Nowadays gene and cell therapy become the basic methods in regenerative medicine. However only few gene and cell products are currently approved for clinical usage. Biosafety problems, complexity of cell and gene technologies and high cost of manufacturing are the main reasons for the slow introduction of such approaches in practical medicine. Treatment of hereditary diseases of the immune system based on the correction of the mutant gene by delivering functional recombinant gene into WBC is the first successfully employed in the clinical practice approach of cell-mediated or ex vivo gene therapy. Earlier we have reported the strategy of the cell-mediated gene therapy based on umbilical cord blood mononuclear cells transduced with adenoviral vectors carrying recombinant genes encoding neurotrophic factors for treatment neurodegenerative diseases, neurotrauma and stroke. Significant disadvantage of this method is the usage of the umbilical cord blood mononuclear cells as a cell carrier for the therapeutic genes. Considering immunodeficiency treatment and our own data we developed a new approach of recombinant gene delivery for personalized ex vivo gene therapy. The method is based on autoinfusion of patient's WBC transduced with recombinant therapeutic genes for correction of certain pathological conditions. In the present study for the first time the human gene-modified leucoconcentrate (GML) producing recombinant reporter gene encoding green fluorescent protein (GFP) was obtained without culturing WBC in vitro. The routine unit of peripheral blood (450 ml) was collected into the plastic blood bag and the leucocyte- and platelet-rich concentrates (50 ml) were obtained by standard method using Macopress Smart (Macopharma, France). Afterwards the equal volume of hydroxyethyl starch 6% was added into the plastic blood bag which was centrifuged (DP-2065 R PLUS, Centrifugal Presvac RV; Presvac, Buenos Aires, Argentina) at 350 rpm for 10 min at 10°C. The obtained supernatant was transferred into the new plastic blood bag using manual plasma extractor FK-01 (Leadcore, Russia) and 200 ml of saline was added into the bag which was centrifuged at 1300 rpm for 10 min at 10°C and the supernatant was expressed out of the bag so that the remaining solution in the bag (30 ml) contained leucoconcentrate (WBC - 45.56 ± 23.93 × 106/ml and RBC - 1.76 ± 3.33 × 109/ml). Transduction of WBC with chimeric adenoviral vector (Ad5/35) carrying GFP gene was performed in the plastic bag with MOI 5 according to the count of WBC in the leucoconcentrate. After transduction for 12 hours, 200 ml of saline was added to the bag with leucoconcentrate, the mixture was centrifuged at 1000 rpm for 10 min at 10°C and the supernatant was squeezed out of the bag. The remained in the bag solution (30 ml) was considered as gene-modified leucoconcentrate carrying GFP gen (WBC - 22.63 ± 8.90 × 106/ml and RBC - 1.77 ± 1.21 × 109/ml). For in vitro study of GFP gene expression the samples of GML-GFP were cultivated for 60 hours after GML-GFP preparation. Fluorescent microscopy in the cytoplasm of the transduced WBC showed specific intensive green fluorescence. Flow cytometry analysis demonstrated that 2.5% of WBC from the GML-GFP efficiently expressed GFP. Thus leucoconcentrate after 72 h of transduction with Ad5/35-GFP with MOI 5 resulted in 2.5% of the GFP-positive cells. Thus the results of this study represent a simple, safe and effective approach for preparation of GML for personalized ex vivo gene therapy aimed at temporary production of the specific recombinant biologically active molecules for pathogenetic therapy of the varied nosological form, such as trauma, ischemic, degenerative, autoimmune, infection and other diseases. This study was supported by the grant of Russian Science Foundation 19-75-10030.

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