Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors

Abstract

On the basis of theoretical structural and comparative studies of various avian leukosis virus SU (surface) envelope proteins, we have identified four small regions (I, II, III, and IV) in their receptor-binding domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of subgroup A, we have constructed a set of SU mutants in which these regions were replaced by the coding sequence of FLA16, a 16-amino-acid RGD-containing peptide known to be the target for several cellular integrin receptors. Helper-free retroviral particles carrying a neo-lacZ retroviral vector were produced with the mutant envelopes. SU mutants in which regions III and IV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the processing and the viral incorporation of SU mutants. When FLA16 was inserted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or II were able to infect avian cells through the subgroup A receptor at levels similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found to promote binding of mammalian cells resistant to infection by subgroup A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment with N-glycosidase F have been used to infect these mammalian cells, and infections have been monitored by neomycin selection. No neomycin-resistant clones could be obtained after infection by viruses with wild-type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of the retroviral vector was found correctly integrated in cell DNA of these colonies. By using a blocking peptide containing the minimal adhesive RGD sequence contained in FLA16, we have shown that preincubation of target cells could specifically inhibit infection by viruses with FLA16.

Gene Transfer Technology in Therapy: Current Applications and Future Goals

“…This approach consists of placing foreign genes into the retroviral envelope in order to confer a cell tropism specific for certain human cell types. The foreign genes used in the early studies to generate hybrid envelopes were: CD4 [80,81], single-chain antibodies [82][83][84], the polypeptide erythropoietin [85], short peptides binding to several integrins [86], and human heregulin [87]. The retroviral systems used in these studies were: avian leukosis virus [80,86], ecotropic MLV [81,82,85,87], spleen necrosis virus [83,84], and amphotropic MLV [88].…”

“…The foreign genes used in the early studies to generate hybrid envelopes were: CD4 [80,81], single-chain antibodies [82][83][84], the polypeptide erythropoietin [85], short peptides binding to several integrins [86], and human heregulin [87]. The retroviral systems used in these studies were: avian leukosis virus [80,86], ecotropic MLV [81,82,85,87], spleen necrosis virus [83,84], and amphotropic MLV [88]. In some cases, there has been a partial success in redirecting the cell tropism of ecotropic retroviruses [81,[83][84][85][86][87][88], but the transduction efficiency is far from being optimal for in vivo applications.…”

“…The retroviral systems used in these studies were: avian leukosis virus [80,86], ecotropic MLV [81,82,85,87], spleen necrosis virus [83,84], and amphotropic MLV [88]. In some cases, there has been a partial success in redirecting the cell tropism of ecotropic retroviruses [81,[83][84][85][86][87][88], but the transduction efficiency is far from being optimal for in vivo applications. A number of more recent reports have shown some improvement of transduction efficiency by chimeric viral particles with altered cell tropism [89][90][91].…”

Gene Therapy for Brain Tumors

“…The anti-angiogenic effect of the conditioned culture medium was absent when wildtype p53 gene expression was switched off in the glioblastoma cells using a tetracycline-regulated promoter unit (see section 2.5) or when mutated p53 had been introduced instead of the wildtype gene. Thus, Van Meir et al (1994) postulated the presence of a glioma-derived angiogenesis inhibitory factor (GD-AIF) upon expression of wildtype p53. This assumption should be explored further as an antiangiogenic gene therapy approach for brain tumor treatment.…”

Prospects for using retroviral vectors for human gene therapy

“…More recently, Kasahara et al [1994] have shown that retroviral virions with a chimeric protein containing the hormone erythropoietin and part of the envelope protein can infect only cells bearing the erythropoietin receptor. Valsesia-Wittmann et al [1994] have shown that retroviral virions with integin-envelope chimeric proteins can specifically infect cells through the integrin receptor. These results suggest, for the first time, that it may be possible to use retroviral vectors to target genes to specific cell types.…”