Development of non-integrative viral vectors for gene therapy of acute intermittent porphyria

Resumen

Acute intermittent porphyria (AIP) is a rare metabolic liver disease characterized by porphobilinogen deaminase (PBGD) deficiency. Clinical features are acute neurovisceral attacks accompanied by overproduction of presumably toxic porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. The principal aims of this study were to assess the protective effects of bone marrow transplantation or PBGD gene transfer mediated by viral vectors into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. On the other hand, mice are not the best model to predict the human immune system reaction against viral vectors. Therefore, immunosuppression studies in order to control the reaction against the vectors were performed in non-human primates (NHP). Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Therefore, PBGD activity normalization in erythroid tissue cannot metabolize the increased levels of porphyrin precursor. Since these precursors are accumulated in the liver, it is the target organ. In order to transfer the PBGD gene to hepatocytes, we developed recombinant adeno-associated vectors (rAAV) and helper-dependent adenovirus (HDA) encoding the human PBGD protein. The cDNA was driven by a liver specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction and progressive loss of large-calibre axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 or HDA injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. Therefore, alternative administration of these three different vectors carrying the same transgene could be an option for the treatment of AIP. Besides, hepatic hPBGD over-expression does not modify cellular homeostasis and restores the metabolic stress generated in the liver of AIP mice associated with the PBGD deficiency. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. We sought to study in non-human primates the feasibility of controlling this response with a three months intensive immunosuppressive (IS) regimen after intravenous administration of rAAV5 vectors or intrahepatic administration of HDA vectors. IS fails to improve AAV5-based liver gene transfer in NHP. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression. On the other hand, immunosuppression during HDA administration permitted the control of the humoral response and maintained PBGD over-expression in NHP during more than one year.