CD25 preselective anti-HIV vectors for improved HIV gene therapy

Abstract

As HIV continues to be a global public health problem with no effective vaccine available, new and innovative therapies, including HIV gene therapies, need to be developed. Due to low transduction efficiencies that lead to low in vivo gene marking, therapeutically relevant efficacy of HIV gene therapy has been difficult to achieve in a clinical setting. Methods to improve the transplantation of enriched populations of anti-HIV vector-transduced cells may greatly increase the in vivo efficacy of HIV gene therapies. Here we describe the development of preselective anti-HIV lentiviral vectors that allow for the purification of vector-transduced cells to achieve an enriched population of HIV-resistant cells. A selectable protein, human CD25, not normally found on CD34+ hematopoietic progenitor cells (HPCs), was incorporated into a triple combination anti-HIV lentiviral vector. Upon purification of cells transduced with the preselective anti-HIV vector, safety was demonstrated in CD34+ HPCs and in HPC-derived macrophages in vitro. Upon challenge with HIV-1, improved efficacy was observed in purified preselective anti-HIV vector-transduced macrophages compared to unpurified cells. These proof-of-concept results highlight the potential use of this method to improve HIV stem cell gene therapy for future clinical applications.

FIG. 1.

Preselective lentiviral vectors and purification of transduced HPCs. (A) A self-inactivating third generation lentiviral vector, CCLc-MNDU3-X-PGK-X2, was utilized to derive the preselective vectors. A 400 bp deletion in the 3’ LTR U3 region is denoted by Δ. The control EGFP+ vector contains an EGFP reporter gene under the control of the MNDU3 promoter and a human CD25 gene under the control of the PGK promoter. The CMAP1 anti-HIV vector contains a triple combination of anti-HIV genes, a human/rhesus macaque chimeric TRIM5α under the control of the MNDU3 promoter, a pol-III U6 promoter-driven CCR5 shRNA, a pol-III U6 promoter-driven TAR decoy, and a human CD25 gene under the control of the PGK promoter. (B) CD34+ HPCs were transduced with the control EGFP+ preselective vector, purified by CD25 immunomagnetic beads, and analyzed by flow cytometry for EGFP and CD25 expression. (C) CD34+ HPCs were transduced with the anti-HIV CMAP1 vector, purified by CD25 immunomagnetic beads, and analyzed by QPCR for vector copy number. All experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; LTR, long terminal repeat; PGK, phosphoglycerate kinase; HPC, hematopoietic progenitor cells; QPCR, quantitative PCR; CMAP1, Cclc-mndu3-anti-hiv-protein-1.

FIG. 2.

Stability of the CMAP1 vector and expression of the anti-HIV genes. Total genomic DNA was extracted from nontransduced (lane 2) and CMAP1 vector-transduced (lane 3) HPC-derived macrophages and analyzed by PCR with vector-specific primers. (A) MNDU3 (forward) and CCR5 shRNA (reverse); (B) TRIM5α (forward) and CCR5 shRNA (reverse); (C) TRIM5α (forward) and CD25 (reverse); and (D) CCR5 shRNA (forward) and CD25 (reverse). The CMAP1 plasmid used in vector production was utilized as a positive control (lane 1). DLa, DNA ladder a (100 bp), DLb, DNA ladder b (1 kb). A schematic of the PCR products is depicted below the gel pictures. (F) Total cellular RNA was extracted from either nontransduced (NT) or CMAP1 vector-transduced HPC-derived macrophages and analyzed by QPCR for the expression of the chimeric TRIM5α, CCR5 shRNA, and TAR decoy. U6 snRNA was used as an internal control.

FIG. 3.

Safety analysis of purified CMAP1-transduced HPCs. (A) HPCs, either nontransduced (NT) or purified CMAP1 vector-transduced, were cultured in semi-solid methylcellulose medium for 10 days and CFUs (BFUs, GEMMS, and GMs) were visualized by microscopy under 10× magnification. (B) Total BFU, GM, and GEMM colonies were counted on day 10 for the NT and purified CMAP1 cultures. (C) Total cells were counted in HPC methylcellulose NT and purified CMAP1 vector-transduced cultures in the absence or presence of IL-2. All experiments were performed in triplicate. Representative cell pictures are displayed. Color images available online at www.liebertpub.com/hgtb

FIG. 4.

Derivation of phenotypically normal macrophages. (A) Macrophages derived from the nontransduced (NT) and purified CMAP1 HPC cultures were visualized by microscopy under 10× magnification. (B) The NT (unshaded) and purified CMAP1 (shaded) macrophages were analyzed by flow cytometry for the cell-surface markers CD14, HLADR, CD4, and CD80. (C) Macrophages, NT, unpurified CMAP1 transduced, and purified CMAP1 transduced were also analyzed by flow cytometry for the expression of CD25 and CCR5. All experiments were performed in triplicate. Representative cell pictures and flow cytometry histogram overlays and cell plots are displayed.

FIG. 5.

Expression of proto-oncogenes in purified CMAP1 macrophages. Cell cultures containing either peripheral blood mononuclear cells (PBMCs), nontransduced (NT) HPC-derived macrophages, or purified CMAP1 vector-transduced HPC-derived macrophages were evaluated by QPCR for their expression of the proto-oncogenes myc, myb, fos, and jun in the presence of IL-2. Experiments were performed in triplicate. Statistical significance (p<0.05) is represented by an asterisk.

FIG. 6.

HIV-1 challenge of CMAP1 HPC-derived macrophages. (A) HPC-derived macrophages, either nontransduced (NT) (♦), unpurified CMAP1-transduced (CMAP1-UP) (▪), or purified CMAP1 (CMAP1-P) (▲)-transduced, were challenged with an R5-tropic BaL-1 strain of HIV-1 at an MOI of 0.05. On various days postinfection, culture supernatants were analyzed for HIV-1 replication by p24 ELISA. (B) On day 28 post-infection, infected cultures were visualized by microscopy under 10× magnification. All experiments were performed in triplicate. Representative cell pictures are displayed.