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CONDITIONAL SILENCING OF HLA EXPRESSION.
Constanca Figueiredo 1, Axel Seltsam MD 1, Peter A. Horn MD 1 and Rainer Blasczyk MD 1. 1 Department of Transfusion Medicine, Hanover Medical School, Hanover, Germany .
HLA polymorphism is the most relevant barrier to the development of cell-based therapies for regenerative purposes. To overcome this limitation, we used RNA interference (RNAi) to specifically knock down HLA class I transcripts.
Regions susceptible to the action of small interfering RNAs (siRNAs) were identified in HLA-A heavy chain and 
2-microglobulin (2m) transcripts to achieve a gene or class I specific HLA silencing, respectively. Lentiviral vectors were designed to express short hairpin RNA sequences (shRNA) targeting HLA-A heavy chain or 2m constitutively or controlled by a doxycycline-inducible promoter system. The level of HLA suppression in HeLa cells and B-LCLs was detected by flow cytometry, real-time RT-PCR and Western Blot. The capacity of this silencing approach to resist IFN-
-mediated HLA class I up regulation was tested in stimulation assays. Complement-dependent cytotoxicity assays were performed to evaluate the protective effect of HLA suppression against immune response.
The transduction of inducible RNAi cassettes containing the sequences for shRNAs targeting 2m suppressed HLA class I expression by up to 90% in HeLa or 60% in B-LCLs in a fully reversible manner. Similarly, conditional delivery of short hairpin RNA targeting HLA-A heavy chain transcripts silenced HLA-A expression by up to 90%. It was shown that HLA silencing was maintained even under inflammatory conditions. In a complement-mediated cytotoxicity assay it was demonstrated that HLA knockdown was extremely effective in preventing antibody-mediated cell lysis.
In conclusion, we demonstrate the feasibility of controlling HLA expression by genetically modifying cell-based therapeutics to overcome the limitations of immunological rejection, bringing cellular therapies closer to reality.