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Optimization of adenoviral transduction of the human muscle tissue for the development of ex vivo gene therapy

The need to improve bone healing permeates the discipline of orthopedic surgery. Osteogenic inducers must be available for sufficient periods of time and in sufficient amounts to promote osteogenesis. One approach to achieving this goal is gene therapy. Using adenoviral gene transfer it is possible to create human skeletal muscle cells that produce osteogenic inducers and subsequently returned those cells at the place of healing. It is essential that gene therapy will be so called „same day“ ex vivo regional gene therapy, with the surgery done within two hours. The aim of the research is to optimize the transduction of grafts of human muscle with a replication defective adenoviral vector constructed to carry the luciferase reporter gene (Ad.Luc 2) in vitro. The transduction efficiency of adenoviral vector in vitro will depend on: a) viral titer, b) period of contact with virus and c) presence of calcium and/or lanthanum ions. First generation adenovirus (ΔE1, ΔE3), serotype 5, carrying firefly luciferase cDNA (Ad.Luc) under the transcriptional control of the human cytomegalovirus early promoter were constructed by cre-lox recombination. Viruses were propagated in 293 cells purified on caesium-chloride gradients, and dialysed against 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 10 mM MgCl2, and 4% sucrose buffer. Viral titres were estimated as 1011 to 1012 particles/ml by optical density and 3x109 plaque forming units (pfu)/ml by standard plaque assay. Muscles were infected with Ad.Luc adenoviral vector and afterwards thouroughly washed with PBS. Incubation was carried out for 72 hours at 37ºC and 5% CO2. After the incubation the luciferase assay and total amount of proteins were determined. The luciferase assay was held out on a luminometer. The light intensity (RLU) is directly proportional to the luciferase concentration. Total amount of proteins was determined by the BCA protein assay. The absorbance was measured at 562 nm on a plate reader. Overall results were expressed as a relative luminescent units per µg of protein (RLU/µg). The samples of muscle tissues were obtained from 30 patients (range: 18–55 years, gender will not have influence on research) with the anterior cruciate ligament injury. All studies were approved by the Etic Committee for Biomedical Research. The optimizations of the transduction is necessary step in the development of the ex vivo gene therapy in orthopedic surgery. Since time is the limiting parameter during surgery we will determine optimal virus concentration and usage of both CaCl2 (5 mM) and LnCl3 (0.2 mM) to help bypass the inefficiency of receptor-dependent uptake of the vector. Adenoviral vectors expressing osteogenic inducers (BMP-2) will be used to evaluate the effectiveness of the developed protocol.

Adenoviral vectors; osteogenic inducers (BMP-2); ex vivo gene therapy; regenerative medicine

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