Pre-Clinical Optimization of Follistatin Plasmid Gene Therapy

for Canine Aging and Degenerative Disease

Delivery Optimization and Biomarker Validation for a Delayed-Start Clinical Trial

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Tristan Roberts, Principal Investigator

Dog Years — San Francisco, CA

February 2026

Requested Funding: $10,000 USD (RSC)

Duration: 6 months

Abstract

Follistatin gene therapy represents a promising multi-mechanism approach to canine aging and degenerative disease, targeting both muscle wasting (via myostatin inhibition) and chronic inflammation (via activin neutralization). Building on a 43-participant human Phase 1 trial that demonstrated safety, improved body composition, and a 7.1-year reduction in epigenetic age, we are preparing a 30–50 dog clinical trial with a delayed-start control design. However, two critical pre-clinical gaps must be addressed before the trial can proceed effectively: optimization of the plasmid–polyethylenimine (PEI) delivery system for reliable intramuscular administration in canines, and validation of a biomarker panel capable of measuring both disease-modifying and aging-related therapeutic responses.

This proposal requests $10,000 to conduct a focused 6-month pre-clinical study with two specific aims: (1) systematically optimize plasmid–PEI complexation parameters—including nitrogen-to-phosphate ratios, preparation protocols, and particle characterization—to ensure consistent and effective gene delivery; and (2) validate a multi-domain biomarker panel spanning inflammatory markers, metabolic parameters, and epigenetic aging clocks for use in the clinical trial. These pre-clinical data will directly inform dosing protocols, endpoint selection, and statistical powering for the full trial, while generating proof-of-concept results suitable for publication and follow-on funding.

Background and Significance

The Aging–Degeneration Nexus in Canines

Aging is the primary risk factor for degenerative disease in companion dogs, just as in humans. Osteoarthritis (OA) affects over 80% of geriatric dogs, driving chronic pain, muscle atrophy, and progressive loss of mobility. Current treatments—NSAIDs, anti-NGF antibodies, joint supplements—manage symptoms without modifying the underlying disease trajectory. Only one disease-modifying drug (Adequan) is approved for canine OA, and its mechanism remains poorly understood. There is a critical unmet need for therapies that address the shared biological drivers of aging and degeneration: chronic inflammation, muscle loss, and metabolic dysfunction.

Follistatin: A Multi-Mechanism Intervention

Follistatin (FST) is an endogenous glycoprotein that binds and neutralizes TGF-β superfamily members, particularly myostatin and activin. This dual inhibition produces complementary therapeutic effects: myostatin blockade promotes muscle hypertrophy and maintenance, while activin neutralization reduces inflammatory signaling in joints and systemic tissues. Published preclinical data demonstrate that follistatin gene therapy reduces synovitis, inhibits cartilage degradation in arthritis models, and mitigates metabolic inflammation in obesity-associated osteoarthritis. Follistatin also promotes brown adipocyte differentiation and thermogenesis, with broader metabolic benefits relevant to age-related metabolic decline.

Human Clinical Foundation

A Phase 1 clinical trial (NCT06411366) conducted by Minicircle Inc. enrolled 43 adults (ages 23–88) and delivered follistatin via minicircle plasmid with PEI-mediated intramuscular injection. Key findings included no serious adverse events, an average gain of 1.96 lbs lean mass with 0.87% body fat reduction, a 7.1-year reduction in extrinsic epigenetic age (as measured by DNA methylation clocks), and anecdotal reports of arthritis symptom improvement in participants with pre-existing joint disease. These results establish both the safety foundation and the biological plausibility for translating this approach to canine degenerative disease and aging.

Canine Preliminary Evidence

A single compassionate-use case in an elderly large-breed dog (Skye) with severe arthritis produced a dramatic and sustained reduction in pain behaviors lasting over six months following follistatin plasmid administration. While anecdotal, this case—combined with published mouse model data showing follistatin gene therapy reduces inflammation and improves joint function in post-traumatic OA—provides strong rationale for a systematic canine study.

Rationale for Pre-Clinical Optimization

Our planned clinical trial (30–50 dogs, delayed-start control design, three-tiered dose escalation) requires two foundational elements that cannot be adequately addressed within the trial itself:

Reliable delivery: Plasmid–PEI complexation is sensitive to preparation conditions, and our preliminary work has identified variability in complex formation that could compromise dosing consistency across trial sites. Systematic optimization of complexation parameters prior to the trial is essential for reproducible intramuscular gene delivery.
Validated endpoints: The trial must simultaneously assess disease modification (arthritis) and biological aging outcomes, but no validated multi-domain biomarker panel exists for this combination in canines. Without pre-validated assays and established baselines, the trial risks being underpowered or collecting data of uncertain reliability.

