Biomedical subjects

Constança Figueiredo

Publications and source records attributed to Constança Figueiredo.

2 recordsLinked to original sources

PubMed2007

Regulating MHC expression for cellular therapeutics.

BACKGROUND: In the past decade, regenerative medicine and cell-based therapies have emerged as new science and technology, with the main goal of repairing, replacing, or regenerating new tissues. A critical issue in this field is the high polymorphism of HLA, which compromises immune acceptance. The lentivirus-mediated delivery of short-hairpin RNAs (shRNAs) has proved to be an efficient method to inhibit the translation of a specific gene. STUDY DESIGN AND METHODS: A lentiviral-based vector system was used for drug-inducible expression of shRNA sequences that target either beta2-microglobulin (beta2m) or HLA heavy-chain transcripts. RESULTS: The transduction of inducible RNA interference cassettes containing the sequences for shRNAs targeting beta2m or HLA heavy chain suppressed HLA class I expression by up to 90 percent in HeLa and B-lymphocyte cell lines as well as in peripheral blood monocytes. The expression of HLA class I antigens was fully restored in these cells after the drug had been discontinued. It was demonstrated that HLA class I knockdown was effective in preventing antibody-mediated cell lysis and CD8+ T-cell response. The residual HLA expression in HLA-silenced cells may provide sufficient protection against natural killer cell-mediated lysis. CONCLUSIONS: These data demonstrate the feasibility of controlling HLA expression by genetically modifying cell-based therapeutics to overcome the limitations of immune rejection, bringing cellular therapies closer to reality.

CD8-Positive T-Lymphocytes↗

PubMed2006

Class-, gene-, and group-specific HLA silencing by lentiviral shRNA delivery.

HLA incompatibility is the most relevant immunologic barrier to cell-based therapies. Improvement of histocompatibility is essential to achieving better survival of allogeneic cells in the foreign organism. RNA interference technology can be used to selectively and stably reduce cellular HLA class I expression. In the present study, we designed small interfering RNA (siRNA) molecules that target either beta2-microglobulin (beta2m) or HLA-A heavy chain transcripts and identified sensitive sites on the target RNAs using an in vitro transcription/translation (IVTT) system. Transfection of siRNA into B-lymphocyte cell lines (B-LCLs) resulted in specific reduction of HLA class I or HLA-A antigen expression by 79% at the mRNA and protein levels. An allele-specific HLA silencing rate of 65% was achieved in a B-LCL heterozygous for HLA-A*24,*68 allospecificities using HLA-A*68-specific siRNA. Lentiviral delivery of short hairpin RNA into HeLa and B-LCL cells resulted in selective and permanent silencing of HLA class I or HLA-A by up to 90% even under inflammatory conditions. In cytotoxicity and proliferation assays, it was demonstrated that HLA class I knockdown was effective in preventing antibody-mediated cell lysis and CD8+ T cell response, while the residual HLA expression in HLA-silenced cells was protective against NK-cell-mediated lysis. The present data strongly suggest that silencing of HLA expression in a class-, gene-, and group-specific manner is an effective approach that may provide a new basis for developing new immunotherapies in the field of regenerative medicine.

B-Lymphocytes↗