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.

FIG. 1

Schematic representation of Sindbis virus envelope (SINDBIS) and chimeric envelope between SINDBIS and the IgG binding domain of protein A (ZZ SINDBIS). Sindbis virus envelope proteins (E3, E2, 6K, and E1) were cloned into an expression vector derived from pHCMV G (27). The envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into virions as envelope proteins. E3 and 6K are leader sequences for E2 and E1, respectively (22). ZZ SINDBIS is a modified Sindbis virus envelope in which the IgG binding domain of protein A (ZZ) was inserted in the E2 region (see Material and Methods). ZZ domain was inserted between amino acids 71 and 74 of E2. The promoter is the cytomegalovirus immediate early promoter, and the intron is intron II of the rabbit β-globin gene. p(A), polyadenylation signal.

FIG. 2

Anti-HLA MAb mediates gene transduction by ZZ SINDBIS pseudotyped HIV-1 vector. 293T cells (HLA antigen positive) and BHK cells (HLA antigen negative) were infected with HRCMVLuc (SINDBIS) and HRCMVLuc (ZZ SINDBIS). The p24 levels of HRCMVLuc (SINDBIS) and HRCMVLuc (ZZ SINDBIS) were 541 and 1,843 ng/ml, respectively. 293T cells (2 × 10⁵) and BHK cells (6 × 10⁴) were infected with 200 μl of each vector for 2 h with or without anti-HLA (1 μg/ml) as described in the text. Four days after infection, the cells were lysed by 200 μl of 1× luciferase lysis buffer (Promega), and 10 μl of each lysate was subjected to a luciferase assay. The luciferase activity of uninfected 293T cells was 1.8 × 10² relative light units. The results are the averages of three independent infections performed in parallel.

FIG. 3

Detection of Sindbis virus envelope and ZZ SINDBIS on virions. Ultracentrifuged and 100-fold-concentrated samples of HIV vector (HRCMVEGFP) pseudotyped by SINDBIS, ZZ SINDBIS, or no envelope (Env) were lysed and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting as described in Materials and Methods. The amount of virus for each sample was normalized by the amount of HIV p24 antigen (90 ng/sample). Viral proteins were detected with anti-Sindbis virus mouse immune ascitic fluid (18). The bands around 60 kDa seen in the no-envelope and SINDBIS samples are nonspecific bands. The upper and lower arrows show bands corresponding to the chimeric protein and the E2 protein, respectively.

FIG. 4

HIV-1 vector (A) and MLV vector (B) can be pseudotyped with ZZ SINDBIS. 293T cells (2 × 10⁵) were infected with 200 μl of HRCMVEGFP (ZZ SINDBIS) or 100 μl of SRαLEGFP (ZZ SINDBIS) (100× concentrated) with or without anti-HLA (1 μg/ml). Three days after infection, EGFP expression was analyzed by flow cytometry. The titers of HRCMVEGFP (ZZ SINDBIS) and SRαLEGFP (ZZ SINDBIS) (100× concentrated) with 1 μg of anti-HLA are 4.5 × 10⁵ and 2.8 × 10⁵ EGFP transduction units/ml, respectively.

FIG. 5

MAbs mediate gene transduction of a heterogeneous cell population by ZZ SINDBIS-pseudotyped HIV vector (ZZ HIV). A mixture of HLtat cells (1.5 × 10⁴) and HLtat CD4 cells (1.5 × 10⁴) was seeded in the same well a day before infection. They were infected with HRCMVEGFP (ZZ SINDBIS) (200 μl, 5 × 10⁴ EGFP transduction units) either alone, with anti-HLA (1 μg/ml), or with anti-CD4 (1 μg/ml) for 2 h. Three days after infection, cells (5 × 10⁵) were stained with anti-CD4–PE. The cells were then analyzed for CD4 and EGFP expression by flow cytometry. CD3- and 28-activated PBMC (1 × 10⁵) were infected with CS-Rh-MLV-E (ZZ SINDBIS) (200 μl, 2 × 10⁵ EGFP transduction units) either alone, with anti-HLA (10 μg/ml), or with anti-CD4 (10 μg/ml). Three days after infection, cells (5 × 10⁵) were stained with anti-CD4–PE and analyzed by flow cytometry. The percentage of EGFP-positive cells in the CD4 positive population was calculated as the number of events in the upper right quadrant divided by the total number of events in the right and left upper quadrants. For the CD4-negative population, the percentage of EGFP-positive cells was calculated by dividing the number of events in the lower right quadrant by the number of events in the right and left lower quadrants.