Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.
Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.
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逆转录病毒介导的人腺苷脱氨酶基因在腺苷脱氨酶缺陷的人的二倍体皮肤成纤维细胞中的有效转移和表达。
DOI：
10.1073/pnas.84.4.1055
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发表时间：
1987
影响因子：
11.1
通讯作者：
Miller,AD
中科院分区：
文献类型：
--
作者：
Palmer,TD;Hock,RA;Osborne,WR;Miller,AD
皮肤成纤维细胞可能被认为是治疗基因的合适接受者，可以治愈多种人类遗传疾病；然而，这些细胞对大多数方法的基因转移都有抵抗力。我们研究了逆转录病毒载体将基因转移到正常人二倍体皮肤成纤维细胞中的能力。携带新霉素或潮霉素 B 抗性基因的逆转录病毒在单次暴露于含病毒的培养基后，使超过 50% 的人类成纤维细胞产生耐药性。这表明效率比其他方法至少提高了 500 倍。通过使用双嗜性逆转录病毒包装细胞，在没有辅助病毒的情况下实现了转移。构建了含有人腺苷脱氨酶 (ADA) cDNA 的逆转录病毒载体，并用于感染 ADA 缺陷患者的 ADA 成纤维细胞。受感染的细胞产生的 ADA 酶比正常个体的成纤维细胞多 12 倍，并且能够快速代谢外源性脱氧腺苷和腺苷，这些代谢物在 ADA 缺陷患者的血浆中积累，是导致这些患者严重联合免疫缺陷的原因。这些实验表明逆转录病毒介导的基因转移到人类成纤维细胞中用于基因治疗的潜力。
Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.