Bahner I, Kearns K, Hao Q L, Smogorzewska E M, Kohn D B
Department of Pediatrics, University of Southern California School of Medicine, California 90027, USA.
J Virol. 1996 Jul;70(7):4352-60. doi: 10.1128/JVI.70.7.4352-4360.1996.
Genetic modification of hematopoietic stem cells with a synthetic "anti-human immunodeficiency virus type 1 (HIV-1) gene" which inhibits replication of HIV-1 may allow production of mature lymphoid and monocytic cells resistant to HIV-1 growth after autologous transplantation. Because productive HIV-1 replication requires binding of the Rev protein to the Rev-responsive element (RRE) within the viral transcripts for the HIV-1 structural proteins, anti-HIV-1 gene products which interfere with Rev-RRE interactions may inhibit HIV-1 replication. One such strategy involves overexpression of the RRE sequences in transcripts derived from retroviral vectors to act as decoys to sequester Rev protein and prevent its binding to the RRE element in HIV-1 transcripts. We developed an in vitro model to test the efficacy of this gene therapy approach in primary human hematopoietic cells. Human CD34+ hematopoietic progenitor cells from normal bone marrow or umbilical cord blood were transduced with retroviral vectors carrying RRE decoy sequences as part of a long terminal repeat-directed transcript expressing the neo gene (L-RRE-neo) or with a control vector expressing only the neo gene (LN). The transduced progenitors were allowed to differentiate into mature myelomonocytic cells which were able to support vigorous growth of the monocytotropic isolate of HIV-1, JR-FL. HIV-1 replication was measured in unselected cell populations and following G418 selection to obtain uniformly transduced cell populations. Inhibition of HIV-1 replication in the unselected cell cultures was between 50.2 and 76.7% and was highly effective (99.4 to 99.9%) in the G418-selected cultures. Progenitors transduced by either the L-RRE-neo vector or the control LN vector were identical with respect to hematopoietic growth and differentiation. These findings demonstrate the ability of an RRE decoy strategy to inhibit HIV-1 replication in primary human myelomonocytic cells after transduction of CD34+ progenitor cells, without adverse effects on hematopoietic cell function.
用一种合成的“抗人类免疫缺陷病毒1型（HIV-1）基因”对造血干细胞进行基因改造，该基因可抑制HIV-1的复制，这可能使自体移植后产生对HIV-1生长具有抗性的成熟淋巴细胞和单核细胞。由于HIV-1的有效复制需要Rev蛋白与HIV-1结构蛋白病毒转录本中的Rev反应元件（RRE）结合，因此干扰Rev-RRE相互作用的抗HIV-1基因产物可能会抑制HIV-1的复制。一种这样的策略涉及在逆转录病毒载体衍生的转录本中过表达RRE序列，以作为诱饵来隔离Rev蛋白并阻止其与HIV-1转录本中的RRE元件结合。我们开发了一种体外模型来测试这种基因治疗方法在原代人造血细胞中的疗效。来自正常骨髓或脐带血的人CD34+造血祖细胞用携带RRE诱饵序列的逆转录病毒载体转导，该序列作为表达新霉素基因（L-RRE-neo）的长末端重复序列定向转录本的一部分，或者用仅表达新霉素基因的对照载体（LN）转导。转导的祖细胞被允许分化为成熟的骨髓单核细胞，这些细胞能够支持HIV-1的嗜单核细胞分离株JR-FL的旺盛生长。在未选择的细胞群体中以及在G418选择后测量HIV-1复制，以获得均匀转导的细胞群体。在未选择的细胞培养物中，HIV-1复制的抑制率在50.2%至76.7%之间，在G418选择的培养物中非常有效（99.4%至99.9%）。用L-RRE-neo载体或对照LN载体转导的祖细胞在造血生长和分化方面是相同的。这些发现证明了RRE诱饵策略在转导CD34+祖细胞后抑制原代人骨髓单核细胞中HIV-1复制的能力，而对造血细胞功能没有不利影响。
