Cell Type–Specific Targeting with Sindbis Pseudotyped Lentiviral Vectors Displaying Anti-CCR5 Single-Chain Antibodies

Abstract

Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the present work, we attempt to generate viral particles for targeting gene delivery. We have used CCR5-positive cells as the target for our strategy. Therefore, we developed a novel Sindbis pseudotyped lentiviral vector where the Sindbis receptor binding envelope protein was modified to directly encode a single-chain antibody fragment (scFv) against the CCR5 chemokine receptor. We have generated two chimeric scFv-Sindbis envelopes, varying the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv. The two chimeric scFv-Sindbis envelopes were successfully incorporated into lentiviral-derived vectors, and the resulting pseudotyped viral particles showed specific targeting to CCR5-expressing cells. However, our data demonstrate that the length of the peptide linker significantly affects the efficiency of infection. Pseudotyped viral particles, which display single-chain antibody fragments with longer peptide linkers, allowed higher titers of infection. The present study can be a model strategy for specific gene delivery mediated by lentiviral vectors pseudotyped with Sindbis envelope displaying scFv that recognizes specific cellular surface proteins. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.

Molecular Engineering of Viral Gene Delivery Vehicles

“…This limitation can be circumvented by coexpressing engineered and wt envelope proteins on virus particles, but with a loss of targeting specificity (111). Alternatively, transduction specificity may be improved by inserting additional cellspecific peptides, such as protease cleavable peptides, between scFv and envelope proteins (113)(114)(115).…”

Lentiviruses with trastuzumab bound to their envelopes can target and kill prostate cancer cells

“…Lentiviruses pseudotyped with the modified Sindbis virus envelope protein have been shown to bind and enter cells when an appropriate monoclonal antibody is attached. 17,18,31,33 We used this approach to show that lentiviruses engineered to express this Fc-binding domain of protein A and then incubated with antibodies to EGFR or HER-2 could readily infect C4-2 ( Figure 5) and LNCaP cells (data not shown). As EGFR is more ubiquitous and often associated with normal epithelia cells, HER-2 is the preferred target antigen for malignant cells.…”

“…Pseudotyping of lentivirus has recently been accomplished using Sindbis virus envelope glycoproteins. 17,18 Sindbis virus belongs to the alphavirus family and is classified within the Togaviridae virus family (genus Alphavirus). 19 The alphaviruses have highly organized icosahedral protein shells with an associated membrane.…”

“…17,32 Through selection of appropriate antibodies, these customized lentiviruses have been shown to be capable of specifically targeting many different cell types. 17,18,31,33 In this study, we bound the therapeutic antibody trastuzumab to lentiviruses pseudotyped with an engineered Sindbis virus envelope protein and then used these vectors to mediate very efficient in vitro and in vivo delivery into androgen-sensitive LNCaP and castration-resistant/ metastatic C4-2 human prostate cancer cells for cellselective expression of either a reporter gene or the therapeutic gene, thymidine kinase, which is able to activate the pro-drug ganciclovir (GCV) into a cytotoxic form.…”

Functional Pseudotyping of Human Immunodeficiency Virus Type 1 Vectors by Western Equine Encephalitis Virus Envelope Glycoprotein

“…Recent examples include glycoproteins from Venezuelan equine encephalitis (VEEV) (10), Semliki Forest (7), Ross River (7,8), and Sindbis (1,14) viruses. In the last case, specificity has been enhanced by inserting various ligand recognition domains within the viral E2 gene (1,14,15). While these pseudotyped particles may broaden the host range of the HIV vector, the titer varies.…”