Hacke K, Treger J A, Bogan B T, Schiestl R H, Kasahara N
Department of Medicine, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA 90095, USA.
Transplant Proc. 2013 Jun;45(5):2040-4. doi: 10.1016/j.transproceed.2013.01.020.
We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.
我们最近开发了一种新颖且高效的策略，该策略专门使用嘌呤类似物6-硫鸟嘌呤（6TG）进行移植前预处理以及次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）缺陷型骨髓（BM）的移植后化学筛选。在小鼠BM移植模型中，联合使用6TG预处理和体内化学筛选始终能使HPRT缺陷型供体BM的植入率>95%，并在原发性和继发性受体中实现组织学和免疫表型正常造血的长期重建，且无明显毒性，也无需任何其他细胞毒性预处理方案。为了将这种联合6TG预处理和化学筛选的策略转化为临床可行的方法，有必要开发对正常造血干细胞（HSC）进行基因改造的方法，使其成为HPRT缺陷型，从而对6TG产生抗性。在此，我们研究了一种通过RNA干扰（RNAi）降低HPRT表达从而赋予原代小鼠BM细胞对6TG骨髓毒性保护作用的策略。相应地，我们构建并验证了一种表达靶向小鼠HPRT基因的短发夹RNA（shRNA）的慢病毒基因转移载体。我们的结果表明，慢病毒载体介导的靶向HPRT的shRNA递送能够有效且长期地降低HPRT表达。此外，在已建立的小鼠细胞系以及原代小鼠BM细胞中，用靶向HPRT的shRNA进行慢病毒转导均与体外对6TG细胞毒性的抗性增强相关。因此，这代表了一种在翻译上可行的方法，可对HSC进行基因工程改造，以实施6TG介导的预处理和体内化学筛选。
