Computational design, construction, and functional assessment of modified FVIII cDNA; for gene therapy purposes in the axis of hemophilia A -Eye disease
Nahid Javanshir1 , Dr. Zahra Soheila Soheili1 *, Dr. Ahmadreza Mehdipour1 , Dr. Hamid Latifi Navid1
- National Institute of Genetic Engineering and Biotechnology, Institute of Medical Biotechnology
Abstract: Hemophilia A is a coagulation disorder caused by a deficiency or dysfunction of factor VIII, related to the X chromosome. The development of coagulation factor concentrates, such as recombinant products, has facilitated the control of excessive bleeding associated with hemophilia A. Eye involvement in hemophilia patients leads to severe spontaneous bleeding and vision loss in the affected eye. Hemophilia patients experienced prolonged bleeding after undergoing surgery for extraocular muscles, enucleation, chalazion, and cataract extraction.
Methods: This research focuses on using computational methods to design a modified factor 8 for hemophilia gene therapy. Our goal is to predict a new structure for factor 8 by analyzing its existing structure and modeling protein-protein interactions. The aim is to create a modified factor 8 with increased expression and secretion compared to the natural factor 8.
Results: In this study for bioengineering of FVIII we considered; promoter engineering to increase transcription, mRNA codon optimization to improve translation, and amino acid replacement to improve secretion. According to the investigations, it is predicted that the natural promoter of factor 8, which is in the region 1175 upstream of the factor 8 gene, is the most efficient region for FVIII expression. Examining the result of the presence of a specific part of the B domain: N-linked oligosaccharides can participate in interactions related to folding inside the ER and also potentially help to facilitate the ER-Golgi transition. Also, in this study, a higher level of expression is possible with changes in GC content, CpG dinucleotide content, Codon usage bias, mRNA secondary structure, premature polyA sites, kozak sequence codon-context, and terminal signals that lead to codon optimization.
Conclusion: Because of the short half-life of factor VIII, repeated intravenous injections are necessary for prophylaxis. This imposes a significant financial burden on patients, their families and the medical system. Gene therapy that leads to permanent therapeutic effects eliminates the need for continuous prophylactic injections for hemophilia A patient. With the advances made in the field of artificial intelligence, the prediction of protein structures and interactions in the genes in question can be very helpful for studies such as gene therapy.
