Antisense Targeting of cFLIP Sensitizes Activated T Cells to Undergo Apoptosis and Desensitizes Responses to Contact Dermatitis

Abstract

Contact dermatitis is the result of inflammatory responses mediated by hapten-specific activated CD8+ and CD4+ T cells. Activation-induced cell death (AICD) is a naturally occurring process regulating the resolution of T-cell responses through decreased expression of the antiapoptotic molecule cellular FLICE inhibitory protein (cFLIP). We show that targeting cFLIP expression in vitro and in vivo, with morpholino antisense applied systemically or topically in conjunction with antigen, sensitizes T cells to undergo "early" AICD resulting in tolerance. Analysis of antisense-treated CD8+ OT-1 splenocytes after co-culture with SIINFEKL-pulsed DCs showed apoptosis occurring in a dose-dependent manner with respect to cFLIP peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) concentration. A transplant acceptance model using male DO.11 donor cells and female BALB/c recipient mice showed that cFLIP antisense treatment could promote antigen tolerance. Hypersensitivity responses induced in mice by the epicutaneous application of the haptens FITC and oxazolone confirmed that topically applied cFLIP antisense could reduce inflammation. Treatment of the skin produced significant reduction in dermatitis and localized infiltration of lymphocytes. Moreover, the treatment was target- and antigen-specific, dose-dependent, and capable of inducing long-lived tolerance. These data suggest that the targeted expression of immune-regulating molecules is possible through the application of antisense to the skin.

Induced IL-10 Splice Altering Approach to Antiviral Drug Discovery

“…Among these is the antisense targeting of EBOV VP35 protein expression, which provided significant protection against lethal challenge. Important to the studies shown here is the capability of a PPMO to deliver antisense into murine leukocytes, both in vitro and in vivo (Marshall et al, 2007;Mourich et al, 2009). The ability to target protein expression in vivo with PPMOs make these particularly useful agents for determining the role of different host immune factors in the pathogenesis of EBOV.…”

Simultaneous Targeting of Two Master Regulators of Apoptosis with Dual-Action PNA– and DNA–Peptide Conjugates

“… 36 , 37 For anti c-FLIP morpholinos, positions in the 5′-UTR and near the AUG start codon have been described as efficient target sites. 38 PNA1–4 conjugates are complementary to different targets around the start codon (−30 to 15, −13 to 3, −5 to 11, and −1 to 15, relative to the start codon). For comparison, we included PNA5 , which is designed to bind within the ISIS 23296 target segment in the c-FLIP coding region.…”

“…ISIS 23296 is an optimized oligophosphorothioate gapmer sequence, which induces RNAs H. Antisense PNAs hinder mRNA translation by steric blockage . Literature evidence suggests that inhibition of protein synthesis by steric block antisense compounds such as PNA, morpholino nucleic acids, and 2′-OMe-RNA occurs more effectively near the site of translation initiation rather than in the coding region targeted by ISIS 23296, indicating that interfering with ribosome assembly is easier than perturbing elongation. , For anti c-FLIP morpholinos, positions in the 5′-UTR and near the AUG start codon have been described as efficient target sites …”

Harnessing programmed cell death as a therapeutic strategy in rheumatic diseases

“…Overexpression of the FADD adapter protein in cultured RA synoviocytes induced apoptosis in these cells, and in a xenograft mouse model of proliferating rheumatoid synovium, ectopic expression of FADD through retroviral transduction lead to a significant reduction of synoviocytes and mononuclear cells 75 . Reducing levels of c-FLIP, another molecule that regulates Fas signaling has been shown to sensitize fibroblast-like synoviocytes from RA patients to Fas-induced apoptosis and sensitized T cells to TCR-induced apoptosis 76,77 . Indirect targeting of Fas through its ligand is another plausible therapeutic mechanism.…”