Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4 T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4 T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated and .

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand () gene with a median survival of 25 years, potentially treatable with CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of in edited healthy donor and patient cells recapitulating the physiological regulation.

Characterization of human mitochondrial ferritin promoter: identification of transcription factors and evidences of epigenetic control.

Mitochondrial ferritin (FtMt) is an iron storage protein belonging to the ferritin family but, unlike the cytosolic ferritin, it has an iron-unrelated restricted tissue expression. FtMt appears to be preferentially expressed in cell types characterized by high metabolic activity and oxygen consumption, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. The human gene (FTMT) is intronless and its promoter region has not been described yet.

Skin fibroblasts from pantothenate kinase-associated neurodegeneration patients show altered cellular oxidative status and have defective iron-handling properties.

Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disease belonging to the group of neurodegeneration with brain iron accumulation disorders. It is characterized by progressive impairments in movement, speech and cognition. The disease is inherited in a recessive manner due to mutations in the Pantothenate Kinase-2 (PANK2) gene that encodes a mitochondrial protein involved in Coenzyme A synthesis.

Oxidative stress and cell death in cells expressing L-ferritin variants causing neuroferritinopathy.

Neuroferritinopathies are dominantly inherited movement disorders associated with nucleotide insertions in the L-ferritin gene that modify the protein's C-terminus. The insertions alter physical and functional properties of the ferritins, causing an imbalance in brain iron homeostasis. We describe the effects produced by the over-expression in HeLa and neuroblastoma SH-SY5Y cells of two pathogenic L-ferritin variants, 460InsA and 498InsTC.

Mitochondrial ferritin limits oxidative damage regulating mitochondrial iron availability: hypothesis for a protective role in Friedreich ataxia.

Mitochondrial ferritin (FtMt) is a nuclear-encoded iron-sequestering protein that specifically localizes in mitochondria. In mice it is highly expressed in cells characterized by high-energy consumption, while is undetectable in iron storage tissues like liver and spleen. FtMt expression in mammalian cells was shown to cause a shift of iron from cytosol to mitochondria, and in yeast it rescued the defects associated with frataxin deficiency.