Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing

Abstract

The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230–232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.

Expression profile of HLA‐B*38:55Q allele

“…Since HLA mismatches may produce severe graft –vs host disease (GvHD) and graft rejection, a misinterpretation of these variants could strongly affect transplant‐related mortality. Hence, several studies proposed assays and methods to discriminate between low expressed or non‐expressed HLA alleles, mainly based on EBV‐transformed cell lines or on recombinant HLA‐molecules' cloning and transfection in established cell lines, not easily reproducible in most laboratories. Gerritsen et al demonstrated that HLA‐A*23:19Q , which has a single polymorphism at position 619 (G > A) compared with HLA‐A*23:01:01 , has no expression by serology and flow cytometry, therefore, it should be reclassified as a null allele .…”

“…Using the same approach, Foll et al reported the HLA‐A*32:11Q as a low expressed variant . More recently, Yin et al demonstrated that HLA‐B*38:68Q is a low expressing allele using the CRISPR/Cas9 gene‐editing technology …”

“…Mutations such as insertions/deletions in HLA sequences can lead to the introduction of stop codons and/or incorrectly spliced products that result in aberrant expressed alleles; numerous alleles have been described having polymorphisms at the exon borders potentially affecting the splicing sites (eg, A*02:01:14Q , A*24:02:03Q , A*33:03:03Q ), and impairing, as a result, the messenger RNA (mRNA) transcription. In our study, the cDNA group‐specific amplification using HLA‐B*38 specific primers proved that the B*38:55Q transcripts were present and unaffected if compared with B*38:01 allele, showing that HLA‐A*38:55Q gene is transcribed and correctly translated, indicating that probably the cause of the low expression level is a post‐translational instability.…”

“…The HLA classes I and II are the most polymorphic genes in the human genome with 24 093 different HLA alleles identified (IPD‐IMGT/HLA database v3.37) . The high level of HLA polymorphism reflects its biological importance to present a large diversity of peptides to the immune system and trigger effective immune responses . In allogeneic hematopoietic stem cell transplantation (HSCT), HLA mismatches between donor and recipient leads to strong alloimmune responses that result in either graft vs host disease (GVHD) or engraftment failure …”

“…The frequency of these questionable alleles has not been well established, but is conceivable that the frequency may be underestimated. Moreover, with the advancement of the full gene HLA sequencing by next‐generation sequencing (NGS) technology, the laboratories are able to obtain high‐resolution HLA typing with additional sequencing informations on exons outside the antigen recognition sites (ARS) and introns, thus, it is likely that more questionable alleles will be discovered …”