SUMMARY Virus entry into cells is typically initiated by binding of virally encoded envelope proteins to specific cell surface receptors. Studying infectivity of lentivirus pseudotypes lacking envelope binding, we still observed high infectivity for some cell types. On further investigation, we discovered that this infectivity is conferred by the soluble bovine protein S in fetal calf serum, or Gas6, its human homologue. Gas6 enhances native infectivity of pseudotypes of multiple viral envelope proteins. Gas6 mediates binding of the virus to target cells by bridging virion envelope phosphatidylserine to Axl, a TAM receptor tyrosine kinase on target cells. Phagocytic clearance of apoptotic cells is known to involve bridging by Gas6. Replication of vaccinia virus, which was previously reported to use apoptotic mimicry to enter cells, is also enhanced by Gas6. These results reveal an alternative molecular mechanism of viral entry that can broaden host range and enhance infectivity of enveloped viruses.

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1-and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.Efficient targeting of specific cells to achieve stable transduction has been attempted by various strategies. Inserting ligands or single-chain antibodies into the retroviral receptor binding envelope subunit has been the most common approach used to alter and/or restrict the host range of retroviral vectors (1,5,7,8,13,14,17,(24)(25)(26). Bridging virus vector and cell by antibodies or ligands is another approach (3,20). In general, most strategies have suffered from inconsistent specificity and low viral titers as a result of modification of the retroviral envelope (1,5,9,13,17,(24)(25)(26). The modified envelope proteins appear to have specific binding activity but have low fusion activity (14, 28), resulting in inefficient entry into cells.The alphavirus Sindbis virus encodes two transmembrane envelope proteins, E1 and E2. E2 is responsible for receptor binding; E1 is responsible for pH-dependent fusion. Unlike retroviruses, the Sindbis virus fusogenic E1 protein can fuse to cells independently of the receptor binding E2 protein (23). Recently, vectors based upon the Sindbis virus RNA genome were constructed whereby the Sindbis virus E2 envelope protein was modified by insertion of an Fc-binding portion (ZZ domain) (12) of protein A (6, 18). These Sindbis virus vectors would bind to and enter cells bearing specific cell surface antigens only in the presence of the appropriate monoclonal antibody (MAb). However, as a lytic RNA virus, Sindbis virus is not suitable for applications requiring stable transduction (6, 21). We tested the possibility that human immunodeficiency virus type 1 (HIV-1)-based vectors could potentially be pseudotyped with Sindbis virus envelope, thereby conferring the targeting properties of the modified Sindbis virus envelope to the HIV-1 vector. MATERIALS AND METHODSPlasmid construction. The expression vector of Sindbis virus envelope protein, plntron SINDBIS, was made by cloning Sindbis virus envelope into pHCMV G (27), replacing the vesicular stomatitis virus G protein. The Sindbis virus envelope fragment was derived from the plasmid TOTO 2000 (kindly provided by Henry Huang). The envelope region of TOTO 2000 was deri...