Albumin Hitchhiking siRNA to Silence MMP13 in a Guinea Pig Model of Post-traumatic Osteoarthritis

Introduction:: Osteoarthritis (OA) is a highly-prevalent, degenerative joint disease associated with aging or injury. OA secondary to injury is known as post-traumatic OA (PTOA). Current treatments focus on pain management, with a distinct lack of disease modifying osteoarthritis drugs (DMOAD). Rather, gold standard corticosteroids temporarily alleviate pain but may actually worsen cartilage thinning and disease progression due to chondrotoxicity.

Matrix metalloproteinase 13 (MMP13) is a potential DMOAD target because it is one of the primary proteases responsible for breakdown of the cartilage extracellular matrix (ECM) during OA pathogenesis. We have shown that local (intra-articular) delivery of nanoparticles for silencing of MMP13 in a mouse model of post-traumatic osteoarthritis (PTOA) helps to protect articular cartilage. ¹ Because ECM degradation byproducts have inflammatory signaling properties that enhance and propagate OA pathogenesis, silencing MMP13 in mice also helps to suppress overall joint inflammation and have holistic joint benefits that includes reduced osteophyte formation, synovial hyperplasia, and subchondral bone changes.

Here, we intravenously apply a carrier-free siRNA conjugate structure that we recently optimized for albumin hitchhiking in vivo.² This structure provides extended circulation time and preferential biodistribution to sites of injury and inflammation. We tested this conjugate technology for silencing of MMP13 in a Dunkin-Hartley Guinea Pig (GP) ACT transection (ACLT) model of PTOA. GPs were chosen as a more translationally-relevant species.³ Results showed that the conjugate effectively distributed to and potently silenced MMP13 in the PTOA joint, with ongoing tests currently looking at long-term efficacy.


Materials and Methods:: We synthesized our siMMP13 conjugate in house with and without the albumin binding end modification (EG18-L)2. Both the parent and conjugate siRNA integrated alternating 2’OMe and 2’F modifications along with phosphorothioate linkages on both ends of both strans to protect against endonucleases and exonucleases, respectively.

GPs underwent an ACL transection surgery and received treatments (10 mg/kg siRNA intravenously) on day 7. A Cy5 labeled nontargeted conjugate was administered one day before sacrifice to show biodistribution via ex vivo IVIS imaging. Knockdown was assessed using qRT-PCR.

Ongoing long-term (2 month) studies include functional readouts to assess pain, limb load bearing, and histological outcomes in the ACLT joint.


Results, Conclusions, and Discussions:: We successfully screened siRNA sequences against GP MMP13 using primary guinea pig knee joint chondrocytes isolated in-house, which yielded a potent, species-specific sequence. We also confirmed that the (EG18)L2 structures are predominantly bound to albumin in human synovial fluid samples, validating the albumin affinity of the structure (nM level K_Ds). In our in vivo ACLT GP study, we detected potent MMP13 mRNA and protein upregulation in the surgical ACLT knee compared to the contralateral control knee. Additionally, the introduction of Cy5-siRNA vs Cy5-siRNA< (EG18L)2 (1 mg/kg) demonstrated that the albumin piggybacking structure, showed higher biodistribution in the PTOA joint and an overall different biodistribution pattern (significantly less kidney filtration) relative to the parent siRNA structure. Cryohistological analyses showed that the albumin piggybacking structure provided significant delivery to both the synovial tissues and the articular cartilage of the ACLT knee. Finally, both qRT-PCR and immunohistochemistry showed potent silencing of MMP13 in the ACLT joint by the albumin hitchhiking siRNAs relative to the ACLT joints of untreated animals. Current progress and data sets are outlined in the two Figures attached.

We currently have an ongoing, longer-term efficacy study that will be included in the conference presetnation. Based on prior results in mice and our GP studies completed to this point, we anticipate that this treatment will reduce joint sensitivity (algometer) and improve limb weight bearing (Bioseb dynamic weight bearing system). We also anticipate that this treatment will lower serum levels of CTX-II (biomarker for collagen degradation) and reduce synovial fluid levels of MP13, CXCL12, C2C (neoepitope created by MMP cleavage of CII113), TNFα, IL-6, and IL-1b. Finally, we will also complete histological scoring of OA progression based on blinded histopathologist analysis of the medial tibial plateau, the primary site of cartilage loss in this model.

Our collective data suggest that MMP13 is a highly promising DMOAD target and that our albumin hitchhiking siRNAs provide an effective technology for potent target gene silencing in a clinically relevant model of PTOA.


Acknowledgements (Optional): :

References (Optional): : 1. Bedingfield, S. K. et al. Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage. Nat. Biomed. Eng. 5, 1069–1083 (2021).

2. Hoogenboezem, E. N. et al. Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy. 2023.02.14.528574 Preprint at https://doi.org/10.1101/2023.02.14.528574 (2023).

3. McAlindon, T. E. et al. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthritis Cartilage 22, 363–388 (2014).