Antibody-Oligonucleotide Conjugates Reduce Trim63 Expression in Skeletal Muscle and Prevent Muscle Atrophy

Abstract

Background

Muscle atrophy is associated with many disease states and can become a debilitating and life-threatening condition with few or no therapeutic options. The muscle-specific E3 ligase muscle RING-finger protein-1 (TRIM63/MuRF1) has been identified as a promising target to treat muscle atrophy. Therapeutic evaluation of TRIM63 is hampered by challenges to target TRIM63 specifically while avoiding the closely related E3 ligases TRIM55/MuRF2 and TRIM54/MuRF3. Small interfering RNA (siRNA) technology can be used to generate specific reduction of messenger RNA (mRNA) transcripts, but delivery of sufficient concentrations of oligonucleotide-based drugs to skeletal muscle has been challenging. We investigated an antibody-oligonucleotide conjugate (AOC) combining the precision of siRNAs and the efficiency of receptor-mediated delivery of oligonucleotides into skeletal muscle (specifically using a transferrin receptor 1 [TfR1] antibody [αTfR1]) to overcome these challenges and efficiently target TRIM63 in muscle.

Methods

Active Trim63 siRNAs were identified by screening in murine C2C12 myotubes and in vivo in wild-type mice. A selected siRNA was then tested in two mouse models of muscle atrophy—hindlimb immobilization and denervation—to evaluate whether siTrim63 AOC can ameliorate muscle atrophy. Muscle weight was assessed at multiple time points to determine the impact of αTfR1 AOC administration. A primate-cross-reactive αTfR1 AOC was administered to cynomolgus monkeys to assess TRIM63 expression across species.

Results

We identified siTrim63 AOCs that reduced Trim63 expression in murine skeletal muscle by > 75% for 16 weeks, and > 50% for 6 months. In the hindlimb immobilization mouse model, siTrim63 AOC resulted in significantly reduced muscle weight loss of 10.1% (**p < 0.01) at 14 days post-immobilization compared to 19.3% (****p < 0.0001) in the control-treated leg. In the denervation model, siTrim63 AOC significantly reduced muscle weight loss to 38.9% (**p < 0.01) at 21 days post-denervation compared to 55.0% (****p < 0.0001) in the control-treated leg. We observed that siTrim63.22 AOCs led to 54% (****p < 0.0001) muscle sparing in denervated hindlimbs measured by leg cross-section size. Treatment of non-human primates (NHPs) with a siTRIM63 AOC reduced TRIM63 expression by 68% (average in two skeletal muscles).

Conclusions

Trim63/TRIM63 expression can be specifically reduced in skeletal muscle by siRNAs targeting Trim63/TRIM63 mRNA when administered systemically as an αTfR1 AOC in mice and NHPs. Our data support the therapeutic hypothesis that TRIM63 inhibition reduces muscle atrophy when administered at or near the time of insult. We provide evidence for the translatability of this strategy to primates.