Summary: Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins from wheat and similar proteins from barley and rye. The inflammatory reaction is controlled by T cells that recognize gluten peptides in the context of human leukocyte antigen (HLA) DQ2 or HLA-DQ8 molecules. The only available treatment for the disease is a lifelong gluten-exclusion diet. We have used RNAi to down-regulate the expression of gliadins in bread wheat. A set of hairpin constructs were designed and expressed in the endosperm of bread wheat. The expression of gliadins was strongly down-regulated in the transgenic lines. Total gluten protein was extracted from transgenic lines and tested for ability to stimulate four different T-cell clones derived from the intestinal lesion of CD patients and specific for the DQ2-α-II, DQ2-γ-VII, DQ8-α-I, and DQ8-γ-I epitopes. For five of the transgenic lines, there was a 1.5–2 log reduction in the amount of the DQ2-α-II and DQ2-γ-VII epitopes and at least 1 log reduction in the amount of the DQ8-α-I and DQ8-γ-I epitopes. Furthermore, transgenic lines were also tested with two T-cell lines that are reactive with ω-gliadin epitopes. The total gluten extracts were unable to elicit T-cell responses for three of the transgenic wheat lines, and there were reduced responses for six of the transgenic lines. This work shows that the down-regulation of gliadins by RNAi can be used to obtain wheat lines with very low levels of toxicity for CD patients.