Addressing these gaps now—before the trial launches—will substantially reduce execution risk, improve data quality, and strengthen the scientific foundation for follow-on funding and regulatory submissions.

Specific Aims

Aim 1: Optimize Plasmid–PEI Delivery System for Canine Intramuscular Administration

Hypothesis: Systematic optimization of nitrogen-to-phosphate (N:P) ratios, preparation protocols, and environmental conditions will yield reproducible plasmid–PEI complexes with consistent particle size, zeta potential, and transfection efficiency suitable for multi-site clinical use.

Experimental Approach

N:P ratio optimization: Test N:P ratios of 4:1, 6:1, 8:1, and 10:1 using follistatin minicircle plasmid (10 μg/mL) with linear PEI (MW 40,000). Characterize resulting complexes via dynamic light scattering (DLS) for particle size distribution and zeta potential.
Preparation protocol standardization: Evaluate the effects of mixing order, incubation time (15, 30, 60 min), temperature (4°C, RT, 37°C), and buffer composition (glucose 5%, saline, HEPES) on complex stability and reproducibility across three independent preparation batches.
Stability assessment: Determine complex stability over 0, 2, 4, 8, and 24 hours post-preparation under anticipated transport and storage conditions, establishing a viable preparation-to-injection window for clinical use.
In vitro validation: Confirm transfection efficiency of optimized complexes in canine primary myocytes or C2C12 cells, measuring follistatin expression via ELISA at 24, 48, and 72 hours post-transfection.

Deliverables

Standardized operating procedure (SOP) for plasmid–PEI complex preparation
Optimal N:P ratio and preparation conditions with reproducibility data
Stability profile defining the preparation-to-injection window
In vitro transfection efficiency benchmarks

Aim 2: Validate Biomarker Panel for Clinical Trial Endpoints

Hypothesis: A multi-domain biomarker panel combining inflammatory markers, metabolic parameters, and epigenetic aging clocks will demonstrate sufficient sensitivity and reliability to detect therapeutic responses in a canine follistatin gene therapy trial.

Experimental Approach

Inflammatory panel validation: Assay canine-specific C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and activin A in serum samples from healthy aged dogs (n=5–10) and dogs with confirmed OA (n=5–10). Establish assay precision (intra- and inter-assay CV), reference ranges, and effect sizes between healthy and arthritic populations.
Metabolic profiling: Validate assays for serum follistatin, myostatin, insulin, glucose, and lipid panels in canine samples. Determine baseline variability and correlations with body condition score, breed, and age.
Epigenetic age assessment: Evaluate canine DNA methylation-based aging clocks using blood samples from the same cohort. Assess reproducibility of epigenetic age estimates and establish baseline age-deviation values for the clinical trial population.
Composite panel analysis: Determine correlations between inflammatory, metabolic, and epigenetic biomarkers to identify redundancies and optimize the final panel for cost-effectiveness and statistical power in the clinical trial.

Deliverables

Validated assay protocols with precision and reference range data for each biomarker
Baseline biomarker profiles for healthy aged vs. arthritic canine populations
Optimized biomarker panel recommendation for the clinical trial
Power analysis informing required sample size for the clinical trial

Timeline

Period	Aim 1: Delivery Optimization	Aim 2: Biomarker Validation
Months 1–2	N:P ratio screening; DLS characterization; preparation protocol testing	Sample collection from aged and OA dogs; assay setup and intra-assay CV testing
Months 3–4	Stability studies; buffer/temperature optimization; batch-to-batch reproducibility	Inter-assay validation; epigenetic clock processing; metabolic panel optimization
Months 5–6	In vitro transfection validation; SOP finalization; preparation of preprint	Composite panel analysis; power calculations; clinical trial protocol refinement; preprint

Milestone check-ins will be posted as progress updates on ResearchHub at the end of each 2-month block.

Budget

Item	Cost	Notes
Plasmid production and PEI reagents	$2,500	Minicircle plasmid prep; PEI (MW 40K); buffers
DLS / zeta potential characterization	$1,200	Core facility or contract lab access
In vitro transfection assays	$1,000	Cell culture, ELISA kits, consumables
Canine serum biomarker assays	$2,500	CRP, IL-6, TNF-α, activin A, follistatin, myostatin ELISAs
Epigenetic age analysis	$1,800	DNA methylation arrays (10–20 samples)
Sample collection and veterinary costs	$500	Blood draws from collaborating vet clinics
Miscellaneous (shipping, consumables)	$500
Total	$10,000

Preliminary Data and Feasibility

This project builds on a substantial foundation of prior work:

