DNA-Inspired Janus Base Nanopieces Deliver mRNA Therapeutics Against the Progression of Cartilage Degradation in Osteoarthritis

Introduction: Osteoarthritis affects more than 32.5 million adults in the U.S., causing pain and discomfort due to deteriorating articular cartilage. Drug-based therapies like IL-1Ra peptide Anakinra have not proven effective in clinical trials, but mRNA therapeutic analogs of these drugs have the potential to create significantly better treatments. However, mRNAs must be able to penetrate thick ECM and overcome endosomal entrapment to deliver to chondrocytes. Current solutions like lipid nanoparticles (LNPs) present issues related to toxicity, immunogenicity, and reduced retention time in cartilage. Spherical LNPs, which have a diameter >50nm, are physically unable to deliver mRNA to cartilage, where the matrix spacing is ~20nm. In this study, we have therefore engineered a new generation of delivery vehicles, called Janus Base Nanopieces (JBNps) to overcome these barriers. JBNps are bundles of self-assembled nanotubes inspired by DNA base pairs which combine with mRNA to form rod-shaped delivery vehicles. Rod-shaped JBNps have a slim profile capable of penetrating cartilage for mRNA delivery, marking a breakthrough over spherical delivery vehicles and indicating their unique suitability for treatment of cartilage diseases. Designed for pore formation and early endosomal escape, JBNps also maximize the bioactivity of degradation-prone mRNA. JBNps additionally have low toxicity and immunogenicity due to their non-covalent structures and DNA-mimicking chemistry. By engineering the sidechain of JBNp, we can also observe an increased retention time of the delivered mRNA in cartilage. With these promising features, we used JBNp to deliver Anakinra encoding mRNA to intra-articular cartilage for treatment in a mouse model of osteoarthritis.

Materials and

Methods: The delivery of JBNp-mRNA was first demonstrated by transfecting human chondrocytes (C28/I2) with eGFP-mRNA, and then by investigating the delivery of JBNp-mRNA-cy5 to the mouse knee joint. Retention of JBNp-mRNA in the ECM of articular cartilage was demonstrated by transfecting bovine cartilage explants with Cy5 mRNA using JBNp, followed by IVIS imaging the Cy5 fluorescence over 24 days. To treat OA, the knee joints of mice with OA as generated via the Destabilization of the Medial Meniscus (DMM) surgery were injected weekly with JBNp-Anakinra mRNAs. The extracted knee joints following this treatment regimen were imaged via Micro-CT, and sections were stained for Safranin O/Fast Green. Behavioral studies were also conducted, including thermal sensitivity (hot plate), mechanical sensitivity (Von Frey) analysis, and gait analysis.

Results, Conclusions, and Discussions: In vitro transfection of eGPF-mRNA by JBNp was confirmed to be achievable via pore formation and early endosomal escape (Fig1B). In vivo delivery of cy5 mRNA loaded JBNp to cartilage was confirmed via IVIS. Ex vivo and in vivo retention studies revealed an extended retention time for JBNps delivered to the intra-articular space (Fig1C-D). Tissue sections stained for Safranin-O/Fast Green and Aggrecan showed an increased presence of healthy cartilage and decrease in cartilage degradation markers in the treatment group (Fig1E). Micro-CT imaging revealed reduced osteophyte formation (Fig1F), while thermal and mechanical sensitivity tests demonstrated lowered withdrawal latency and 50% withdrawal threshold (Fig1G). Gait analysis further showed increased paw area and stance length in the treatment group (Fig1H-I).

We have herein presented a new class of delivery vehicles that can deliver an mRNA therapeutic with better efficacy than other drug-based therapeutics and LNPs. Our rod-shaped JBNps are capable of escaping the early endosome via pore formation and delivering mRNA both in vitro and in vivo, with an enhanced cartilage-retention time. In an OA mouse model, JBNps were shown to penetrate the ECM and cartilage of the knee joint, and therapeutically deliver IL-1Ra encoding mRNA against the progression of OA, showing a decreased rate of cartilage degradation and improvement in thermal and mechanical sensitivity. Our JBNp platform has thusly proven its candidacy as a promising solution with a demonstrated ability to deliver therapeutics against novel biological targets for OA.

Acknowledgements (Optional):