Although T cell responses to peptides derived from α-, γ- and ω-gliadins as well as from HMW- and LMW-glutenins have been described, various studies have indicated that the α-gliadins are among the most immunogenic regarding CD 
. A crucial step towards the elimination of gluten toxicity would thus be the elimination of T cell stimulatory α-gliadin sequences. Our extensive genetic analysis of over 3000 α-gliadin transcripts from different bread wheat accessions showed a high heterogeneity of the α-gliadins genes and considerable differences in the number of T cell stimulatory sequences encoded by the various α-gliadin genes. We identified three major factors determining these differences: i) the length of tandem repeats of antigenic sequences; ii) natural amino acid substitutions that affect the antigenicity of T cell epitopes, and iii) amino acid deletions that eliminate the antigenicity of T cell epitopes.
The α-gliadins from locus Gli-D2
generally encode several copies of both the DQ2-Glia-α1 and DQ2-Glia-α2 epitopes in addition to the DQ2-Glia-α3 and DQ8-Glia-α1 epitopes, Gli-A2
genes usually encode only the DQ2-Glia-α1 and DQ2-Glia-α3 epitopes while the Gli-B2
genes encode no or at most one DQ2-Glia-α1 epitope, next to the DQ8-Glia-α1 epitopes. The 33-mer sequence with 6 T cell stimulatory sequences 
was only found in a minority of the α-gliadins analyzed, all of which are expressed from Gli-D2
. Many natural occuring amino acid substitutions affecting the antigenicity of the canonical α-gliadin peptides were identified. Typical examples are the proline to serine substitution at p8 in the DQ2-Glia-α2 epitope and the arginine to proline substitution at p2 in the DQ2-Glia-α3 epitope, that both completely eliminate the T cell stimulatory properties of these peptides.
The analysis of gluten extracts from the diploid wheat varieties underscores these observations and provides a molecular basis for the previous observation that A-genome diploid T. monococcum
varieties lack the DQ2-Glia-α2 epitope 
. All α-gliadins from this genome encode an altered version of the DQ2-Glia-α2 epitope with a serine at p8 that fails to induce T cell responses. We observed that a similar substitution eliminates the T cell stimulatory properties of the DQ2-Glia-α1 and DQ2-Glia-α3 epitopes. The more variable reaction pattern of T-cells and antibodies towards gluten extracts of the S-genomes reflects the higher level of genetic variation in these outcrossing species. These α-gliadins contain another variant of the DQ2-Glia-α1 and DQ2-Glia-α2 region that does not incude the A-genome specific serine at p8. Furthermore, amino acid deletions in the canonical α-gliadin peptides prohibit binding to HLA-DQ2 and hence T cell recognition. A typical example is the deletion of the glutamine at p4 in the DQ2-Glia-α1 epitope which generates a peptide that no longer binds to HLA-DQ2, presumably due to defective docking of the anchor residues into their respective pockets in the HLA-DQ2 molecule. Our results confirm previous observations 
that the α-gliadin locus Gli-D2
encodes the most toxic α-gliadins while substantially less toxicity is associated with those from Gli-A2
, but also provide the molecular basis for these differences.
Unfortunately, due to the complexity of both the Gli-2
gene family and the wheat genome, it will be a difficult task to generate tetraploid pasta and hexaploid bread wheat that is entirely safe for consumption by all CD patients by conventional breeding methods. Our results now provide a rationale for an alternative approach as we demonstrate that by the introduction of naturally occurring amino acid substitutions the toxicity of all four T cell epitopes in α-gliadins can be eliminated. Using novel methods such as zinc finger nucleases 
we can introduce the underlying SNPs as specific mutations into the α-gliadin genes of wheat to eliminate toxicity completely. Technically this will not be easy, but our results indicate precisely the three complementary sets of actions that need to be performed.
