疗后，55%的小鼠骨髓中表达MGMT(P140K)。在约50%的定向骨髓来源祖细胞中观察到前病毒整合，对前病毒插入位点的分析表明每个转导的祖细胞集落最多有两个整合。用亚髓细胞消融疗法选择的转导骨髓细胞在移植后长达6个月内重建了二级受体小鼠。相比之下，对原受体小鼠进行髓细胞消融治疗后，只有25%存活。造血干细胞被转导，因为存活动物的骨髓细胞用MGMT(P140K)阳性细胞重建了二级受体长达5至6个月。
亚髓细胞消融治疗后，MD9-P140K转导的骨髓细胞在体内的选择比髓细胞消融治疗更有效。这些数据表明，必须存在临界数量的转导干细胞，才能产生足够数量的基因改造后代，以抵御与髓细胞消融治疗相关的急性毒性。
