Antibody-Directed Targeting of Retroviral Vectors via Cell Surface Antigens

Abstract

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...

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.…”

“…26 Dubuisson and Rice 30 showed that the receptor-binding properties of Sindbis virus could be disrupted by insertion of short peptides (11 aa) into defined regions of the E2 glycoprotein but with retention of all other replication functions. An IgGbinding domain (ZZ domain) of Staphylococcus aureus protein A has been inserted within the E2 glycoprotein 31 and lentivirus or Sindbis vectors have been constructed with this modified envelope. By incubating with any IgG antibody, the ZZ domain and the Fc region of IgG form a tight complex.…”

“…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.…”

Surface-engineering of lentiviral vectors

“…Likewise, screening of a large panel of pseudotyped vectors established the superiority of the gibbon ape leukemia virus (GALV) and the cat endogenous retroviral glycoproteins (RD114) for transduction of progenitor and differentiated hematopoietic cells [22,34,[42][43][44][45]. Finally, owing to the capacity of the influenza virus, Sindis virus, MLV and GALV glycoproteins, among others, to pseudotype MLV and lentiviral vectors [22,25,46], several Engineering Lentiviral Vectors S85 approaches can be undertaken to modify their tropism in order to modify the host range of vectors (see below).…”

“…Non-retroviral glycoproteins include those derived from vesiculoviruses [9,10], lyssaviruses [11,12], arenaviruses [13], hepadnaviridae [14], flaviviridae [15,16], paramyxoviridae [17,18], orthomyxoviruses [19][20][21][22], filoviruses [17,23], and alphaviruses [24][25][26]. These foreign glycoproteins can be distinguished by the cell-surface receptors they bind, their architecture and topology and their cell entry properties.…”