New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

Abstract

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 α3, α4, and α5 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.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

sgRNA identification and design and dual plasmid cloning strategy to achieved CRISPR/Cas9 correction. In the upper lane of panel a, the sgRNA is represented in red and blue: PAM in blue and in red the mutated nucleotide for the COL4A5 CRISPR/Cas9 design. Below, the same design is reported for the COL4A3 variant. Plasmid functionality is based on the cloning of two plasmids for CRISPR/Cas9 components delivery. In the first plasmid, the sgRNA is under the control of Human U6 Promoter (b), while the mCherry Red/Green Reporter System is under the control of a CMV Promoter. The Cas9 plasmid is cloned with spCas9 endonuclease gene under control of a CMV promoter flanked by two Self-Cleaving Cas9 for auto-inactivation (c). Representative FACS analysis on HEK293T cells after 48 h of transfection with reporter harboring the wild type sequence and mutated reporter, for COL4A3 and COL4A5 variants, respectively. The population of cells mCherry+/GFP+ is gated in the UR quadrant. The double fluorescence is present only in the cells co-transfected with Donor/Reporter plasmid with specific variant and spCas9 (41.9% and 27.6% for COL4A3 and COL4A5, respectively) demonstrating the specificy of sgRNA (d)

Fig. 2

Transfection efficacy in urine-derived podocyte-lineage cells. a Type I (Upper left panel) and Type II (Upper right panel) urinary cell colonies (arrowheads) at day 4. At day seven the isolated cells start to aggregate in egg shape conformation (Bottom left panel). On the eighth day, cells starts to have clonal expansion and they can splitted (Bottom right panel) Scale bars, 400 μm. b Both plasmids were transfected on cells in clonal growth stage (Bottom panel) and mCherry and GFP fluorescence was observed in vivo through fluorescence microscope. cRepresentative FACS analysis of GFP expressing. Cells were transfected either with Donor/Reporter plasmid alone and sgRNA/Cas9 coupled with the Donor/Reporter plasmids. The cells were analyzed by FACS 48 h after transient transfection. The percentage of GFP + cells is indicated in the upper right quadrant (UR). At least three independent experiments were performed. The average population in the UR quadrant was 0.2% for Reporter alone; 21.7% for Cas9 + Donor/Reporter

Fig. 3

CasAnalyzer results on editing efficiency on COL4A5 podocyte-lineage cells. CasAnalzyer Tool operating on the CRISPR/Cas9 treated sample and control has reported a HDR of 58.8% in patients’ mutated podocyte-lineage cells. IGV Visualization Software for the variant shows how there is a substantial loss of heterozygosity with the restoration of the wild-type base, confirming the functionality of the system

Fig. 4

CasAnalyzer results on editing efficiency on COL4A3 podocyte-lineage cells. The CasAnalzyer Tool performed on the CRISPR/Cas9 treated sample and control has reported a HDR of 44.2% in patients’ mutated podocyte-lineage cells. IGV Visualization Software for this variant also exhibited the loss of heterozygosity with the restoration of the wt base in COL4A3 gene