New insights into pathogenesis of acute porphyria attacks and proof-of-concept of augmenting hepatic PBGD as etiological treatment

Abstract

Acute Intermittent Porphyria (AIP) is a rare disease caused by a hepatic deficiency of porphobilinogen deaminase (PBGD) and is characterized by acute neurovisceral attacks. Currently, available treatments for AIP are not optimal and do not cover the entire spectrum of symptoms and presentations of the disease; thus, research for the development of more efficient drugs is essential. So, unlike approved treatments, our line of research is based on increasing protein PBGD levels in the liver as an etiological solution to the disease. AIP affects central, autonomic and peripheral nervous system. However, many aspects of the onset and course of this disease are still unknown. Recent research led by our group, reported some alterations in the central nervous system (CNS) such as decreased cerebral blood flow during porphyria crises and brain ventricle enlargement in patients suffering recurrent porphyria attacks. Hence, the first aim of this thesis was to analyze these pathological features in a porphyric mouse model and the effect of increasing hepatic PBGD activity on these CNS implications. In recent years, messenger RNA (mRNA) based treatments have revolutionized the approach to this type of diseases, thus the administration of human PBGD (hPBGD) mRNA could be a promising therapeutic approach. mRNA therapy is based on modified RNA sequences that are delivered into lipid nanoparticles (LNPs) that prevent mRNA degradation and target the liver. It is worth noting that the administration of hPBGD mRNA allows expression of the therapeutic protein as rapid as hours, a crucial feature to stop the acute attack as soon as the first symptoms appear. In addition, the LNPs can be readministered, allowing single or repeated administrations depending on the clinical status of the patient. In rare diseases, recruiting patients for clinical trials is difficult. For this reason, additional preclinical efficacy and safety studies are required, preferably in large animal models. Thus, the therapeutic effect of hPBGD mRNA was assayed in a new model of acute attack in rabbits. The second aim of this thesis was the biochemical and clinical characterization of the new rabbit model, as well as the therapeutic effect of the hPBGD mRNA against the acute attack in this model. As another therapeutic option, our group generated a recombinant protein formed by linking Apolipoprotein AI (ApoAI) to the N-terminus of human PBGD (rhApoAI-PBGD) to transfer PBGD protein into liver cells. Intravenous or subcutaneous administration of rhApoAI-PBGD showed sustained efficacy against AIP attacks in the mouse model. However, large-scale production under good manufacturing practices (GMP) conditions is challenging for enzyme replacement therapies. For that purpose, the third aim of this thesis was to develop a large-scale production and purification process of rhApoAI-PBGD protein in CHO-K1SP cells, a cell line used for the production of recombinant proteins. As result of this thesis, we concluded that: - Augmenting PBGD activity in the liver could be an etiological treatment for AIP as it restores regulation of hepatic heme synthesis pathway and protects against disturbances associated with acute attacks on central, peripheral and autonomic nervous systems on mouse and rabbit models. - Messenger RNA-based therapy in the variegate porphyria rabbit model showed more efficient protection against the acute attack than hemin, the standard of care for acute hepatic porphyrias, and rapid therapeutic action to treat acute metabolic decompensations. Thus, it is a promising strategy for etiological treatment of acute porphyria attacks.