PROJECT SUMMARY/ABSTRACT Deficiency of clotting factor VIII (FVIII) causes the bleeding disorder hemophilia A (HA), an X chromosome-linked bleeding defect affecting 1/5,000 males world-wide. Although bleeding episodes are prevented by prophylactic administration of FVIII preparations or novel molecules that bypass FVIII, there is no effective long-term cure. Gene therapy through adeno-associated virus (AAV)-mediated ectopic FVIII expression in hepatocytes has emerged as a promising therapeutic approach for HA, but has met with two hurdles: 1) A requirement for very high vector doses to drive FVIII expression in hepatocytes; and 2) Declining therapeutic transgene expression over time accompanied with transient liver damage after vector injection, both of which may be directly linked to the fact that FVIII is prone to misfolding, aggregation and retention in the endoplasmic reticulum (ER). We demonstrated that increased FVIII synthesis does not equate to increased FVIII secretion. Since isolation of the FVIII gene in 1984, we have discovered many detrimental cellular responses as a consequence of heterologous expression of wildtype FVIII or B-domain deleted FVIII (BDD), presently the FVIII derivative in clinical gene therapy studies for HA. These responses include: 1) Formation toxic aggregates in the ER; 2) Hepatosteatosis; 3) Activation of an innate inflammatory response; 4) Activation of the ER unfolded protein response that leads to cell death; 5) Production of detrimental reactive oxygen species; and 6) Potential development of hepatocellular carcinoma (HCC). Moreover, we and the Xiao lab (Project 2) discovered two BDD variants that efficiently fold and are secreted from the cell. “F309S” BDD-FVIII has a single amino acid change at Phe309 to Ser so it does not aggregate in the ER. Another BDD-FVIII variant “X5”, which exchanges 5 amino acids in human BDD for corresponding porcine residues, is also secreted more efficiently than wildtype BDD-FVIII. We hypothesize that: a. FVIII expressed in hepatocytes by AAV delivery forms aggregates in the ER and activates stress responses resulting in cell death and potential HCC; and b. Novel FVIII molecules that more efficiently fold and are secreted will exhibit less toxicity in vivo are improved options for HA gene therapy. These hypotheses will be tested in 3 aims: 1. Delineate the cellular responses induced by AAV-BDD expression in hepatocytes of mice and define their pathophysiological consequences, especially HCC development; 2. Fully evaluate the potential of BDD variants F309S and X5 as more efficacious, safe and durable options for HA gene therapy; and 3. Characterize innate responses upon FVIII expression in primary human hepatocytes in humanized mice (Core 2). Our in- depth understanding of these issues should encourage the design of more optimal AAV-BDD vectors for HA gene therapy (Project 2) and increase our understanding of the impact of cellular stress and systemic inflammatory responses ass...