Back to Search Start Over

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes.

Authors :
Valentina Perri
Marsha Pellegrino
Francesca Ceccacci
Anita Scipioni
Stefania Petrini
Elena Gianchecchi
Anna Lo Russo
Serena De Santis
Giovanna Mancini
Alessandra Fierabracci
Source :
PLoS ONE, Vol 12, Iss 4, p e0175784 (2017)
Publication Year :
2017
Publisher :
Public Library of Science (PLoS), 2017.

Abstract

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Subjects

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
19326203
Volume :
12
Issue :
4
Database :
Directory of Open Access Journals
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
edsdoj.07e3160907ee4d81a8f125229f3a9a55
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pone.0175784