Anirudh Jain
Background:
Rheumatoid arthritis (RA) is an inflammatory and debilitating disease of the joint affecting 1% of the population in the United States. Rheumatoid arthritis affects the lining of joints, causing a painful swelling that can eventually result in bone erosion and joint deformity. It is an autoimmune disease which occurs when the immune system mistakenly attacks the body's tissues. Interleukin-17 (IL-17) is a protein mediator called cytokine produced by immune cells. It has been shown that IL-17 is a key cytokine promoting arthritis while another cytokine, interferon-gamma (IFN-gamma) is limiting the disease. Current treatment with drugs that suppress immune system does not achieve sustained remission and not all patients respond.
There is clearly an unmet need for developing therapies that can induce sustained remission. The central hypothesis of this proposal is that RA pathogenesis is mediated by Th17 cells. Retinoic acid related orphan receptor C2 (RORC2) gene product. RORγt is a pivotal molecule that is the master transcription factor for differentiation of Th17 cells which produce several inflammatory cytokines, including IL-17, IL-17F, IL-22 and IL-21. Experiments designed in this proposal are focused on suppressing Th17 cells by targeting on RORγt.
Still need to add a paragraph of T-Cell differentiation and how Th17 cells and its cytokines are produced.
Abundant evidence indicates a critical role of CD4+ T cells in the pathogenesis of RA.
However, not all CD4+ T cell subsets but Th17 cells are pathogenic in RA. The protein CD7 is expressed on all CD4+ T cells including Th17 cells making it an ideal aptamer target for Th17 cells. Besides the signature cytokine, IL-17A (commonly referred to as IL-17), Th17 cells also produce IL-17F, IL-21 and IL-22 which are all implicated in chronic inflammation [1]. Th17 cells isolated from early RA patients potently stimulate RA synovial fibroblasts to produce IL-6 and IL-8 and matrix metalloproteinases. Interestingly, these Th17 cells produce more IL-17 upon interaction with RA synovial fibroblasts in an autocrine manner [2]. Furthermore, cytokine network in RA blood and joints are in favor of maintenance of Th17 cells [3,4]. These data affirm the pathogenic role of Th17 cells. Early phases of clinical trials with blocking IL-17 activity have yield promising therapeutic effect in RA. However, the rather moderate efficacy of anti-IL-17 antibodies suggests that other Th17 cytokines such as IL-17F, IL-21 and IL-22 are also important in the pathogenesis of RA. Based on these observations, we argue that targeting
Th17 cells rather than targeting IL-17 alone is a more effective approach to therapy of RA.
RORyt is a nuclear transcription factor. The intracellular location of RORyt makes it impossible for conventional therapeutic such as monoclonal antibodies or fusion proteins to access. There is no small molecule known to suppress RORyt. However, small interfering RNA
(siRNA) targeted approach offers a possibility to block RORyt expression. siRNA specific to a given mRNA is delivered intracellularly and selectively to degrade the complementary mRNA,

Anirudh Jain providing a precise blockade of a gene product. siRNA have some distinct advantages over conventional drug therapies, that is, siRNA targeting is highly sequence specific with a larger number of accessible targets and is rapid manufacturing. This technology has been evaluated in several preclinical to phase II clinical trials for diseases ranging from asthma, cancer to viral infections [5]. However, the major hurdle for widespread use of siRNA as a therapeutic agent is the inefficient intracellular siRNA delivery to target sites or cells in the body.
Aptamers were single-stranded nucleic acids that bind to molecular targets with high affinity and specificity due to their stable three dimensional shapes [6]. Aptamers are typically
20–100 nucleotides in length and can be selected from libraries (up to 10~15 unique sequences) to bind with high affinity to a wide array of protein families to modulate the protein function similar to antibodies [7]. Comparing to antibodies, aptamers possess some unique characteristics that derive from their nucleic acid composition. For instance, using chemical substitutions and other modifications, aptamers elicit minimal immunogenicity in vivo relative to antibodies. Their relatively small physical size shows improved transportation and better tissue penetration.
Straightforward chemical synthesis makes them amenable to backbone modification and rapid in vitro selection. Thereby aptamers can be reproducibly and economically synthesized in a large scale for clinical applications. A pegylated RNA aptamer against vascular endothelial growth factor (VEGF), pegaptanib has been approved by FDA to treat wet age-related macular degeneration [8] and many other aptamers are being explored for therapeutic purposes.
This proposal explores the in vivo therapeutic efficacy of siRNA targeting on RORyt in
CD7 Th17 to suppress proinflammatory cytokines. The success of these experiments will have great impact in the field of siRNA mediated therapy, in particular in CD4+ T cell mediated conditions. The data obtained from this project will provide valuable information for design of clinical studies with similar approach.
Proposal:
The aim of this project is to create an effective therapeutic treatment for Rheumatoid
Arthritis. This proposal aims to accomplish this by creating and measuring the efficacy of an aptamer-siRNA chimera on RORC2 gene suppression and in vitro application. Aptamers are single chain RNA or DNA nucleotides which exhibit high specificity and affinity in binding to a variety of protein ligands and are sometimes referred as “nucleotide antibodies”. In addition to being developed as therapeutic agents themselves, aptamers are excellent carriers for cell type and tissue specific drug delivery. A chimera of CD7 aptamer-RORγt siRNA is currently being constructed through the SELEX Process. The efficacy of the chimera will be tested for suppression of RORC2 in Th17 cells in vitro .
Methods:
Creation and Testing of the CD7 Aptamer:

