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Despite recent improvements in the engineering of viral envelope proteins, it remains a significant challenge to create lentiviral vectors that allow targeted transduction to specific cell populations of interest. In this study, we developed a simple 'plug and play' strategy to retarget lentiviral vectors to any desired cell types through in vitro covalent modification of the virions with specific cell-targeting proteins (CTPs). This strategy exploits a disulfide bond-forming protein-peptide pair PDZ1 and its pentapeptide ligand (ThrGluPheCysAla, TEFCA). PDZ1 was incorporated into an engineered Sindbis virus envelope protein (Sind-PDZ1) and displayed on lentiviral particles while the TEFCA pentapeptide ligand was genetically linked to the CTP. Her2/neu-binding designed ankyrin repeat proteins (DARPin) were used as our model CTPs. DARPin-functionalized unconcentrated lentiviral vectors harboring Sind-PDZ1 envelope protein (Sind-PDZ1-pp) exhibited >800-fold higher infectious titer in HER2 + cells than the unfunctionalized virions (8.5 × 10 6 vs. <10 4 IU/mL). Moreover, by virtue of the covalent disulfide bond interaction between PDZ1 and TEFCA, the association of the CTP with the virions is nonreversible under non-reducing conditions (e.g. serum), making these functionalized virions potentially stable in an in vivo setting.

Previous studies conducted in our laboratory with Sindbis viral vectors in animal models demonstrated excellent in vivo targeting of tumor cells and significant reduction of metastatic implant size. To explore the influence of Sindbis strain on these factors, we constructed new plasmids from the wild-type Ar-339 Sindbis virus strain and compared their sequences. We found differences in the replicase and envelope proteins between JT, HRSP, and Ar-339 sequences. We made chimeras combining both strains and studied their efficiency in SCID mice bearing tumor xenograft using IVIS in vivo imaging techniques. We found that JT envelope proteins targeted tumors more efficiently than those of Ar-339, while the Ar-339 replicase showed increased efficacy in tumor reduction. To determine which residues are responsible for tumor targeting, we made mutants of Ar-339 E2 envelope protein and tested them by IVIS imaging in ES-2 tumor-bearing and tumor-free mice. The change of only one amino acid from E70 to K70 in Ar-339 E2 suppressed the ability to target metastatic tumor implants in mice. A K70 and V251 double E2 mutant did not reverse the loss of targeting capability. Only the mutant with JT E2 and Ar-339 helper targeted tumor, though with less intensity.