Human Phase 1 trial (NCT06411366): 43 participants received follistatin minicircle plasmid via PEI-mediated intramuscular injection with no serious adverse events. Demonstrated improvements in body composition and a mean 7.1-year reduction in extrinsic epigenetic age. These data establish the core safety and biological activity profile of the intervention.
Canine case study: A single elderly large-breed dog with severe OA showed sustained pain reduction lasting >6 months following follistatin plasmid administration, providing proof-of-principle for canine translation.
Published preclinical evidence: Follistatin gene therapy mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet–induced obesity mouse models (Science Advances, 2020), and follistatin pretreatment alleviates synovitis and cartilage degeneration in carrageenan-induced arthritis models.
Technical experience: The PI has hands-on experience with plasmid–PEI complexation, having prepared and administered treatments in the human trial as Research Coordinator at Minicircle Inc. Current work at Dog Years has identified specific complexation variability issues that this proposal directly addresses.

Translational Pathway and Future Directions

This pre-clinical study is designed as the foundational step toward a clearly defined translational pathway:

Immediate next step: Data generated here will directly inform the design and execution of a 30–50 dog clinical trial with delayed-start control design and three-tiered dose escalation, for which a full protocol has been developed and regulatory planning (FDA pre-INAD, IACUC) is underway.
Regulatory strategy: By demonstrating that follistatin gene therapy can modify arthritis—a well-defined disease indication—we establish a viable regulatory pathway for gene therapy that also addresses biological aging, circumventing the challenge that “aging” alone is not currently recognized as an approvable indication.
Publication plan: Results will be preregistered on ResearchHub and submitted as a Registered Report. The optimized delivery protocol and validated biomarker panel will be published as open-access resources for the broader aging and gene therapy research communities.
Follow-on funding: Pre-clinical data from this study will strengthen applications for seed funding ($1–3M) currently in preparation to support the full clinical trial, as well as future NIH SBIR/STTR applications.

The dual focus on aging and disease modification positions this work at the intersection of two major research frontiers. Canine models offer unique translational advantages, as companion dogs share human environments, develop similar age-related diseases naturally, and have well-characterized aging biology, making them an ideal bridge between preclinical research and human therapeutic development.

Open Science Commitment

This project is fully aligned with ResearchHub’s open science mission:

Preregistration: The study protocol will be preregistered on ResearchHub prior to data collection, including specific hypotheses, analytical plans, and decision criteria.
Open data: All raw and processed data—including DLS measurements, assay validation parameters, biomarker concentrations, and epigenetic age estimates—will be deposited in an open-access repository.
Open protocols: The optimized plasmid–PEI SOP and validated biomarker panel will be published as freely available methods papers, enabling reproducibility by other research groups.
Progress updates: Bi-monthly progress updates will be shared via ResearchHub’s Updates tab, including both positive results and negative findings.
Preprint: A preprint of final results will be uploaded to ResearchHub upon study completion.

Investigator Qualifications

Tristan Roberts is CEO and Principal Investigator at Dog Years, a biotech startup developing follistatin gene therapy for canine applications. He served as Research Coordinator at Minicircle Inc., where he helped execute the Phase 1 human follistatin clinical trial (NCT06411366) and has negotiated exclusive licensing rights for veterinary applications of the technology. His academic background includes cognitive psychology and neuroscience at Reed College, with undergraduate research in neural signal processing. He brings direct hands-on experience in plasmid–PEI preparation, clinical trial coordination, and regulatory planning (FDA pre-INAD, IACUC documentation) to this project.

Key collaborators: Emmanuel Bijaoui (Canine Research Specialist), Brandy Nunez, DVM (Veterinary Specialist), Andreas Stuermer, PhD (Gene Therapy Expert), and Juan Antonio Armas (Data Analyst).

Selected References

Minicircle Inc. Phase 1 Clinical Trial. ClinicalTrials.gov Identifier: NCT06411366.
Li X, et al. Follistatin-mediated skeletal muscle hypertrophy via PI3K/Akt/mTOR signaling. Biosci Rep. 2014;34(5):e00143.
Braga M, et al. Follistatin promotes adipocyte differentiation, browning, and energy metabolism. J Lipid Res. 2014;55(3):375-384.
Tang R, et al. Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet–induced obesity. Sci Adv. 2020;6(19):eaaz7492.
Jones KL, et al. Follistatin alleviates synovitis and articular cartilage degeneration induced by carrageenan. Int J Inflam. 2014;2014:959271.
Winbanks CE, et al. Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin. J Cell Biol. 2012;197(7):997-1008.
Dog Aging Project Consortium. The companion dog as a model for the longevity dividend. Cold Spring Harb Perspect Med. 2023.

Pre-Clinical Optimization of Follistatin Plasmid Gene Therapy

Pre-Clinical Optimization of Follistatin Plasmid Gene Therapy

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Request for Proposals - Aging and Longevity

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