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Review
. 2013 Jan;8(1):107-21.
doi: 10.2217/fmb.12.122.

Library screening and receptor-directed targeting of gammaretroviral vectors

Peter M Mazari  1 , Monica J Roth
Affiliations
Review

Library screening and receptor-directed targeting of gammaretroviral vectors

Peter M Mazari et al. Future Microbiol. 2013 Jan.

Abstract

Gene- and cell-based therapies hold great potential for the advancement of the personalized medicine movement. Gene therapy vectors have made dramatic leaps forward since their inception. Retroviral-based vectors were the first to gain clinical attention and still offer the best hope for the long-term correction of many disorders. The fear of nonspecific transduction makes targeting a necessary feature for most clinical applications. However, this remains a difficult feature to optimize, with specificity often coming at the expense of efficiency. The aim of this article is to discuss the various methods employed to retarget retroviral entry. Our focus will lie on the modification of gammaretroviral envelope proteins with an in-depth discussion of the creation and screening of envelope libraries.

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Figures

Figure 1
Figure 1. Approaches to alter retroviral receptor usage
(A) Entry of a gammaretrovirus. The envelope (Env) protein consists of a trimer of the SU and TM proteins. The VRA and VRB function in receptor binding. The host cell receptor is depicted as a multiple transmembrane protein (Table 1). (B) Viral pseudotyping. The Env protein from an alternative virus (schematically depicted as sindbis virus) can associate with retroviral particles. The pseudotyped Env will bind to its cognate receptor, which is not limited to multiple transmembrane proteins [–40]. (C) Antibody conjugation systems. Antibodies can be nonspecifically bound to modified Env proteins to direct viral entry. For sindbis virus, the mutation of the receptor-binding domains and insertion of the protein A ZZ domain allows for association with IgG molecules. Antibody binding to antigen delivers viral particles to cells [–61]. (D) DNA shuffling. Combinations of related viral species (schematically shown using a six-color gradient) are mixed during PCR, allowing for the generation of complex chimeras derived from mixed portions of all of the parental sequences. These chimeras are then selected for properties including altered receptor recognition or protein stability [93]. (E) Randomized VRA or molecular replacements. For feline leukemia virus, substitutions within the VRA region are known to alter receptor usage. By randomizing 11 amino acids within the VRA, libraries of random Env proteins are generated and have been screened for functional entry into cells. The cognate receptors need to be identified. This method has identified novel Env/receptor pairs [,,,–100,103]. Alternatively, through the use of molecular modeling, specific substrates have been engineered into the VRA region of murine leukemia virus, allowing entry through the somatostatin receptor [84]. (F) Insertion of additional domains to either target or block the wild-type Env receptor-binding domain. For blocking domains, the cleavage by a host cell protease results in the release of the virus in the vicinity of the targeted cell [–89]. Additional binding domains function to bind alternative receptors, but can allow the wild-type Env to function as a trigger for membrane fusion. (G) Domain substitutions. Binding domains can be used to substitute for a large section of the surface subunit protein. Examples of these types of substitutions include single-chain antibodies [,–55]. (H) Complementation studies. Viral entry involves more than receptor binding and requires a complex series of conformational changes to allow for membrane fusion. Viruses capable of binding, but not fusion, can be complemented with alternative fusogenic Env proteins [57,81,82]. SU: Surface subunit; TM: Transmembrane subunit; VRA: Variable region A; VRB: Variable region B; VRC: Variable region C; ZZ: IgG binding domain.
Figure 2
Figure 2. Generation of feline leukemia virus envelope variable region A 11-amino acid randomized library and the selection of isolates in tissue culture for functional entry
Individual steps are shown schematically. The FeLV-A Env is first modified to remove a 14-amino acid region encoding the receptor-binding domain and replaced with a stuffer fragment encoding back-to-back BbsI type 2 restriction sites. This modified vector is used to create the library, randomizing 11 amino acids of the receptor-binding domain through ligation of three oligonucleotides that regenerate the BbsI overhangs. The expression vector is murine leukemia virus based, and expresses both the env gene and a selectable marker (neo). Viral particles are assembled through introduction into producer cells expressing the gag and pol genes. Viruses released can be used directly to screen for viral entry. Alternatively, the genes can be transferred using VSV-G-pseudotyped virus at a low MOI to establish a constitutive producing library. Viruses released from the constitutive library can be used for screening gene delivery on the target cells of interest. Productive infection is scored by the transfer of neoR, after selection in G418. Env isolates are subsequently identified through PCR analysis of the env gene and further characterized for tropism and receptor usage. FeLV-A: Feline leukemia virus-A; LTR: Long terminal repeat; MOI: Multiplicity of infection; TGA: Stop codon; VRA: Variable region A; VSV-G: Vesicular stomatitis virus; WPRE: Woodchuck post-transcriptional regulatory element.
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