Development and in vitro efficacy of transgenic stem cell platform for hemophilia A gene therapy

Hemophilia A affecting approximately %80 of patients with hemophilia, is an X-linked recessive inherited bleeding disorder caused by deficiency of Factor VIII (FVIII) protein. While patients are presented with intra-articular and intramuscular bleeding, the degree of factor deficiency determines the frequency and severity of bleeding. Plasma- derived therapies and recombinant factor concentrates are used to control and treat the bleeding symptoms. The objective of this study is to design a gene therapy for FVIII deficiency, which is responsible for Hemophilia A. For this purpose, a lentiviral vector with EF-1 alpha promoter, B domain deleted Factor VIII gene sequence, and CD45 R0 truncated surface marker (EF1α - FVIII.CC - CD45R0t) was designed. The goal of the current study is to develop a gene therapy that can be secured from the immune response by transducing mesenchymal stem cells, can be tracked in vivo due to its surface marker (CD45R0t) and can provide non - toxic and functional FVIII secretion. After the characterization and quality control tests of FVIII encoding lentivirus, transduction efficiency with mesenchymal stem cells, FVIII persistence, and expression analyzes were performed. The presence of the FVIII protein was also confirmed by the Western Blot method. The lentivirus (LTR sequence) developed has been tested to be non-replicative, safe by polymerase chain reaction (PCR) analysis. Time-dependent alterations of FVIII secretion was evaluated using the Elisa method. The functionality of the factor VIII protein was demonstrated using mixing test through activated partial thromboplastin time (aPTT) and FVIII levels. There was no statistically significant difference in FVIII secretion over time, signifying that stable levels of FVIII were achieved (p > 0.05). In the mixing test analyses, FVIII deficient human plasma products with and without gene therapy were compared in terms of aPTT and FVIII levels. When aPTT levels were evaluated; it was observed that mean aPTT levels of the group that did not receive gene therapy was 92.69 ± 11.38 seconds, while the mean aPTT levels of the group that received the gene therapy were decreased to 38.60 ± 13.38 seconds (p < 0.001). When FVIII levels were evaluated, it was found that the mean FVIII level of the group that did not receive gene therapy was 0.41 ± 0.03 IU/dL, while the mean FVIII levels of the group that received gene therapy increased to 25.41 ± 33.38 IU/dL (p < 0.01). As an alternative to previous studies, the current study introduces a new method in Hemophilia A gene therapy, the first steps to contribute to in vivo animal models and clinical trials have been taken.