Abstract

Abstracts

Stem cell derived cellular therapeutics:
Controlling MHC expression

Constanca Figueiredo, Axel Seltsam, Rainer Blasczyk, and Peter A. Horn

Over the last decade, regenerative medicine and cell-based therapies have emerged as a new science and technology, whose main goal is to repair, replace or regenerate new tissues. A critical issue in this field is the high polymorphism of HLA which compromises immune acceptance. As RNA interference (RNAi) is capable of effectively knocking down specific transcripts, RNAi was used to selectively reduce cellular HLA class I and class II expression. For this purpose, small interfering RNAs (siRNAs) were designed and chemically synthesised to target β2-microglobulin (β2m) as well as the HLA-A heavy chain or HLA-DR alpha chain transcripts. Sensitive sites in the target RNAs were identified using an in vitro translation system. The best siRNAs were used for knockdowns in B-LCL, HEK and HeLa cell lines. In order to achieve a stable reduction of HLA expression, constitutive and conditional lentiviral expression vectors were constructed encoding the sequences for short hairpin RNAs. The transduction of drug-inducible RNAi cassettes containing the sequences for shRNAs targeting β2m or HLA-A heavy chain suppressed HLA class I expression by up to 90% in HeLa and B-LCL cell lines as well as in peripheral blood monocytes. Also, HLA-DR expression was reduced by up to 90% in IFN-γ stimulated HeLa cells, 57% in B-LCLs and 86% in peripheral blood monocytes, upon expression of HLA-DRA specific shRNAs. The expression of HLA class I and HLA class II 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, while the residual HLA expression in HLA class I-silenced cells was protective against NK cell-mediated lysis.
These data 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.

Biomaterials and DDS technologies to realize advanced medical therapy

Yasuhiko Tabata

As the third choice following the reconstruction surgery and organ transplantation, a new therapeutic trial based on the natural potential to induce tissue regeneration, has been expected. For successful therapy of tissue regeneration, it is necessary to make use of cells, the scaffold of cell proliferation and differentiation, and signaling molecules (growth factors and genes) or their combination. However, only by using cells, it is practically difficult to induce tissue regeneration. Therefore, a biomedical technology or methodology to create a local environment which enables cells to promote their proliferation and differentiation and consequently induce tissue regeneration, is highly required. If cells around a tissue defect have an inherent regeneration ability, tissue regeneration will be induced only by supplying a cell scaffold of biomaterials to the defect. For the tissue site of poor regeneration potential, combination with cells or signaling molecules is required. One practically possible way to enhance the in vivo efficacy of signaling molecules is to make use of drug delivery system (DDS). We have explored biodegradable hydrogels for the controlled release of bioactive growth factors or genes and succeeded in regeneration repairing of various tissues with or without the combination of cells. Some clinical trials for the growth factor-induced regeneration of blood vessels, bone, and skin dermis have been started to demonstrate the superior therapeutic availability. In this seminar, several experimental data of tissue or organ regeneration were overviewed to emphasize scientific and clinical significance of biomaterial and DDS technologies in advanced medical therapy.