Walsh C E, Liu J M, Miller J L, Nienhuis A W, Samulski R J
Clinical Hematology Branch, NHLBI/NIH, Bethesda, Maryland 20892.
Proc Soc Exp Biol Med. 1993 Dec;204(3):289-300. doi: 10.3181/00379727-204-43665.
Gene transfer of human globin genes into human pluripotent stem cells via viral vectors may soon be realized. The high level of globin gene expression believed to be required for the treatment of severe hemoglobinopathies necessitated the inclusion of cis-acting sequences (LCR). Retroviral vectors containing the LCR elements are prone to rearrangement, low titer, and poor expression. Inclusion of a "minilocus" containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction. Recombinant AAV is among the newest of genetic transfer vectors. This once obscure virus possesses unique properties that distinguish it from all other vectors. Its major advantage is the lack of pathogenicity in humans. Wild-type AAV has the unusual ability to selectively integrate into the mammalian genome at a specific region, thus reducing the concern for genomic disruption and insertional mutagenesis. The ability of AAV to carry regulatory elements without interference from the viral template may enable greater control of transferred gene expression. Disadvantages currently include the inferior packaging systems which yield low numbers of recombinant virions which are contaminated with wild-type adenovirus. The small AAV genome that can be packaged (approximately 5 kb) rules out its use for transfer of larger genes. Recombinant AAV viruses do not appear to demonstrate the same site-specific genomic integration as wild-type viruses. Elucidation of the mechanism of site-specific integration should prove useful in the development of safe vectors for gene transfer as well as provide insight into the nature of DNA recombination in humans.
通过病毒载体将人类珠蛋白基因转移到人类多能干细胞中可能很快就能实现。治疗严重血红蛋白病被认为需要高水平的珠蛋白基因表达，因此必须包含顺式作用序列（LCR）。含有LCR元件的逆转录病毒载体容易发生重排、滴度低且表达不佳。包含一个与珠蛋白基因相连的含有四个HS位点的“微型基因座”，在移植小鼠中导致了更高的表达，但前病毒仍会发生重排，而且这些实验对于人类骨髓干细胞转导有何意义尚不清楚。重组腺相关病毒（AAV）是最新的基因转移载体之一。这种曾经鲜为人知的病毒具有使其有别于所有其他载体的独特特性。其主要优点是对人类无致病性。野生型AAV具有在特定区域选择性整合到哺乳动物基因组中的非凡能力，从而减少了对基因组破坏和插入诱变的担忧。AAV携带调控元件而不受病毒模板干扰的能力，可能使对转移基因表达的控制更强。目前的缺点包括包装系统较差，产生的重组病毒粒子数量少且被野生型腺病毒污染。可包装的小AAV基因组（约5 kb）排除了其用于转移较大基因的可能性。重组AAV病毒似乎没有表现出与野生型病毒相同的位点特异性基因组整合。阐明位点特异性整合机制，对于开发安全的基因转移载体应是有用的，同时也能深入了解人类DNA重组的本质。
