Co-display of RDTR and SCF on LVs allows transduction of immature c-Kit⁺hCD34⁺ cells. (A) Schematic representation of the LVs displaying the RD114 glycoprotein, SCF, and TPO. A TPO truncated form, 171 aa long, was fused to the N-terminus of the influenza HA glycoprotein (TPOHA). SCF was also fused to the N-terminus of the HA glycoprotein, which allowed efficient functional incorporation on LVs (SCFHA). Because these chimeric HA glycoproteins demonstrated a reduced infectivity, we needed to coexpress an additional fusion competent glycoprotein, RD114. The cytoplasmic tail of RD114 was exchanged for that of the MLV glycoprotein, resulting in a mutant RDTR, which allowed efficient incorporation on HIV vectors as described previously.¹⁷  (B) Immunoblots of LV particles displaying RDTR together with TPOHA, SCFHA, or both chimeric glycoproteins at their surface. LVs were purified over a sucrose cushion by ultracentrifugation. The upper part of the membrane was stained with Abs against the surface domain of RD114, the middle part with Abs against the influenza HA glycoprotein to detect the TPOHA and SCFHA chimeric envelopes. HIV-1 capsid (CA) was detected to assess equivalent loading of vectors. (C) CD34⁺ CB cells were incubated with RDTR/TPO-displaying (RDTR/TPOHA), RDTR/SCF-displaying (RDTR/SCFHA), or RDTR/SCF/TPO-displaying (RDTR/TPOHA/SCFHA) LVs in the presence or absence of RetroNectin. As controls, CD34⁺ cells were incubated with LVs displaying RDTR in the presence of recombinant cytokines with or without RetroNectin at MOI = 100. At day 3 after transduction, cells were evaluated for CD34⁺ surface marker and GFP expression by FACS (means ± SD, n = 6). (D) CD34⁺ CB cells were incubated with HIV vectors co-displaying the RDTR envelope with TPOHA, SCFHA, or both cytokines displaying glycoproteins in the absence of RetroNectin. Counterpart transductions with VSV-G/TPOHA, VSV-G/SCFHA, or VSV-G/SCFHA/TPOHA were performed. Transductions were performed at a MOI = 100 or MOI = 1. The percentage of GFP⁺CD34⁺ cells was analyzed by FACS (means ± SD, n = 4). CB hCD34⁺ cells were pre-incubated with increasing concentrations of rSCF (E) or empty SCFHA displaying lentiviral particles (F) as indicated on the x-axis. We indicated in the x-axis the functional SCF equivalent (= ng SCF activity; see “Determination of activity of cytokine-displaying vectors”). Subsequently, incubation with RDTR/SCFHA-LVs at MOI = 10 was performed. The percentage of GFP⁺CD34⁺ cells was analyzed 3 days after transduction by FACS (means ± SD, n = 3). (G) CD34⁺ CB cells were incubated with RDTR/SCFHA co-displaying vectors; RDTR-LVs in the presence of rSCF, SCFHA, or SCFHAX single-displaying vectors; or a mixture of RDTR single-displaying LVs (RDTR) and SCFHA single-displaying vectors (SCFHA). SCFHAX contains an inactivated cleavage site, making the glycoprotein incompetent for cell fusion. All transductions were performed at MOI = 10 in the presence and absence of RetroNectin. At day 5 after transduction, cells were evaluated for CD34⁺ surface marker and GFP expression by FACS (means ± SD, n = 3). (H) Immunoprecipitation of RDTR/SCFHA-LVs with anti-HA Abs followed by immunoblot detection of RDTR (left) and immunoprecipitation of RDTR/SCFHA LVs with anti-RD114 Abs followed by detection of SCFHA by Western blot (right) are shown. The positions of RDTR and SCFHA are indicated.