(F-225) CDK inhibition enhances intramuscular expression of self-amplifying RNA encoding monoclonal antibody

Introduction:: Nucleic acid encoding neutralizing antibodies can be an effective and rapid countermeasure against disease outbreaks. Although promising, our report demonstrating the potential of self-amplifying replicon RNA (repRNA) encoding the monoclonal antibody (mAb) ZIKV-117 to prevent Zika virus (ZIKV) infection in mice, highlighted several barriers to achieving therapeutic levels by intramuscular (IM) injection. Specifically, we found that repRNA-driven antiviral responses significantly limit transgene expression. Thus, we hypothesized that co-delivery of innate immune response inhibitors with repRNA could enhance transgene expression in transfected cells.


Materials and Methods:: We screened a library of 1876 small molecule kinase inhibitors in A549-Dual® cells pre-treated with exogenous interferon (IFN) to identify hits that rescue and enhance repRNA translation of the reporter gene nanoluciferase (nLuc) under selective pressure from type-I IFN. Candidate compounds were encapsulated in our proprietary cationic oil-in-water emulsion formulation, LION^TM, and co-delivered with varying doses of repRNA expressing the reporter protein SEAP or the ZIKV neutralizing human mAb ZIKV-117¹ by IM route in BALB/c mice. Serum samples collected at various time points were assayed for protein (SEAP or ZIKV-117) levels and IFN-⍺2.


Results, Conclusions, and Discussions:: A total of 99 compounds (out of 1,876) enhanced nLuc expression by at least 100-fold, out of which 10 compounds, all CDK inhibitors, were advanced for in vivo evaluation based on potency ranking. In vivo evaluation of the 10 candidate compounds co-delivered with LION/repRNA-SEAP identified Dinaciclib, a potent inhibitor of CDK1/2/5/9, as the lead compound for further evaluation. In BALB/c mice (6-8 weeks old females, n=3/group), IM administration of 10 µg repRNA-SEAP formulated with LION-encapsulating Dinaciclib (Dinaciclib-LION) resulted in 2-3-fold greater serum SEAP levels compared to mice treated with repRNA-SEAP/LION, without Dinaciclib. In subsequent studies, BALB/c mice (6-8-weeks old females, n=5/group) were given, IM, 25 µg repRNA-ZIKV-117 formulated either with LION only, Dinaciclib-LION for co-delivery, or LION with Dinaciclib administered separately by intravenous (IV) route (LION + Dinaciclib). Both Dinaciclib-LION and LION + Dinaciclib treatments resulted in a statistically significant ( >3-fold) increase in total serum ZIKV-117 concentration (AUC) compared to the LION, without Dinaciclib treatment (Figure 1B). Notably, serum ZIKV-117 concentration peaked on day-3 post-treatment with Dinaciclib-LION, compared to day-6 with control LION or LION + Dinaciclib treatment (Figure 1A). Interestingly, serum IFN-⍺2 levels were significantly reduced in mice that received Dinaciclib via IV route, indicating systemic suppression of innate immune response. Co-delivery of Dinaciclib-LION with repRNA restricted Dinaciclib activity locally in the muscle, without causing systemic innate immune suppression, as seen by the unaltered serum IFN-⍺2 levels. Using a high-throughput library screening approach, the present study identifies a previously unknown effect of CDK inhibition on repRNA transgene expression. Encapsulation of the CDK inhibitor Dinaciclib in our cationic nanocarrier LION for co-delivery with repRNA-ZIKV-117 produced a dual effect. Firstly, it reduced the time to reach peak antibody expression in serum from day-8 to day-3, indicating a rapid onset of therapeutic response, and secondly, it increased the overall magnitude of expression by 3-fold. Previous studies have shown that Dinaciclib can inhibit the translation of type-I IFN genes², which could potentially explain the enhanced repRNA-encoded protein expression observed in this study.The findings provide valuable insights into the potential use of Dinaciclib as an enhancer of repRNA-based therapeutics.


Acknowledgements (Optional): :

References (Optional): : 1. Sapparapu, G. et al. Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice. Nature 540, 443–447 (2016)

2. Cingöz, O. & Goff, S. P. Cyclin-dependent kinase activity is required for type I interferon production. Proc National Acad Sci 115, 201720431 (2018)