i) In the Gli-B2-derived α-gliadins analyzed, none of the DQ2 epitopes are present due to a single amino acid deletion in the region encoding the DQ2-Glia-α1 and DQ2-Glia-α2 epitopes, which generates a peptide that has decreased binding affinity for HLA-DQ2 and is no longer recognized by T cells. Therefore, a single amino acid substitution in the DQ2-Glia-α3 epitope will result in a peptide that has completely lost HLA-DQ2 binding properties and T cell stimulatory properties. To eliminate the remaining DQ8-Glia-α1 epitope in some Gli-B2 α-gliadins, a single glutamine to arginine substitution, which naturally occurs in the α-gliadins from the A-genome, would suffice. Such a minimally genetically modified B-genome α-gliadin gene would thus no longer encode any T cell stimulatory peptides. Moreover, by starting with an α-gliadin gene in which the sequence of the p31-43 peptide is naturally altered (for example the gene encoding protein no. 9 in SI 2), the chance of innate immune stimulation by a protein derived from such a gene would also be minimized.
ii) For Gli-A2 α-gliadins, the approach to eliminate toxicity would be to introduce two proline to serine substitutions at p8 in the DQ2-Glia-α1 and DQ2-Glia-α3 epitopes present. As these α-gliadins genes encode a shorter version of the immunodominant 33-mer in which the DQ2-Glia-α-2 epitope is already non-functional, and contain a version of the DQ8-Glia-α1 epitope that has no T cell stimulatory properties, these two substitutions would completely remove toxicity in proteins encoded by such modified genes.
iii) Regarding the Gli-D2
α-gliadins, we found that the proline to serine substitutions at p8 completely abrogated the in vitro T cell stimulatory properties of the 33-mer peptide and of an elongated version of the 33-mer that also encodes the DQ2-Glia-α3 epitope. This result underscores our previous observation that most T cell stimulatory gluten peptides have a proline at p8 
. However, to render α-gliadins from the D-genome non-toxic, up to seven substitutions need to be introduced in a single gene.
An alternative approach is to design safe α-gliadin genes that can subsequently be introduced into celiac disease safe cereals such as rice or maize, for the production of gluten proteins. As such modified proteins will be very similar to existing α-gliadins they will most likely have indistinguishable technological properties. Thus, such gluten proteins could enhance the baking properties of these cereal crops, or they could be extracted from these crops and used as an ingredient to generate novel high quality foods for celiac disease patients. For the generation of high quality cereals that can replace wheat-based products the simple introduction of detoxified α-gliadins is unlikely to be sufficient, as baking quality is mostly determined by the HMW and LMW glutenin proteins. Therefore, additional studies will have to investigate how other gluten proteins can be detoxified as there is substantial evidence that these contain T cell stimulatory peptides as well. In previous studies we provided evidence that such epitopes can be found in the γ-gliadins as well as in the LMW- and HMW-glutenins 
and others have extended these observations 
. In particular, we found that T cell responses to LMW-glutenins were found in children while these are much less frequent in adults 
. Moreover, in a recent study a highly antigenic ω-gliadin peptide was described 
that is identical to an antigenic hordein-derived peptide reported by us earlier 
. In essence this hordein/omega peptide is a sequence variant of the DQ2-Glia-α1 peptide and also carries a proline at the p8 position 
. It is therefore feasible that the toxicity of this peptide can be eliminated by a proline to serine substitution at p8 as well. Preliminary results show that amino acid substitutions similar to those that destroy the T cell stimulatory properties in α-gliadins might also be effective for γ-gliadin derived epitopes (Salentijn et al, in prep), but it is likely that for the LMW- and HMW-glutenins other approaches will be required. This will be the subject of further studies.
In conclusion, we have demonstrated that by utilizing naturally occurring amino acid substitutions the toxicity of the four T cell epitopes in α-gliadins can be eliminated. Such modified proteins will most likely display indistinguishable technological properties. Thus, our results provide a rational approach to eliminate CD related toxicity from α-gliadins, which represents a first but crucial step towards the realization of safe gluten containing food products for CD patients.