CALM-AF-AI: Counteracting Age-related Loss of Muscle With AAV-Follistatin Combined With Angiogenesis-Inducing VEGF Plasmid Gene Therapy

Overview

This Phase 1/2a, open-label, non-randomized study is designed to evaluate the safety and tolerability of intramuscular AAV9-Follistatin gene therapy administered either as monotherapy or in combination with a VEGF-encoding plasmid. Secondary objectives include the assessment of preliminary signals of biological and functional activity, including changes in skeletal muscle mass and performance.

Detailed description

Approximately 12 participants (with a potential expansion to 18-21) will be sequentially assigned to three cohorts: low-dose AAV-Follistatin monotherapy (n=3), high-dose AAV-Follistatin monotherapy (n=3), or combination therapy (AAV-Follistatin + VEGF plasmid, n=6). A cautious 3+3 dose-escalation design with sentinel dosing will be employed.

All investigational products are administered via intramuscular injection into large skeletal muscles. In Cohorts 1 and 2, AAV-Follistatin is administered once on Day 1. In Cohort 3, VEGF plasmid is administered on Day 1 and Day 12 (±2 days), followed by AAV-Follistatin on approximately Day 27 (±3 days), corresponding to 15 ± 1 days after the second VEGF plasmid dose.

Rapamycin will be administered for approximately two months to mitigate immune responses to the AAV vector. Participants will undergo regular safety monitoring, including clinical assessments, laboratory testing, and strength evaluations.

The study enrolls adults aged 35-75 years with evidence of age-related muscle decline, who are in generally stable health and able to provide informed consent and comply with study procedures. Eligible participants must demonstrate low or acceptable antibody titers to the AAV vector and hold Próspera ZEDE eResidency or Physical Residency.

Key exclusion criteria include: uncontrolled significant medical conditions; active or recent malignancy; clinically relevant immune disorders or current immunosuppressive therapy; pregnancy or breastfeeding; prior exposure to AAV-based gene therapy; or recent participation in other investigational studies.

Screening (up to 7 days) includes medical history, physical examination, laboratory tests, and baseline muscle assessments (e.g., DXA, strength, and functional testing). The core study period lasts approximately 90 days (for Group 3 = 120 days), with frequent safety assessments and functional evaluations. Participants may opt into extended safety follow-up at approximately 6 and 12 months.

Participation involves potential risks, including:

* Immune reactions to AAV or follistatin (e.g., flu-like symptoms, elevations in liver enzymes), monitored and managed per protocol; * Risks associated with VEGF plasmid (e.g., transient limb pain or swelling); * Adverse effects related to rapamycin (e.g., mucositis, metabolic changes, increased infection risk); * Unknown or rare risks inherent to gene therapy. Independent safety oversight and predefined stopping rules are in place.

Direct clinical benefit cannot be guaranteed. Participants may experience improvements in muscle mass, strength, endurance, or functional performance; however, this is an early-phase trial primarily designed to assess safety and feasibility. The study may contribute to the development of future therapies for age-related muscle decline.

Participants should anticipate approximately three months of active participation, with optional follow-up extending to 12 months.

Interventions

• Genetic AAV9-Follistatin gene therapy
  One-time intramuscular administration of an adeno-associated virus, serotype 9, (AAV9) vector encoding human follistatin.
• Genetic VEGF Plasmid
  Intramuscular supercoiled plasmid DNA gene therapy encoding vascular endothelial growth factor (VEGF).

Primary outcome measures

• Number of participants with treatment-emergent adverse events (TEAEs), including serious adverse events (SAEs) [Time frame: Day 1 through Day 90 after AAV administration]
• Number of participants with dose-limiting toxicities (DLTs) [Time frame: Day 1 through Day 21 after AAV administration]

Secondary outcome measures (4)

• Number of participants with predefined clinically significant laboratory abnormalities [Time frame: Day 1 through Day 90 after AAV administration]
• Number of participants with new-onset symptomatic heart failure or arrhythmias (CTCAE Grade ≥2) [Time frame: Day 1 through Day 90 after AAV administration]
• Number of participants with injection site reactions [Time frame: Day 1 through Day 90 after AAV administration]
• Number of participants who discontinue study treatment due to adverse events [Time frame: Day 1 through Day 90 after AAV administration]

Eligibility criteria

Inclusion criteria

• Voluntary written informed consent obtained prior to any study-related procedures
• Ability to read, understand, and sign the Informed Consent Form and reliably complete required study documents
• Willingness to undergo medical intervention, including genetic therapy, and to comply with the visit schedule and all study procedures
• Commitment to maintain a stable medication and supplement regimen throughout the study, with no initiation of new medications, supplements, or performance-enhancing substances unless approved by the Investigator
• Men and women aged 35-75 years
• Body mass index (BMI) between 18.0 and 35.0 kg/m² at screening
• Active Prospera ZEDE eResidency or Physical Residency
• Stable comorbid conditions for at least 3 months prior to screening
• Postmenopausal status (women)
• Willingness to use reliable contraception for 6 months following therapy
• Low or undetectable antibody titers to AAV9 (≤1:100 by ELISA)

