Antibody-directed lentiviral gene transduction in early immature hematopoietic progenitor cells

Abstract

Background: The specific and efficient transduction of retroviral particles remains problematic for in vivo and ex vivo gene therapy studies, where the targeting cell population is a heterogeneous bulk population.

Methods: Pseudotyping lentiviral particles with Sindbis virus envelope (Env) proteins modified with an immunoglobulin Fc-binding domain presents a method of selecting cells within a mixed population through antibody (Ab)-mediated targeting. Conditions were tested for targeted lentiviral gene delivery to hematopoietic progenitor cells via Ab-conjugated envelopes independent of CD34.

Results: Conditions to optimize the efficiency of gene delivery were established using the ABCG2 multidrug resistance protein, associated with stem cell phenotypes, as the cell surface target. By varying the proportion of ABCG2 expressing cells in a population, ABCG2-targeted gene delivery was detectable by flow cytometry when ABCG2(+) cells comprised greater than 5% of the population. Conditions that increased the efficiency of gene transfer, including cholesterol independent Env proteins and pH, increased nonspecific gene delivery. The feasibility of this cell-Ab-virus sandwich system in targeting transduction in a mixed population was tested in cells derived from human cord blood (CB). Conjugation of viral particles with anti-CD133 and anti-ABCG2 hematopoietic stem cell-associated Ab resulted in targeted gene transfer into early immature hematopoietic progenitor cells. Enhancement was found when the hematopoietic progenitor cells were enriched from CB cells via the depletion of lineage(+) committed cells.

Conclusions: Gene transfer to lineage(-) early immature hematopoietic progenitors from human umbilical CB was obtained using CD133, ABCG2 or HLA-1 antibodies conjugated to lentiviruses pseudotyped with modified Sindbis viral Env proteins.

Figure 1

Expression of ABCG2. Cell surface expression of ABCG2 as determined by flow cytometry in the presence of antibody 5D3. Panel A. Saos2 cells were transduced with empty pcDNA vector designated Saos2-pcDNA. Panel B. Human osteosarcoma cell line Saos2 transduced with pcDNA vector containing ABCG2 gene (named Saos2-ABCG2). Cells with high expression of ABCG2 were sorted by flow cytometry, yielding 97% ABCG2⁺ of the cells. Panel C. Overlay of expression of Saos2-pcDNA and Soas2-ABCG2 cells.

Figure 2

Specificity of targeted transduction of m168-pseudotyped lentiviral vector on the host cells. Saos2-ABCG2 cells and Saos2-pcDNA cells were infected by virus bearing m168 in the following conditions: no conjugation with antibody, conjugated with anti HLA class I antibody, clone BXP3 of anti-ABCG2 antibody and clone 5D3 of anti-ABCG2 antibody. In every group, 1 × 10⁵ cells were infected with virus at a MOI of 1 via antibody (1 µg/ml) if used. The MOI of 1 was based on infection of 293T cells using the HLA ABC antibody. Lentiviral particles were generated following the protocol published [37]. m168, HIV-1 expression vector and packaging vector were co-transfected in a total amount of 15 µg at a ratio of 1:2.5:2.5 (m168: HR’CMVGFPW: pCMVd8.2dVPR) in 4 × 10⁶ 293T cells. The results are shown as %GFP⁺ detected by flow cytometry 3 days post-infection.

Figure 3

Targeting transduction of m168-pseudotyped virus in a mixed cell population. Saos2-ABCG2 and Saos2-pcDNA cells were mixed in different ratios with the total number as 2 × 10⁵ cells. The mixed populations were infected with viruses bearing m168 conjugated with the 5D3 anti-ABCG2 antibody. Three days post-infection, the cells were stained with PE-conjugated anti-ABCG2 antibody and the %ABCG2⁺GFP⁺ cells are shown in the top right of each plot.

Figure 4

Transduction of m168-pseudotyped virus packaging the GFP gene was performed on CB MNCs with or without lineage depletion. The infection was directed by anti-HLA class I, anti-ABCG2, anti-CD133 and in the absence of antibody conjugate. The cells were also infected by VSV-G pseudotyped virus. Following overnight infection, the cells were plated in methylcellulose medium for 14 days. The numbers of resultant colonies and GFP⁺ colonies were scored under bright-field (panels A, C, and E) and fluorescence (panels B, D and F) microscope, respectively. Representative pictures are shown. Panel A and B: BFU-E with surrounding variable sized non-hemoglobinized cells transduced with CD133 Ab-conjugate. Panel C and D: BFU-E with surrounding variable sized non-hemoglobinized cells transduced with ABCG2 Ab-conjugate. Panels E and F, CFU-GM transduced with VSV-G pseudotyped particles.