In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene
In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene
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DOI：
10.1172/jci37506
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发表时间：
2009-07-01
影响因子：
15.9
通讯作者：
Sorrentino, Brian P.
中科院分区：
文献类型：
--
作者：
Larochelle, Andre;Choi, Uimook;Sorrentino, Brian P.
使用HSC的基因治疗的主要限制是低基因转移效率和大多数治疗基因不能赋予基因校正的细胞选择性优势。体内富集基因修饰细胞的一种方法是在逆转录病毒载体中包括耐药性基因，例如DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶（MGMT*）的P140 K突变体。我们用编码MGMT* 和荧光标记的慢病毒载体转导的CD 34（+）细胞移植5只恒河猴，有或没有同源框B4（HOXB 4），一种有效的干细胞自我更新基因。移植后几个月，淋巴和骨髓谱系中的转基因表达和共同整合位点证实了长期再生HSC的转导。然而，所有的动物都表现出基因标记的淋巴和骨髓细胞后，O-6-苄基鸟嘌呤（BG）和替莫唑胺（TMZ）管理只是一个短暂的增加。在1只动物中，用MGMT* 慢病毒载体转导的细胞在BG/TMZ的多个疗程后得到保护和扩增，从而提供TMZ的最大耐受剂量的显著增加。使用1，3-双-（2-氯乙基）-1-亚硝基脲（BCNU）的额外化疗周期导致基因标记水平的类似增加，但引起高水平的非造血毒性。在MGMT* 载体中包含HOXB 4在化疗治疗后没有导致基因标记或HSC扩增的实质性增加。因此，我们的数据表明，慢病毒介导的基因转移在移植的造血干细胞可以提供在体内的祖细胞的化学保护，虽然选择的长期再增殖的造血干细胞没有看到。
Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34(+) cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O-6-benzylguanine (BG) and temozolomide (TMZ) administration. In I animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.