AAV-mediated FOXG1 gene editing in human Rett primary cells.

Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20-35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

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

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.

An efficient method to enrich for knock-out and knock-in cellular clones using the CRISPR/Cas9 system.

Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 nuclease (CRISPR/Cas9) and Transcription Activator-Like Effector Nucleases (TALENs) are versatile tools for genome editing. Here we report a method to increase the frequency of Cas9-targeted cellular clones. Our method is based on a chimeric construct with a Blasticidin S Resistance gene (bsr) placed out-of-frame by a surrogate target sequence. End joining of the CRISPR/Cas9-induced double-strand break on the surrogate target can place the bsr in frame, thus providing temporary resistance to Blasticidin S: this is used to enrich for cells where Cas9 is active. By this approach, in a real experimental setting, we disrupted the Aicda gene in ~70% of clones from CH12F3 lymphoma cells (>40% biallelically). With the same approach we knocked in a single nucleotide to reconstruct the frame of Aicda in these null cells, restoring the function in ~37% of the clones (less than 10% by the standard approach). Targeting of single nucleotide changes in other genes yielded analogous results. These results support our enrichment method as an efficient tool in genome editing.

Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc).

Multiple sclerosis (MS) is a chronic auto-immune disease characterized by a damage to the myelin component of the central nervous system. Self-antigens created by aberrant glycosylation have been described to be a key component in the formation of auto-antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS-specific auto-antibodies, and it is actively used in diagnostics and research to monitor and quantify MS-associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His-tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide-clone interaction were calculated obtaining a value of KD in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies.