4d), indicating transduction did not affect cell proliferation or multi-lineage differentiation potential

4d), indicating transduction did not affect cell proliferation or multi-lineage differentiation potential. a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human cDNA into mouse hematopoietic stem/progenitor cells (HSPCs) followed by bone-marrow transplantation corrected GM-CSF signaling in AMs and lung disease in cDNA driven by an internal elongation factor 1 (short; EFS) promoter (Lv.EFS.CSF2RAcoop). This article reports the results of nonclinical, safety and function studies for this vector in cultured macrophages and primary human cells. Material and Methods Cell culture Murine Ba/F3 cells were cultured in RPMI1640 (GIBCO; Life Technologies, Paisley, United Kingdom) supplemented with 10% fetal calf serum (FCS), 100?IU/mL of penicillin/streptomycin (PAA, Pasching, Austria), and 2?ng/mL of mIL-3 (Peprotech, Hamburg, Germany) on suspension culture plates. Murine alveolar macrophage (mAM) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, 10?IU/mL of penicillin/streptomycin, 2?mM HEPES (PAA) on adherent culture plates. All cell lines were cultured in standard conditions at 37C and 5% CO2. Human CD34+ cells were isolated from umbilical cord blood (purchased from Hannover Medical School). All donors have given informed consent. After gradient centrifugation of peripheral blood mononuclear cells (PBMCs), CD34+ cells were enriched from PBMCs by magnetic separation using CD34+ MicroBead kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells were cultured in StemSpan (STEMCELL Technologies, Vancouver, Canada) containing 100?IU/mL of penicillin/streptomycin, 2?mM of L-glutamine (Thermo Fisher Scientific, kb NB 142-70 Waltham, MA), 100?ng/mL of hSCF, 100?ng/mL of hFlt3l, and kb NB 142-70 50?ng/mL of hTPO (Peprotech) at 37C and 5% CO2. For differentiation toward macrophages, CD34+ cells were transferred to RPMI1640 containing 10% FCS, 100?IU/mL of kb NB 142-70 penicillin/streptomycin, 100?ng/mL of hM-CSF, 100?ng/mL of hGM-CSF, 100?ng/mL of hFlt3l, 20?ng/mL of hIL-3, and 20?ng/mL of hIL-6 (Peprotech) for at least 10 days. Lentiviral vector construction and production Codon optimization of was performed based on PUBMED “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_006140″,”term_id”:”1824163938″,”term_text”:”NM_006140″NM_006140. Codon-optimized cDNA was flanked by AgeI and SalI restriction sites and synthesized by GeneScript (Piscataway, NJ). Using restriction digestion (AgeI, SalI), cDNA was inserted into a third-generation self-inactivating lentiviral backbone (pRRL.cPPT.EFS.GFP), which was used as a control vector throughout the experiments. The final vector pRRL.cPPT.EFS.CSF2RAcoop (Lv.EFS.CSF2RAcoop) was sequence verified by DNA sequencing (GATC, Konstanz, Germany). For production of viral particles, a transient four-vector transfection of HEK293T cells was used, as previously described.13 HEK293T cells were cultured in DMEM (PAA) containing 10% FCS, 100?IU/mL penicillin/streptomycin, 20?mM of HEPES (PAA), and 25?M of chloroquine (SigmaCAldrich, Steinheim, Germany). Cells were transfected using calcium phosphate precipitation in the presence of 8?g/mL of gag/pol, 5?g/mL of pRSV-Rev, 5?g/mL of lentiviral vector plasmid, and 1.5?g/mL of vesicular stomatitis virus glycoprotein (VSVg). Viral supernatants were harvested 36 and 48?h post transfection, filtered, and concentrated by ultracentrifugation (Becton Dickinson, Krefeld, Germany) for 16?h at 14,000 and 4C. Viral titers were determined by several dilutions on SC-1 fibroblasts and flow cytometry analysis. Lentiviral transduction For lentiviral transduction, 100,000?mAM or Ba/F3 cells were transferred to respective culture medium containing 4?g/mL of protamine sulfate. Viral transduction was performed for 24?h. Thereafter, cells were washed and transferred back to standard culture medium. Transduction efficiency was analyzed 72?h after transduction using flow cytometry. CD34+ cells were transduced using RetroNectin? (Takara Bio, Inc., Shiga, Japan), with a multiplicity of infection (MOI) of 20, according to Kit the manufacturer’s instructions. Generation of mAM cell lines The mAM cell line is a murine AM cell line previously derived from GM-CSF-deficient mice.14 The mAM-hGM-R cell line is a murine AM cell line expressing functional human GM-CSF receptors previously derived from mAM cells by retroviral-mediated expression of normal human GM-CSF – and -subunits (MIEG3-vectors, respectively).1 The mAM-hPAP cell line is a murine AM cell line expressing vectors, respectively).1 Cell sorting Before sorting, Ba/F3 cells were stained with CD116 PE antibody for 45?min at 4C and separated on a XDP flow cytometer (Beckman Coulter, kb NB 142-70 Krefeld, Germany). Single cells from high, medium, and low expressing fractions were sorted and cultured, as previously described. Cell cytology Approximately 50,000 cells were re-suspended in 200?L of phosphate-buffered saline and centrifuged onto glass slides using a medite Cytofuge? (medite, Burgdorf, Germany) at 600 for 7?min. Glass slides were subsequently stained using Pappenheim staining. hGM-CSF-dependent survival assay After Ba/F3 cells had been cultured in X-VIVO 15 (Lonza, Basel, Switzerland) for 24?h without.