Anti-Collagen Type I (native) polyclonal antibody

Alport syndrome (AS) is an inherited genetic disorder characterized by range of alterations from glomerular basement membrane abnormalities up to end-stage renal disease. Pathogenic variants in the collagen alpha 3, alpha 4, and alpha 5 encoding genes are causative both of the autosomal dominant and of the X-linked forms of AS. Podocytes are the only renal cells that are able to produce the COL(IV)a3-a4a5 heterotrimer. We have previously demonstrated how it is possible to isolate podocyte-lineage cells from urine of patients, providing an easily accessible cellular model closer to the podocytes' physiological conditions. Taking advantage of disease-relevant cell lines, we employed a two-plasmid approach in order to achieve a beneficial and stable variant-specific correction using CRISPR/Cas9 genome editing. One plasmid carries a Donor DNA and a reporter system mCherry/GFP to track the activity of Cas9 in cells. The other plasmid carries a self-cleaving SpCas9 and the variant-specific sgRNA. We have analyzed two stable podocyte-lineage cell lines, harboring a variant in the X-linked COL4A5 (p.(Gly624Asp)) and in the autosomal COL4A3 gene (p.(Gly856Glu)). We have achieved reversion of variants greater than 40% with undesired insertions/deletions lower than 15%. Overall, we have demonstrated a new gene therapy approach directly on patients' cells, key players of Alport pathogenesis, and we have reverted COL4 causative variants towards the wild type state. These results, in combination with preclinical models, could open new frontiers in the management and the treatment of the disorder.

Collagen IV nephropathies (COL4Ns) comprise benign familial microscopic hematuria, thin basement membrane nephropathy (TBMN), X-linked Alport syndrome (AS) and also autosomal recessive and dominant AS. Apart from the X-linked form of AS, which is caused by hemizygous mutations in the COL4A5 gene, the other entities are caused by mutations in the COL4A3 or COL4A4 genes. The diagnosis of these conditions used to be based on clinical and/or histological findings of renal biopsies, but it is the new molecular genetics approach that revolutionised their investigation and proved particularly instrumental, especially, in many not so clear-cut cases. More recently, the spectrum of COL4N has expanded to include late onset focal segmental glomerulosclerosis (FSGS) that develops on top of TBMN in later life. Also, other reports showed that some patients with a primary diagnosis of familial FSGS proved to have variants in COL4 genes. In the presence of a renal biopsy picture of FSGS and in the absence of either electron microscopy studies or molecular genetic studies that point to TBMN and COL4N, the patient and his family may be mistakenly diagnosed with hereditary FSGS leading to unnecessary further investigations, erroneous family counselling and improper corticosteroid treatment. TBMN is a frequent finding in the general population, and according to several recent reports, it may be the underlying cause and the explanation for many familial and sporadic cases of late-onset FSGS with non-nephrotic proteinuria. This is an important new finding that needs widespread recognition. It is anticipated that the molecular genetic analysis with next generation sequencing will certainly offer timely correct diagnosis. (C) 2015 S. Karger AG, Basel