Oral administration of chitosan/DNA nanoparticles containing DNA coding for FVIII and FC IgG fragment or IL-10 for the modulation of immune responses to FVIII in hemophilia mice

Abstract

Hemophilia A is an X-linked bleeding disorder, which occurs due to deficiency of clotting protein FVIII. Current treatment involves regular lifelong infusion of recombinant or plasma-derived FVIII protein, which is suboptimal, invasive and very expensive. One of the biggest challenges of the current therapy is the recognition of FVIII by immune system as a foreign substance, leading to the development of neutralizing antibodies, which makes further administration of FVIII ineffective. Therefore, an alternative cost effective and safe treatment to Hemophilia A is highly desirable. We propose chitosan-mediated oral non-viral gene therapy as a safe and costeffective strategy for immune modulation in severe hemophilia A cases. Having a strong affinity for DNA and forming nanoparticles, chitosan-DNA complexes protect plasmid DNA fi-om degradation by the low pH envu-onment of the stomach and from nucleases in the GI tract; further, safe re-administration of nanoparticles is possible. We also propose to include in the plasmid DNA coding for the Fc fragment of IgG heavy chain region, which contains Tregitope sequences, T cell epitopes that specifically activate regulatory T cells or anti-inflammatory cytokine IL-10 that may modulate the immune response against FVIII protein. Therefore, it is hypothesized that orally administered chitosan/DNA nanoparticles will lead to clinically relevant amount of FVIII in the host without an immune response, providing long term, cost-effective and safe treatment for hemophilia A. For this study we aimed to: I) optimize nanoparticles in terms of effective gene expression in vitro; 2) treat hemophilic mice orally with chitosan nanoparticles containing DNA coding for FVIII and for immunomodulatory elements (Fc fragment of IgG and IL-10) to induce tolerance to FVIII. We found that the key factors contributing to the effectiveness of gene expression in chitosan/DNA nanoparticles are the type of chitosan, the charge ratio (N/P) and the DNA concentration. In vivo, the optimized nanoparticles containing FVIII+Fc DNA significantly decreased antibody formation specific to F V I I I in prophylactic group of mice, and in 4/6 mice receiving nanoparticles containing FVIII+IL-10. It is tempting to hypothesize that this strategy might also be extended to modulate immune responses to other antigens.