The overall objectives of the proposed research are to investigate the biochemistry, molecular pathology, and potential therapy of two inborn errors of heme biosynthesis: acute intermittent porphyria (AIP), an autosomal dominant hepatic porphyria due to the half-normal activity of hydroxymethylbilane synthase (HMBS), and congenital erythropoietic porphyria (CEP), an autosomal recessive disorder due to the markedly deficient activity of uroporphyrinogen III synthase (UROS). Recombinant human HMBS isozymes (housekeeping and erythroid-specific) and UROS will be purified to homogeneity for antibody production and NMR studies to characterize the structure and reaction mechanism of UROS and its interaction with HMBS in a putative cytosolic complex or """"""""metabolon"""""""" for the efficient conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB) to the cyclic tetrapyrrole, uroporphyrinogen (UROgen) III. Fluorescent antienzyme antibodies will be used to determine if HMBS and UROS co-localize in a cytosolic complex, hypothetically on the surface of mitochondria. For precise diagnosis and structure/function studies, denaturing HPLC (dHPLC) methods will be developed to expedite detection of HMBS and UROS mutations causing AIP and CEP, respectively. For AIP, efforts will determine if the life-threatening, acute neurologic attacks can be prevented by liver-targeted gene therapy. Recombinant adeno-associated viral (rAAV) vectors containing hepatic regulatory elements and the murine HMBS eDNA will be evaluated in HEK-293 and Hepa 1-6 cells. The optimally expressing vector(s) with various envelope serotypes will be injected into the portal vein of the HMBS-deficient mice (""""""""AIP mice"""""""") and their ability to prevent phenobarbital-induced acute porphyric attacks will be assessed by monitoring plasma and urinary 5'-aminolevulinic acid (ALA) and PBG levels. Optimal gene delivery, hepatic expression and persistence, and possible hepatic toxicity will be determined. For CEP, a viable UROS knock-in mouse will be generated based on human CEP mutations with residual activity. These mice will be used to evaluate bone marrow transplantation (BMT) and hematopoietic stem cell (HSC) gene replacement, the latter employing lentiviral vectors containing the murine erythroid-specific UROS promoter and other erythroid enhancer elements to drive erythroid expression. The effectiveness (expression, persistence, toxicity, etc) of these vectors to correct the murine CEP biochemical/clinical abnormalities will be evaluated.
Showing the most recent 10 out of 56 publications
