CCR5 as a natural and modulated target for inhibition of HIV

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection-this without detrimental effects to the host-and transplantation of CCR5-delta32 stem cells in a patient with HIV ("Berlin patient") achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells. We have developed a dual therapeutic anti-HIV lentiviral vector (LVsh5/C46) that down-regulates CCR5 and inhibits HIV-1 fusion via cell surface expression of the gp41-derived peptide, C46. This construct, effective against multiple strains of both R5- and X4-tropic HIV-1, is being tested in Phase I/II trials by engineering HIV-resistant hematopoietic cells.

Figure 1

Lentiviral construct LVsh5/C46. LVsh5/C46 is a self-inactivating (SIN) lentiviral vector that lacks all viral coding sequences (gag, pol, and env genes deleted) which may give rise to the formation of replication-competent retrovirus or immunogenic peptides and is also devoid of all retroviral enhancer-promoter sequences that are known to be involved in insertional mutagenesis by related gammaretroviral-derived vectors. The internal promoters were chosen from human genes that show expression in hematopoietic stem/progenitor cells as well as T lymphocytes and macrophages, as required for anti-HIV therapy. Promoters were also chosen to direct appropriate levels of gene expression so as not to interfere with endogenous cellular processes. 5' LTR (long terminal repeat), derived from HIV-1 with the U3 region replaced with the cytomegalovirus (CMV) promoter/enhancer; 3' LTR, derived from HIV-1 with a 133bp deletion in the U3 region; cPPT, central polypurine tract; H1, human H1 RNA promoter; Ubc, human ubiquitin promoter; WPREmt, mutant woodchuck hepatitis virus post-transcriptional response element.

Figure 2

LVsh5/C46 introduction into target cells and simultaneous expression of C46 and knockdown of CCR5. Peripheral blood mononuclear cells (PBMC) were transduced with LVsh5/C46 (lower panel), or left untransduced (upper panel). C46 was detected by 2F5 monoclonal antibody, and CCR5 was detected by staining with anti-CD195 (CCR5) antibody. Cells from 3 independent donors are shown. Donor 2 was homozygous for CCR5-delta32 genotype and expresses no CCR5.

Figure 3

Schematic of the process for engineering protection from HIV-1 into human recipients via LVsh5/C46 mediated modification of CD4⁺ T lymphocytes and CD34⁺ HSPC. 1. Apheresis, small or standard volume respectively, to obtain CD4⁺ T cells or CD34⁺ hematopoietic stem cells; 2. Cell isolation using CliniMACS and DynaMag CTS bead separation; 3. Lentiviral vector transduction with LVsh5/C46 in appropriate cytokines—CD3/CD28 bead stimulation and IL2 for T cells, stem cell factor, thrombopoietin and FLT-3 ligand for hematopoietic stem cells; 4. Harvest of transduced cells and freezing; 5. Following release testing, transplantation of genetically modified cells. Busulfan (4 mg/kg or 8 mg/kg) is administered pre cell infusion to make bone marrow space for introduced CD34⁺ HSPC.