Ponnazhagan S, Nallari M L, Srivastava A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5120.
J Exp Med. 1994 Feb 1;179(2):733-8. doi: 10.1084/jem.179.2.733.
We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human alpha-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neoR) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that express high levels of alpha-globin mRNA. Clonal populations of neoR cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and alpha-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus-infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense alpha-globin sequences showed no inhibition of expression of the endogenous alpha-globin gene, the SV40 promoter and the alpha-globin gene promoter-driven antisense alpha-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of beta-thalassemia.
由于游离α-珠蛋白的积累会缩短β地中海贫血患者红细胞的寿命，我们试图研究基于腺相关病毒2（AAV）的载体抑制人类α-珠蛋白基因产物过量产生的有效性，以开发一种针对β地中海贫血的治疗方法。我们构建了重组AAV病毒粒子，其分别包含由疱疹病毒胸苷激酶（TK）启动子、SV40早期基因启动子和人类α-珠蛋白基因启动子驱动的反义方向的人类α-珠蛋白基因序列，以及一个作为选择标记的对新霉素（neoR）具有抗性的细菌基因。这些重组病毒粒子用于感染表达高水平α-珠蛋白mRNA的人类红白血病细胞系（K562）。在用新霉素类似物药物G418选择后获得了neoR细胞的克隆群体。对从这些细胞中分离的总基因组DNA样本进行Southern印迹分析，以记录转导的neo和α-珠蛋白基因的稳定整合。还通过Northern印迹分析了从模拟感染和重组病毒感染培养物中分离的总细胞RNA样本。虽然TK启动子驱动的反义α-珠蛋白序列未显示对内源性α-珠蛋白基因表达的抑制，但SV40启动子和α-珠蛋白基因启动子驱动的反义α-珠蛋白序列在转录水平分别将该组成型过表达基因的表达抑制了约29%和91%。这些研究表明利用基于AAV的反义基因转移方法治疗β地中海贫血具有可行性。
