Silencing RORγt in Human CD4+ T cells with CD30 aptamer-RORγt shRNA Chimera

Abstract

Targeting specific T cell subtypes and intervening in their function are emerging a critical strategy for treatment of autoimmune diseases. Here we report that an RNA CD30 aptamer was utilized to deliver short hairpin RNA (shRNA) to CD30 + T cells to target retinoic acid receptor-related orphan receptor gamma t (RORγt), leading to impaired expression of RORγt and suppression of IL-17A and IL-17F. A DNA template consisting of CD30 aptamer and RORγt shRNA sequences was synthesized and was transcribed CD30 aptamer-RORγt shRNA chimera (CD30-AshR-RORγt). Insertion of 2′-F-dCTP and 2′-FdUTP was incorporated during CD30-AshR-RORγt transcription to increase its resistance to RNase. CD30-AshR-RORγt was specifically up-taken by CD30 + Karpas 299 cells, but not by Jurkat cells which lack CD30. It was also up-taken by activated, CD30 expressing human CD4 + T cells, but not by resting CD4 + T cells. The RORγt shRNA moiety of CD30-AshR-RORγt chimera was cleaved and released by Dicers. Then, CD30-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and activated human CD4 + T cells. Consistently, silence of Th17 cell differentiation and IL-17A and IL-17F synthesis with CD30-AshR-RORγt was demonstrated in activated human CD4 + T cells from healthy donors and RA patients. CD30-AshR-negative control chimera and prostate specific membrane antigen (PSMA)-AshR-RORγt had no significant impact on the expression of RORγt or IL-17A and IL-17F. These data present a novel strategy for shRNA delivery using CD30 RNA aptamers to down-regulate CD30 + Th17 cells and can be developed as a targeted therapy for treating Th17 cell mediated conditions.

Aptamer-Based Targeted Delivery of Functional Nucleic Acids

“…In 2010, Kim et al reported an anti-PSMA aptamer-functionalized polyplex for the targeted co-delivery of Bcl-xL shRNA and doxorubicin to LNCaP cells, demonstrating the selective inhibition of PSMA-expressing prostate cancer cells . In addition to cancer cell targeting, Shi et al reported a CD30 aptamer–RORγt (related orphan receptor gamma t) shRNA chimera to mediate the expression of RORγt, mainly expressed in immune cells and known to regulate circadian rhythm, IL-17A, and IL-17F in CD30 + T cells, providing a potential approach for the treatment of autoimmune inflammatory diseases . Similar to the design shown in Figure d, Sanati et al developed AS1411 aptamer-decorated micelles loaded with survivin shRNA for the specific inhibition of tumor growth in the C26 tumor-bearing mouse model …”

“…81 In addition to cancer cell targeting, Shi et al reported a CD30 aptamer−RORγt (related orphan receptor gamma t) shRNA chimera to mediate the expression of RORγt, mainly expressed in immune cells and known to regulate circadian rhythm, IL-17A, and IL-17F in CD30 + T cells, providing a potential approach for the treatment of autoimmune inflammatory diseases. 82 Similar to the design shown in Figure 4d targeting various human cancers. 84,85 The C/EBPα (CCAAT/ enhancer-binding-protein-alpha), 86,87 DPYSL3 (dihydropyrimidinase-related protein 3), 88 and XIAP (X-linked inhibitor of apoptosis protein) 89 genes have been investigated as targets by aptamer−saRNA chimeras for the targeted treatment of pancreatic cancer.…”

The Interleukine-17 Cytokine Family: Role in Development and Progression of Spondyloarthritis, Current and Potential Therapeutic Inhibitors

“…Among this diversity, there are aptamers specific to different cytokines [215] and their receptors, including IL-17A and IL-17RA (see Table 4). 2'-F-RNA aptamer AptAF42dope1 primary human foreskin fibroblast BJ cells [217] DNA aptamers M2 and M7 imiquimod induced psoriasis mouse model [218] IL-17RA DNA aptamer RA10-6 mouse model of osteoarthritis [219] Liposomes + antisense oligonucleotide imiquimod induced psoriasis mouse model human cytokine-induced psoriasis skin model [220] Th17 cells CD4 aptamer + RORγt shRNA CD4+ cells [221] CD30 aptamer + RORγt shRNA CD30+ and CD4+ cells [222] An IL-17A-specific 2'-fluoro-modified RNA aptamer Apt21-2 was successfully used for blocking IL-17A interaction with IL-17RA in vitro [216]. The aptamer was also tested in model animals and provided an improvement in symptoms in rheumatoid arthritis and experimental autoimmune encephalomyelitis mouse models.…”

“…For instance, a CD4-specific RNA aptamer provided targeted delivery of RORγt shRNA into CD4+ cells [221]. Simultaneously, a CD30-binding aptamer was conjugated to RORγt shRNA for IL-17 downregulation [222]. RORγt (retinoic acid-related orphan receptor γt) is the master transcription factor that controls differentiation of Th17 cells and synthesis of Th17 cytokines.…”

Leveraging Exosomes as the Next-Generation Bio-Shuttles: The Next Biggest Approach against Th17 Cell Catastrophe

“…Apart from the covalent binding method, physical methods can opt as one of the options for allowing the binding of ligand moieties to exosome membranes. Attachment of CD30 aptamer to exosomes by hydrophobic interactions can be employed for Th17 pathological condition as CD30 is actively expressed by activated T cells [187] (Figure 3). on their surface using different sorting modules that result in a sustained response against Th17 cells.…”