A new nucleic acid-based agent inhibits cytotoxic T lymphocyte-mediated immune disorders.

BACKGROUND Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and graft-versus-host disease (GVHD) are distinct immune reactions elicited by drugs or allogeneic antigens; however, they share a pathomechanism with the activation of cytotoxic T lymphocytes (CTLs). CTLs produce cytotoxic proteins, cytokines, chemokines, or immune alarmins, such as granulysin (GNLY), leading to the extensive tissue damage and systemic inflammation seen in patients with SJS/TEN or GVHD. Currently, there is no effective therapeutic agent specific for CTL-mediated immune disorders. OBJECTIVES By targeting GNLY(+) CTLs, we aimed to develop a nucleic acid-based agent consisting of an anti-CD8 aptamer with GNLY small interfering RNA (siRNA). METHODS We performed systematic evolution of ligands using exponential enrichment to select and identify effective anti-CD8 aptamers. We developed an aptamer-siRNA chimera using a "sticky bridge" method by conjugating the aptamer with siRNA. We analyzed the inhibitory effects of the aptamer-siRNA chimera on CTL responses in patients with SJS/TEN or GVHD. RESULTS We identified a novel DNA aptamer (CD8AP17s) targeting CTLs. This aptamer could be specifically internalized into human CTLs. We generated the CD8AP17s aptamer-GNLY siRNA chimera, which showed a greater than 79% inhibitory effect on the production of GNLY by drug/alloantigen-activated T cells. The CD8AP17s aptamer-GNLY siRNA chimera decreased cytotoxicity in in vitro models of both SJS/TEN (elicited by drug-specific antigen) and GVHD (elicited by allogeneic antigens). CONCLUSIONS Our results identified a new nucleic acid-based agent (CD8 aptamer-GNLY siRNA chimera) that can significantly inhibit CTL-mediated drug hypersensitivity, such as that seen in patients with SJS/TEN, as well as the alloreactivity seen in patients with GVHD. This study provides a novel therapeutic strategy for CTL-mediated immune disorders.

[1]  Yuan-Tsong Chen,et al.  Shared and restricted T-cell receptor use is crucial for carbamazepine-induced Stevens-Johnson syndrome. , 2011, The Journal of allergy and clinical immunology.

[2]  Yuan-Tsong Chen,et al.  A marker for Stevens–Johnson syndrome , 2004 .

[3]  A. Krensky,et al.  Biology and clinical relevance of granulysin. , 2009, Tissue antigens.

[4]  T. Ganz,et al.  An antimicrobial activity of cytolytic T cells mediated by granulysin. , 1998, Science.

[5]  Yong Wang,et al.  Cell type–specific delivery of siRNAs with aptamer-siRNA chimeras , 2006, Nature Biotechnology.

[6]  Daniel G. Anderson,et al.  Knocking down barriers: advances in siRNA delivery , 2009, Nature Reviews Drug Discovery.

[7]  M. Bevan,et al.  CD8 lineage commitment in the absence of CD8. , 1997, Immunity.

[8]  B. Julian,et al.  OKT3 first-dose reaction: association with T cell subsets and cytokine release. , 1991, Kidney international.

[9]  H. Salmasian,et al.  Corticosteroid regimens for treatment of acute and chronic graft versus host disease (GvHD) after allogenic stem cell transplantation. , 2010, The Cochrane database of systematic reviews.

[10]  T. Morio,et al.  Analysis of serum granulysin in patients with hematopoietic stem‐cell transplantation: Its usefulness as a marker of graft‐versus‐host reaction , 2006, American journal of hematology.

[11]  E. Coene,et al.  Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ , 2005, Journal of Virology.

[12]  John J Rossi,et al.  Cell-specific aptamer-mediated targeted drug delivery. , 2011, Oligonucleotides.

[13]  B K Jakobsen,et al.  Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. , 2000, Immunology today.

[14]  K. Ravichandran,et al.  Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity , 2004, Journal of Virology.

[15]  Yuan-Tsong Chen,et al.  Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis , 2008, Nature Medicine.

[16]  Todd M. Allen,et al.  Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras. , 2011, The Journal of clinical investigation.

[17]  R. Zamoyska,et al.  The CD8 coreceptor revisited: one chain good, two chains better. , 1994, Immunity.

[18]  Shui-Tein Chen,et al.  HLA-B*1502-bound peptides: implications for the pathogenesis of carbamazepine-induced Stevens-Johnson syndrome. , 2007, The Journal of allergy and clinical immunology.

