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Methotrexate resistance gene transfer in stem cells.

机译：甲氨蝶呤抗性基因在干细胞中的转移。

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摘要

Gene modification of hematopoietic stem cells (HSCs) has the potential to cure genetic, malignant and acquired diseases. Despite success in pre-clinical gene therapy studies, achieving genetic correction or a therapeutic response in humans has been challenging. HIV-1-based lentivirus vectors have come to the forefront of pre-clinical studies due to their ability to more effectively transduce quiescent HSCs. Drug resistance gene expression coupled to chemotherapy after HSC transplantation may support in vivo selection of gene-modified cells while protecting the patient from chemotoxicity. We hypothesized that lentivirus-mediated transfer of a methotrexate (MTX) resistance gene, Tyr22-dihydrofolate reductase (Tyr22-DHFR), into stem cells would support long-term stable gene expression in vivo and protect hematopoietic daughter cells from MTX toxicity. To test our hypothesis, we first generated high-titer lentivirus vectors expressing Tyr22-DHFR and green fluorescent protein (GFP) in different genetic configurations, and then compared MTX resistance, enzyme activity and GFP fluorescence in mouse and human cell lines including human embryonic stem cells (hESCs). Tyr22-DHFR-HSCs protected transplanted mice from MTX myelotoxicity, and conferred a significant survival advantage compared to MTX treated GFP-HSC transplanted mice. To assess the feasibility of a physiologic scale-up in a large animal model, we demonstrated DHFR-GFP expression in canine CD34+ cells and long-term engraftment of gene-modified cells in vivo. MTX administration increased gene-marking in the peripheral blood of one dog, without causing cytopenia. We also defined the optimal priming of HSCs (c-G-CSF/c-SCF BM), transduction conditions and MTX tolerated doses in dogs. Finally, we present a novel application of selective expansion of hESCs-derived cells in mouse xenografts. Methotrexate-resistant (MTXr)-DHFR hESCs gave rise to MTXr-GFP+ teratomas, indicating that gene-modified cells retain their pluripotency during MTX treatment. MTXr-hESCs placed in stromal cell co-culture differentiated into GFP+ hematoendothelial cells, including CD34+CD45 + subsets, which subsequently gave rise to MTXr-hematopoietic colony forming cells (CFCs). Finally, we showed that MTX administration of mice bearing hESC xenografts supported in vivo selection of Tyr22-DHFR-hESC-hematopoietic cells and increased engraftment of gene-modified cells in the bone marrow of treated mice. Taken together, these results show that lentivirus vectors effectively transduce MTXr-DHFR into HSCs, thereby preventing life-threatening myelotoxicity (as observed in our mouse studies), and supporting long-term engraftment of gene-modified cells in vivo. These studies mark significant progress of MTX resistance gene therapy toward clinical trials in humans.
机译：造血干细胞（HSC）的基因修饰具有治愈遗传，恶性和获得性疾病的潜力。尽管在临床前基因疗法研究中取得了成功，但在人类中实现基因校正或治疗反应仍具有挑战性。基于HIV-1的慢病毒载体因其能够更有效地转导静态HSC的能力而成为临床前研究的前沿。 HSC移植后与化疗偶联的耐药基因表达可支持体内选择基因修饰的细胞，同时保护患者免受化学毒性。我们假设慢病毒介导的甲氨蝶呤（MTX）抗性基因Tyr22-二氢叶酸还原酶（Tyr22-DHFR）进入干细胞的转移将支持体内长期稳定的基因表达，并保护造血子细胞免受MTX毒性。为了检验我们的假设，我们首先生成了高滴度慢病毒载体，它们以不同的遗传结构表达Tyr22-DHFR和绿色荧光蛋白（GFP），然后在小鼠和人类细胞系（包括人类胚胎干）中比较了MTX的耐药性，酶活性和GFP荧光细胞（hESC）。与经MTX处理的​​GFP-HSC移植小鼠相比，Tyr22-DHFR-HSC可以保护移植的小鼠免受MTX的骨髓毒性，并具有显着的生存优势。为了评估在大型动物模型中进行生理放大的可行性，我们证明了DHFR-GFP在犬CD34 +细胞中的表达以及体内基因修饰细胞的长期植入。施用MTX可增加一只狗外周血中的基因标记，而不会引起血细胞减少症。我们还定义了狗中HSC（c-G-CSF / c-SCF BM）的最佳启动，转导条件和MTX耐受剂量。最后，我们提出了在小鼠异种移植物中选择性扩增hESCs衍生细胞的新应用。耐甲氨蝶呤（MTXr）-DHFR hESCs产生了MTXr-GFP +畸胎瘤，表明基因修饰的细胞在MTX处理期间保留了其多能性。放置在基质细胞共培养物中的MTXr-hESCs分化为GFP +血内皮细胞，包括CD34 + CD45 +亚群，随后产生了MTXr-造血集落形成细胞（CFC）。最后，我们显示了对携带hESC异种移植物的小鼠进行MTX支持在体内选择Tyr22-DHFR-hESC造血细胞，并增加了基因修饰细胞在治疗小鼠骨髓中的植入。综上所述，这些结果表明，慢病毒载体可有效地将MTXr-DHFR转化为HSC，从而防止威胁生命的骨髓毒性（如在我们的小鼠研究中观察到的），并支持基因修饰的细胞在体内的长期移植。这些研究标志着MTX抗性基因疗法在人类临床试验中的重大进展。

著录项

  • 作者

    Gori, Jennifer Leah.;

  • 作者单位

    University of Minnesota.;

  • 授予单位 University of Minnesota.;
  • 学科 Biology Molecular.;Biology Microbiology.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 168 p.
  • 总页数 168
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;微生物学;
  • 关键词

  • 入库时间 2022-08-17 11:39:01

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