Albina Khairetdinova

Coagulation factor VIII (FVIII) is a key therapeutic protein for hemophilia A, an inherited bleeding disorder caused by FVIII deficiency or dysfunction. The development and production of FVIII-based therapeutics remains challenging due to the intrinsic complexity of FVIII biosynthesis, folding, secretion, and functional stability. Advances in the understanding of the FVIII life cycle, as well as studies of its natural homologs, have created opportunities to develop improved FVIII variants. In parallel, therapeutic strategies to restore FVIII activity have evolved from replacement therapy with exogenous FVIII to gene-based delivery of a functional F8 copy. Among these approaches, AAV-mediated gene therapy has emerged as a clinically viable platform with a generally favorable safety profile, prompting the development of multiple therapeutic candidates for hemophilia A. This review summarizes established and current knowledge of FVIII structure, biosynthesis, and immunology in the context of developing effective and clinically applicable AAV-mediated therapies for hemophilia A. Particular attention is given to engineered FVIII variants and the latest clinical findings in AAV gene therapy, as well as to long-term efficacy and safety.

The creation of new proteins by combining natural domains is a commonly used technique in protein engineering. In this work, we have tested the possibilities and limitations of using circular homo-oligomeric Sm-like proteins as a basis for attaching other domains. Attachment to such a stable base should bring target domains together and keep them in the correct mutual orientation. We chose a circular homoheptameric Sm-like protein from Sulfolobus acidocaldarius as a stable backbone and the apical domain of the GroEL chaperone protein as the domain of study. This domain by itself, separated from the rest of the GroEL molecule, does not form an oligomeric ring. In our design, the hyperstable SacSm held the seven ADGroELs together and forced them to oligomerize. The designed hybrid protein was obtained and studied with various physical and chemical methods. Stepwise assembly and self-organization of this protein have been shown. First, the SacSm base was assembled, and then ADGroEL was folded on it. Functional testing showed that the obtained fusion protein was able to bind the same non-native proteins as the full-length GroEL chaperone. It also reduced the aggregation of a number of proteins when they were heated, which confirms its chaperone activity. Thus, the engineering path we chose made it possible to create an efficient thermostable chaperone. The result obtained shows the productivity of the way we chose for the creation and stabilization of oligomeric proteins.