Pictures were scanned with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Cytotoxicity and anti-HIV activity of GPI-anchored bifunctional inhibitors in human primary CD4+ T cells Human primary CD4+ T cells were enriched from peripheral blood mononuclear cells (PBMCs) by negative selection with CD4+ T Cell Isolation Kit (Miltenyi Biotec) and maintained in complete RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (HyClone) and 150 IU/ml human rIL-2 (Peprotech). By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed MYO9B a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and QX77 its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches. KEYWORDS: HIV, gene therapy, broadly neutralizing antibodies (bNAbs), fusion inhibitory peptide, glycosylphosphatidylinositol (GPI) Introduction Over 37 million people are currently living with human Immunodeficiency virus (HIV), and about 1.8 million new infections per year continues to fuel the HIV pandemic. Antiretroviral therapy (ART) can efficiently suppress HIV replication in treated individuals and QX77 has dramatically reduced the morbidity and mortality associated with AIDS-related illness. Unfortunately, due to the establishment of a reservoir of quiescent, infected CD4+ T cells that harbour replication-competent virus, discontinuation of daily ART would lead to rapid viral rebound and continued disease progression, whereas lifelong treatment often results in cumulative toxicities and drug resistance [1,2]. Therefore, therapeutic interventions that can achieve a sterilizing or functional cure are among the top priorities of the HIV field. The cure or functional cure of the Berlin patient and London patient through transplantation of allogeneic hematopoietic QX77 stem cells (HSCs) harbouring a naturally occurring mutation in the HIV coreceptor CCR5 (CCR532) suggests that infusion of HIV-resistant cells could be a viable treatment approach [3,4]. Currently, there are tremendous works to engineer CCR5 mutations into autologous cells using gene-editing technologies; however, the allogeneic transplantation of CCR5 gene-edited HSCs has so far achieved only very limited therapeutic efficacy in patients [5C7]. For example, Xu actin antibody (Sigma). After three washes with TBS-Tween 20, the membrane was incubated QX77 with IRDye 680LT goat-anti-rabbit IgG, IRDye 680LT goat-anti-mouse IgG, or IRDye 800CW goat-anti-human IgG for 2 h at room temperature. Images were scanned with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Cytotoxicity and anti-HIV activity of GPI-anchored bifunctional inhibitors in human primary CD4+ T cells Human primary CD4+ T cells were enriched from peripheral blood mononuclear cells (PBMCs) by negative selection with CD4+ T Cell Isolation Kit (Miltenyi Biotec) and maintained in complete RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (HyClone) and 150 IU/ml human rIL-2 (Peprotech). CD4+ T cells were stimulated by mixing with anti-CD3/anti-CD28-coated magnetic beads (Gibco) at a bead-to-cell ratio of 1 1:1. After 24 h of stimulation, 2 105 of CD4+ T cells were transduced with the CMI02 and CMI06 vectors at a multiplicity of infection (MOI) of 60, spinoculated at 2000 X g for 2 h at 32C, and incubated at 37C for 4 h. Then, the culture medium was replaced with fresh complete RPMI 1640 medium. 72 h after transduction, the cells were de-beaded and applied to detect the transduction efficiency by FACS analysis. Cell viability of transgene-transduced primary CD4+ T cells was measured using Cell Counting Kit-8 (Abbkine). QX77 Briefly, the cell concentration was adjusted to 3105/ml, and 100 l of cell suspension were seeded to each well of a 96-well plate and then cultured at 37C for.