Maxwell I H, Maxwell F, Rhode S L, Corsini J, Carlson J O
University of Colorado Cancer Center, Denver 80262.
Hum Gene Ther. 1993 Aug;4(4):441-50. doi: 10.1089/hum.1993.4.4-441.
Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or beta-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (-) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.
基于自主细小病毒LuIII基因组构建的重组体，是通过用与病毒P4启动子相连的荧光素酶或β - 半乳糖苷酶报告基因取代感染性克隆（pGLu883）中的病毒编码序列而构建的。在用这些构建体中的任何一个与提供LuIII非结构蛋白和衣壳蛋白的质粒共转染的细胞中，重组基因组发生切除和复制。通过检测暴露于该培养基的受体细胞中的报告基因活性可知，转导病毒粒子在共转染细胞的培养基中积累。转导活性可被抗LuIII血清中和。在共转染HeLa、293或NB324K细胞后，观察到复制型DNA和转导病毒粒子的产生，效率依次增加。当重组基因组与辅助构建体中病毒基因侧翼序列存在同源性时，有时会观察到具有复制能力的细胞病变病毒的伴随产生。通过从辅助构建体中去除左端同源性可将其降至最低；通过这种方法，获得了不含具有复制能力病毒的荧光素酶转导病毒制剂。用这些制剂，我们在受体HeLa细胞中观察到荧光素酶表达（3天后下降）持续长达7天。重组病毒DNA与链特异性荧光素酶探针的杂交表明两条链均被包装（如LuIII报道的那样），但（ - ）链过量几倍。我们认为转导自主细小病毒在基因转移应用中将会有用，可能包括仅需要瞬时表达时的基因治疗。
