Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

Fibroblasts were isolated from skin punch biopsies, as previously described (Jing et al., 2014) . In brief, dermal and epidermal layers were dissociated after overnight incubation of biopsy tissue with Dispase (BD). The dermis was minced and cultured in complete DMEM-Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Gibco), and 55 µM 2-ME (Sigma-Aldrich) to allow fibroblasts to grow out. Where indicated, human primary fibroblasts were treated with 100 IU/ml recombinant human IFNalpha-2b (Intron A; Merck) overnight to induce MDA5 expression. Fibroblasts isolated from a healthy normal donor or the patient's mother were used for IFIH1 genome editing. Cells from the patient's mother were used to generate wild-type/ null, mutant (c.1093A>G, p.K365E)/null, and null/null genotypes. U6gRNA-Cas9-2A-GFP plasmids (HS0000455078; Sigma-Aldrich) were transfected into the fibroblasts using the P3 Primary Cell 96-well Nucleofector kit with Nucleofector Program 96-DT-130 (Lonza). 3 d after transfection, cells highly expressing GFP were single-cell sorted into 96-well plates using a FAC SAria Fusion cell sorter (BD) and cultured until confluent. Cells from the patient's mother, or from a healthy normal donor that had not undergone genome editing were also single-cell sorted and cultured in parallel for use as controls. Genotypes for each genome-edited clone were screened by agarose gel electrophoresis of PCR amplified products using forward primer 5′-AAA GGG GAA ATA CGG AAT TGG-3′ and reverse primer 5′-GAG TCA ATG ACA CAA ATG CCA TC-3′, followed by confirmation by Sanger dideoxy sequencing. Three to five clones each of IFIH1 genotypes wild-type/null, mutant/null, wild-type/mutant, and null/null were selected and expanded for experiments.