Development of combination therapy using a mCRP aptamer and Leflunomide for precision medicine-based rheumatoid arthritis treatment

Rheumatoid arthritis (RA) is characterized by inflammation and bone destruction. Disease-modifying antirheumatic drugs (DMARDs), are used to alleviate bone erosion and prevent joint damage by inhibiting the function of inflammatory factors or immune-related cells. Leflunomide, as one of the DMARDs, is widely used in RA treatment in China. However, clinical data shows that only 40-50% of RA patients have good response to it.

Recently, we found that RA patients responded to Leflunomide in attenuating bone erosion dependent on the basal serum CRP levels before treatment. CRP is synthesized and secreted by the liver, which is commonly considered as an inflammation biomarker. However, it has been considered to be involved in mediating the occurrence and development of RA. Further, it has been reported that CRP could directly regulate osteoclastic activity. We found that RA patients with good response to Leflunomide showed relatively low basal serum CRP (CRP low), whereas the others with limited response demonstrated high basal CRP (CRP high).

Using rat collagen-induced arthritis (CIA) model, we observed the similar relationship between the high basal CRP and the limited response to Leflunomide. The CRP high CIA rats were treated with liver-targeted CRP siRNA in combination with Leflunomide or not. Our results showed that CRP siRNA monotherapy partially attenuated bone erosion when compared to a non-sense siRNA. When combined with Leflunomide, CRP silencing compensated the limited efficacy of Leflunomide, leading to adequate attenuation of bone erosion in the CRP high CIA rats. Based on these findings, we concluded that high CRP could directly induce bone erosion in CRP high RA independent of inflammation induced osteoclast activation pathways (Liang, Li et al. 2019).

CRP has two isoforms: native CRP (nCRP) and monomeric CRP (mCRP). nCRP is inefficient for osteoclastic activity. However, nCRP dissociates into mCRP upon entering inflammatory sites and mCRP regulates osteoclastic activity in a RANKL-dependent manner. mCRP inhibits RANKL-induced osteoclastic differentiation by neutralizing RANKL but directly induces osteoclastic differentiation via FcγRs signaling in the absence of RANKL in vitro. Therefore, the overall effects of mCRP on osteoclastic activity in vivo might be dependent on the ratio of mCRP to RANKL. We found that there was excessive molar concentration of mCRP (over 10,000-fold) than RANKL in synovial fluid of both CRP high RA patients and CIA rats (Liang, Li et al. 2019), indicating that mCRP could be a specific therapeutic target.

Aptamers are RNA or ssDNA, which specifically bind to targets. The advantages of aptamers include easy chemical synthesis, simple modification, low cost, low toxicity and convenient storage when compared to antibodies. We screened an mCRP-specific aptamer AptmCRP3, which inhibited mCRP-induced osteoclast differentiation in vitro by blocking the interaction between mCRP and FcγRs. In CIA rats with CRPhigh , immunofluorescence results showed that AptmCRP3 and mCRP are well co-localized in the joints, indicating that AptmCRP3 can recognize mCRP in vivo.

Thus, we hypothesize that AptmCRP3 monotherapy could partially attenuate bone erosion and combination of AptmCRP3 with Leflunomide could compensate the limited efficacy of Leflunomide, leading to adequate attenuation of bone erosion in CRP high RA. To test this hypothesis, CRP high rats with established CIA will be employed to determine (1) dose-response pattern and dose interval of AptmCRP3; (2) effects of AptmCRP3 monotherapy and combination of AptmCRP3 with Leflunomide on bone erosion; (3) toxicity of monotherapy and combination of AptmCRP3 with Leflunomide. This proposal would present a precision medicine-based therapeutic strategy for RA. Combination of Leflunomide with AptmCRP3 will be a promising approach for adequately attenuating bone erosion in CRP high RA.