Expression of human factor VIII by splicing between dimerized AAV vectors.

Area of Science:

• Gene Therapy
• Molecular Biology
• Virology

Background:

• Adeno-associated virus (AAV) vectors are promising for hemophilia gene therapy.
• AAV's limited packaging capacity (5 kb) hinders the delivery of large therapeutic genes like factor VIII (F8) cDNA (7 kb).
• Previous strategies using single AAV vectors for B-domain-deleted F8 (BDD-F8) were constrained by regulatory element size.

Purpose of the Study:

• To evaluate the feasibility of split AAV vector technology for enhanced F8 gene expression.
• To overcome AAV packaging limitations for delivering the full-length F8 gene.
• To assess the efficacy of AAV heterodimerization for functional factor VIII production.

Main Methods:

• Developed two complementary AAV vectors: a 5' vector with regulatory elements and F8 exons 1-12, and a 3' vector with the remaining F8 cDNA.
• Utilized split AAV vector technology for head-to-tail dimerization upon coinfection.
• Assessed F8 protein production in 293 and HepG2 cells and in immunodeficient mice following intraportal injection.

Main Results:

• Coinfection with both 5' and 3' AAV/F8 vectors resulted in functional human factor VIII protein production (120 mU/ml).
• Neither vector alone produced detectable factor VIII protein.
• Demonstrated correct vector dimerization and spliced BDD-F8 mRNA expression via PCR and RT-PCR.
• In vivo studies in mice showed sustained factor VIII levels (2% of normal) for four months.

Conclusions:

• Split AAV vector technology effectively expands AAV packaging capacity for F8 gene delivery.
• AAV heterodimerization is a viable strategy for expressing functional factor VIII.
• This approach holds potential for improving hemophilia gene therapy by enabling the delivery of larger therapeutic constructs.