Anirudh Jain
A CD7 aptamer-RORγt will be selected through the Systematic Evolution of Ligands by
Exponential Enrichment (SELEX) process. To briefly describe the SELEX process, first a random pool of single stranded RNA is exposed to the target molecule (CD7) then all the nonbinding RNA are removed and the remaining RNA are then reverse transcribed to single stranded DNA (ssDNA). The ssDNA is then amplified through Polymerase Chain Reaction
(PCR) and then the resulting ssDNA is transcribed back into RNA. This whole procedure constitutes one round of the SELEX method. To select for high affinity binding RNA, each round the ratio of the target molecule to the RNA is increased and the incubation time is decreased. Binding capacity and specificity of the CD7 aptamer will be assessed using flow cytometry and Karpas299 cells. Karpas299 cells express CD7 [24] that bound to CD7 aptamer with an identical staining pattern to anti-CD7 antibody. An Enzyme Linking Immuno Sorbent
Assay (ELISA) assay will also be used to measure dose response of the aptamer.
Creation and Testing of the CD7 aptamer-siRNA chimera:
A CD7 aptamer-RORC2 siRNA chimera will generated. Complementary double-stranded
DNA template including the sequences for CD7 aptamer and RORC2 siRNA sense strand will be generated by PCR. The resultant PCR product will be used as template for RNA transcription.
The transcribed RNA (containing aptamer and sense chain of siRNA) will be purified. Finally, the aptamer containing the sense strand of siRNA will be annealed with the antisense strand of siRNA to form the aptamer-siRNA chimera. The binding capacity of the aptamer-sRNA chimera will be confirmed by staining Karpas299 cells in flow cytometry. Efficiency of the chimera on suppressing RORC2 gene expression will be tested in Karpas299 cells and CD4
+
T cells.
Karpas299 cells expressed high levels of RORC2 gene upon stimulation with PMA and PHA and produced IL-17 accordingly which will hypothetically be suppressed by the CD7 aptamer-
RORC2 siRNA chimera.

Anirudh Jain
Literature Cited:
[1] P. Miossec, T. Korn, V.K. Kuchroo, Interleukin-17 and type 17 helper T cells, N Engl J Med
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[2] J.P. van Hamburg, P.S. Asmawidjaja, N. Davelaar, A.M. Mus, E.M. Colin, J.M. Hazes, R.J.
Dolhain, E. Lubberts, Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17A production,
Arthritis Rheum 63 (2010) 73-83.
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Rodrigues, J. Polido-Pereira, M.V. Queiroz, H.S. Rosario, M.M. Souto-Carneiro, L.
Graca, J.E. Fonseca, Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis, Arthritis Res Ther 12 (2011) R196.
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Laurindo, K.B. Elkon, Regulation of IL-17 in Rheumatoid Arthritis., Arthritis Rheum. 54
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[6] P.R. Bouchard, R.M. Hutabarat, K.M. Thompson, Discovery and development of therapeutic aptamers, Annu Rev Pharmacol Toxicol 50 (2010) 237-257.
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[8] C. Campa, S.P. Harding, Anti-VEGF compounds in the treatment of neovascular age related macular degeneration, Curr Drug Targets 12 (2011) 173-181.