Biology subjects

Gross, K. Y.

Publications and source records attributed to Gross, K. Y..

3 recordsLinked to original sources

bioRxiv2026

Mismatch repair dissection by in vivo RNAi reveals dose-dependent modulators of somatic instability and proteome remodeling in Huntingtons disease

Human and mouse genetics have established mismatch repair (MMR) as a central mediator of somatic repeat expansion, a key pathogenic process in Huntingtons disease (HD) and related disorders. How individual MMR components function within the intact mammalian brain and interact with broader cellular networks remains poorly understood. We screened more than 500 chemically stabilized siRNAs targeting 10 MMR genes and used interventional RNAi in the Q111 HD mouse model to systematically dissect MMR function in vivo. MSH3 and PMS1 emerged as the most dose-sensitive regulators of somatic expansion but displayed markedly different effects on proteome stability. Quantitative proteomics generated an in vivo atlas of MMR component abundance and cross-regulation in the mammalian CNS, uncovering extensive connectivity between DNA repair, transcriptional regulation, chromatin remodeling, and mitochondrial biology. Together, these findings establish a systems-level framework linking MMR biology to neuronal function and offer mechanistic insight into selective neuronal vulnerability in HD.

neuroscience↗

bioRxiv2024

Divalent siRNA for prion disease

Prion protein (PrP) lowering is effective in animal models of prion disease and is being tested clinically in prion disease patients, but there remains a need for more potent PrP-lowering drug candidates. Inspired by the reported potency and duration of action of divalent short interfering RNA (siRNA), a new oligonucleotide drug modality for the central nervous system, we sought to discover and develop a new PrP-lowering drug candidate. Herein we identify a mouse Prnp-targeting divalent siRNA molecule, 1682-s4, that lowers PrP to 49% residual brain expression in wild-type mice, and, in the context of intracerebral infection with Rocky Mountain Laboratory (RML) prions, achieves a 2.7-fold increase in survival time with pre-symptomatic chronic treatment and 64% increase in survival time with a single dose after symptom onset. We describe the generation of two transgenic mouse lines, Tg25109 and Tg26372, expressing the full human PRNP gene and its non-coding sequence, and demonstrate their utility for in vivo discovery of potent human PRNP-targeting oligonucleotides. We discover siRNA sequence 2439 against human PRNP and compare its potency in different divalent siRNA chemical scaffolds. We determine that both the fixed UU tail and extended nucleic acid linkages of scaffold s4 contribute to superior potency compared to other scaffolds tested, offering 9.4 and 15.9 percentage points respectively of additional PrP knockdown. A single dose of 348 {micro}g of 2439-s4 lowered whole brain hemisphere human PrP in transgenic mice to 17% residual after 30 days, while 52 {micro}g lowered PrP to 49% residual. 1-2% of the dose of 2439-s4 delivered into cerebrospinal fluid is retained in the brain, and the median effective tissue concentration is estimated at 1.2 micrograms per gram of tissue. Good Laboratory Practices toxicology studies identified no significant liabilities, and the U.S. FDA has cleared an Investigational New Drug application to bring 2439-s4 into clinical trials.

neuroscience↗

bioRxiv2024

Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

Small interfering RNA (siRNAs) hold immense promise for treating cardiac and muscular diseases, but robust and scalable delivery to these tissues remains a challenge. Recent advances in delivery strategies to muscle include conjugation of biologics (antibody/antibody fragments, peptides), which are currently in clinical development. However, the manufacturing of biologic-siRNA conjugates is a challenging and complex process. By contrast, lipophilic siRNAs are readily chemically synthesized at scale and support sufficient cardiac and skeletal muscle delivery. In this work, we refine siRNA design elements to enhance potency and durability and support clinically relevant silencing in muscle. Applying this strategy for siRNAs targeting myostatin (MSTN), a key target in muscle-wasting conditions, we show that a single subcutaneous dose in mice achieved robust and durable silencing ([~]80% inhibition up to 6 weeks, [~]30% at 14 weeks). Biweekly dosing resulted in >95% reduction of circulating MSTN for half a year, with no observed systemic or target-related toxicity. MSTN inhibition resulted in muscle growth and increased lean muscle mass, correlating with improved grip strength. Interestingly, the functional impact on muscle growth and strength significantly outlasts the target silencing, suggesting extended pharmacological effects. Systemic administration was equally efficacious in all muscle groups tested, including skeletal muscle, heart, tongue and diaphragm. The informational nature of the muscle-active chemically defined siRNA scaffold was confirmed by demonstrating muscle and heart efficacy with three additional targets. Our findings pave the way for potent and long-lasting gene modulation in muscle using chemically defined, lipophilic siRNAs, offering a new avenue for treating muscular diseases.

pharmacology and toxicology↗