Complement-protected amphotropic retroviruses from murine packaging cells

The application of retroviruses generated from murine cells for in vivo gen e therapy is restricted primarily because of the rapid inactivation of thes e viruses by the human complement system, To circumvent this disadvantageou s property of murine retroviruses we have generated infectious amphotropic retroviruses that exhibit strong protection against human complement attack , The membrane of these viruses contains a fusion protein, DAFF2A that is c omposed of the catalytic domain of the human complement regulatory protein (CRP) decay-accelerating factor (DAF) and the envelope protein of the ampho tropic murine leukemia virus (MuLV) 4070A (EnvA), The fusion of two other C RPs, MCP and CD59, to the same amphotropic Env moiety did not lead to equiv alent results, The fusion protein DAFF2A was stably expressed in mouse NIH 3T3-based helper cells and independently identified with either alpha-DAF M Ab or alpha-Env PAb on the cell membrane, Western blot analysis confirmed t he expected molecular weight of the fusion protein. Viral titers obtained f rom NIH 3T3 helper cell pools were 5 x 10(5) CFU for wild-type amphotropic EnvA virus and 1 x 105 CFU for DAFF2A virus, respectively, By blocking the catalytic domain of DAF by pretreatment with alpha-DAF MAb DAFF2A, recombin ant virions could be converted to wild-type with respect to sensitivity aga inst human serum, Since the method for producing virions that are protected against human serum should be applicable to any cell type it offers a nove l tool for human in vivo gene therapy.