[19]  J. Ferrara,et al.  Advances in the clinical management of GVHD. , 2008, Best practice & research. Clinical haematology.

[20]  E. Holler,et al.  Graft-versus-host disease , 2009, The Lancet.

[21]  J. Rossi,et al.  Aptamer-targeted cell-specific RNA interference , 2010, Silence.

[22]  John J. Rossi,et al.  The promises and pitfalls of RNA-interference-based therapeutics , 2009, Nature.

[23]  Yuan-Tsong Chen,et al.  Direct interaction between HLA-B and carbamazepine activates T cells in patients with Stevens-Johnson syndrome. , 2012, The Journal of allergy and clinical immunology.

[24]  Takashi Shimizu,et al.  Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1 , 2002, Immunology.

[25]  W. Shlomchik,et al.  Graft-versus-host disease , 2007, Nature Reviews Immunology.

[26]  H. Hakonarson,et al.  Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection. , 2011, The American journal of pathology.

[27]  Z. Tian,et al.  A Novel NF-κB Binding Site Controls Human Granzyme B Gene Transcription1 , 2006, The Journal of Immunology.

[28]  M. Mockenhaupt.  The current understanding of Stevens–Johnson syndrome and toxic epidermal necrolysis , 2011, Expert review of clinical immunology.

[29]  P. Hari,et al.  NF-kappaB as a target for the prevention of graft-versus-host disease: comparative efficacy of bortezomib and PS-1145. , 2006, Blood.

[30]  Nicki Panoskaltsis,et al.  Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. , 2006, The New England journal of medicine.

[31]  Anthony D. Keefe,et al.  Aptamers as therapeutics , 2010, Nature Reviews Drug Discovery.

[32]  J. Tschopp,et al.  Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. , 1998, Science.

[33]  Weihong Tan,et al.  Identification of liver cancer-specific aptamers using whole live cells. , 2008, Analytical chemistry.

[34]  B. Williams,et al.  Activation of the mammalian immune system by siRNAs , 2005, Nature Biotechnology.

[35]  Young Jik Kwon,et al.  Efficient and targeted delivery of siRNA in vivo , 2010, The FEBS journal.

[36]  Transfection optimization for primary human CD8+ cells. , 2011, Journal of immunological methods.

[37]  Y. Tay,et al.  Stevens-Johnson syndrome and toxic epidermal necrolysis: efficacy of intravenous immunoglobulin and a review of treatment options. , 2009, Singapore medical journal.

[38]  Paul M. Allen,et al.  Specificity of T-cell alloreactivity , 2007, Nature Reviews Immunology.

[39]  John J. Rossi,et al.  Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells , 2009, Nucleic acids research.

[40]  J. Serody,et al.  Ex vivo Inhibition of NF‐κB Signaling in Alloreactive T‐cells Prevents Graft‐Versus‐Host Disease , 2009, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[41]  P. Zou,et al.  A comparative study of outcomes of idarubicin- and etoposide-intensified conditioning regimens for allogeneic peripheral blood stem cell transplantation in patients with high-risk acute leukemia , 2009, Acta Pharmacologica Sinica.

[42]  Dinshaw J. Patel,et al.  A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia , 2010, Nucleic acids research.

[43]  P. Demoly,et al.  Epidemiology of hypersensitivity drug reactions , 2005 .

[44]  Yuan-Tsong Chen,et al.  HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Jian Fei Wang,et al.  CD8 T Cell-Mediated Killing of Cryptococcus neoformans Requires Granulysin and Is Dependent on CD4 T Cells and IL-151 , 2002, The Journal of Immunology.

[46]  W. Krzyzosiak,et al.  Sequence-non-specific effects of RNA interference triggers and microRNA regulators , 2009, Nucleic acids research.

[47]  E. Mariani,et al.  Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants , 2001, Clinical Diagnostic Laboratory Immunology.

[48]  M. Sarwal,et al.  Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation. , 2001, Human immunology.

[49]  R. Stoltenburg,et al.  SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. , 2007, Biomolecular engineering.

[50]  Wan Ariffin Bin Abdullah.  Singapore Med J , 1993 .

[51]  W. Pichler,et al.  Drug hypersensitivity reactions involving skin. , 2010, Handbook of experimental pharmacology.

[52]  F. Flowers,et al.  Practical guidelines for the management of toxic epidermal necrolysis and Stevens–Johnson syndrome , 2007, International journal of dermatology.

[53]  Konan Peck,et al.  Identification and characterization of oligonucleotides that inhibit Toll-like receptor 2-associated immune responses , 2022 .