Molecular and cellular studies reveal folding defects of human ornithine aminotransferase variants associated with gyrate atrophy of the choroid and retina

The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments

Human neural stem cell transplantation in ALS: initial results from a phase I trial

We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanit\ue0 and the competent Ethics Committees and was monitored by an external Safety Board

Human Neural Stem Cell-Based Drug Product: Clinical and Nonclinical Characterization

Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials