D'Costa Jenice, Harvey-White Judith, Qasba Pankaj, Limaye Advait, Kaneski Christine R, Davis-Warren Alberta, Brady Roscoe O, Bankiewicz Krys S, Major Eugene O, Arya Suresh K
Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
J Med Virol. 2003 Oct;71(2):173-82. doi: 10.1002/jmv.10467.
Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by alpha-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer.
慢病毒载体是将基因导入分裂细胞和非分裂细胞（包括神经元细胞和干细胞）的主要候选载体。出于安全性考虑，HIV-2慢病毒载体可能比HIV-1慢病毒载体更适合用于人类基因转移。在HIV-1核心中进行交叉包装的HIV-2载体可能更安全。这些载体在疾病模型中的疗效证明将验证它们的实用性。帕金森病和法布里病为验证提供了极佳的模型。帕金森病是大脑中多巴胺能神经元的局灶性变性，产生神经递质多巴胺的能力逐渐丧失。目前的治疗方法是给予递增剂量的左旋多巴，但会带来毒性。我们在此探讨这样一个假说：该途径中的关键酶芳香酸脱羧酶（AADC）的基因转移将使神经元细胞更有效地将左旋多巴转化为多巴胺。另一方面，法布里病是一种单基因遗传病，其特征是α-半乳糖苷酶A（AGA）缺乏，导致包括成纤维细胞在内的几种细胞类型中糖脂蓄积。这两种疾病的临床前研究动物模型均已具备。我们设计了带有AADC转基因的单顺反子HIV-1和HIV-2载体，以及带有AGA和嘌呤霉素抗性转基因的单顺反子和双顺反子HIV-2载体。它们用HIV-2核心或HIV-1核心进行包装（杂交载体）。AADC基因在神经元细胞中的基因转移赋予了转导细胞将左旋多巴有效转化为多巴胺的能力。同样，AGA载体诱导法布里成纤维细胞产生高水平的AGA酶，并导致细胞内糖脂的快速清除。单顺反子和双顺反子载体均有效。因此，在双顺反子载体下游插入第二个基因并无有害影响。此外，自包装载体和交叉包装的杂交载体在基因转移方面均有效。