Exclusion criteria

• Pregnancy, breastfeeding, or intent to become pregnant; premenopausal status (unless ≥12 months amenorrhea or FSH ≥30 IU/L)
• Subjects who have a history of alcohol or drug abuse within 1 year of study entry
• Initiation of prohibited medications, supplements, or interventions during the study period that may confound efficacy or safety assessments
• Active malignancy or ANY history of cancer
• Strong family history of cancer in first-degree relatives (≥2 relatives with cancer diagnosed <60 years) OR known hereditary cancer syndrome (BRCA1/2, Lynch syndrome, Li-Fraumeni, FAP, HNPCC) without genetic counseling and enhanced surveillance clearance
• Clinically significant cardiovascular disease, including:
• Any history of stroke, transient ischemic attack (TIA), myocardial infarction (MI), or unstable angina (regardless of time since event)
• Diagnosed coronary artery disease (CAD) documented by coronary angiography or stress testing
• Significant atherosclerotic disease at any location (stenosis ≥50% in carotid, femoral, or other major arteries)
• Prior coronary revascularization (percutaneous coronary intervention \[PCI\] or coronary artery bypass grafting \[CABG\])
• Prior valvular repair or replacement
• History or current diagnosis of heart failure
• Uncontrolled hypertension (SBP >140 mmHg or DBP >85 mmHg despite treatment)
• Left ventricular ejection fraction (LVEF) <50%, QTc ≥480 ms, or severe valvular heart disease
• Ventricular arrhythmias requiring chronic drug treatment or implantable cardioverter-defibrillator
• Presence of pacemaker or persistent left bundle branch block (LBBB)
• Known diagnosed cardiomyopathy of any etiology
• Significant left ventricular hypertrophy, defined as maximal left ventricular wall thickness ≥15 mm in any segment at end-diastole (by echocardiography or cardiac MRI)
• Known or suspected hypercoagulable state/thrombophilia, including any of the following:
• Any history of venous thromboembolism (DVT, PE, or thrombosis at any site), particularly if:
• Unprovoked, or
• Associated with only minor provoking factors (e.g., minor surgery, combined oral contraceptives, short-term immobilization)
• Recurrent thrombotic events, including recurrent superficial venous thrombosis
• Thrombosis at unusual sites, including but not limited to:
• Mesenteric, portal, or splenic vein thrombosis
• Cerebral venous sinus thrombosis
• Hepatic vein thrombosis (Budd-Chiari syndrome)
• Renal vein thrombosis
• Retinal vein thrombosis
• Superior vena cava thrombosis not related to central venous catheterization
• Strong family history of venous or arterial thrombosis at a young age in first-degree relatives
• History of recurrent pregnancy loss or severe obstetric complications suggestive of a hypercoagulable state
• High-degree myopia (≥ -6.0 diopters) or pathological myopia without ophthalmologic clearance
• Any history of retinal detachment, vitreous hemorrhage, or retinal vascular disease (diabetic retinopathy, retinal vein occlusion, age-related macular degeneration with neovascularization)
• Use of systemic anti-VEGF therapy (e.g., bevacizumab)
• Current use of prohibited medications or supplements (see Section 4.3)
• Fasting plasma glucose ≥7.0 mmol/L (≥126 mg/dL) at screening
• HbA1c ≥6.5% (≥48 mmol/mol) at screening
• Current diabetes mellitus (any type)
• History of peptic ulcer disease within 12 months
• Known osteoporosis (DXA T-score ≤ -2.5 at the hip or spine)
• Severe pulmonary disease, including COPD or restrictive lung disease (FVC <49% predicted)
• Advanced renal disease (CKD stage 3-5, eGFR <60 mL/min/1.73 m²) or dialysis dependence
• Chronic liver disease or hepatic impairment:
• Any history of cirrhosis, chronic viral hepatitis (HBV, HCV), autoimmune hepatitis, or cholestatic liver disease
• Active hepatitis or evidence of hepatic decompensation
• ALT or AST >1.5× upper limit of normal (ULN)
• Total bilirubin >1.5× ULN (unless due to Gilbert's syndrome)
• Non-alcoholic steatohepatitis (NASH) and clinically significant liver fibrosis
• Active cholecystitis, symptomatic gallbladder disease (e.g., biliary colic), or any other clinically significant hepatobiliary abnormality
• Neurodegenerative, neuromuscular, psychiatric, or movement disorders associated with functional or cognitive impairment (e.g., dementia, Parkinson's disease)
• History of drug-induced myopathy or rhabdomyolysis
• Elevated creatine kinase (CK) at screening CK >1.0× ULN confirmed on two separate occasions at least 48 hours apart
• Exception: Transient CK elevation within 72 hours of strenuous exercise, intramuscular injections, or trauma is permitted if CK normalizes (≤1.0× ULN) on repeat testing before baseline visit
• Systemic connective tissue diseases (CTDs), including:
• Systemic lupus erythematosus (SLE), SLE overlap syndromes, or drug-induced SLE
• Mixed connective tissue disease (MCTD)
• Systemic sclerosis (SSc, scleroderma): diffuse, limited, or sine scleroderma
• Inflammatory myopathies: polymyositis (PM), dermatomyositis (DM), inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), or overlap myositis
• Primary Sjögren's syndrome requiring systemic immunosuppression (corticosteroids >10 mg/day prednisone equivalent, DMARDs, or biologics)
• Rheumatoid arthritis with extra-articular manifestations or requiring biologic DMARDs
• Undifferentiated connective tissue disease (UCTD) meeting ≥2 CTD classification criteria
• Current or recent (within 3 months) use of immunosuppressive agents, including corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, IVIG, or rituximab
• Acute bacterial, fungal, or viral infection, fever, or receipt of live vaccines within 30 days prior to screening
• Infectious disease exclusions, including:
• Active hepatitis B infection or reactivation risk (HBsAg positive, HBV DNA detectable, or isolated anti-HBc without anti-HBs)
• Hepatitis C infection (detectable HCV RNA or treatment within 6 months)
• HIV infection
• Active or latent tuberculosis (positive QuantiFERON-TB Gold Plus ≥0.35 IU/mL)
• Acute herpesvirus infection, defined as
• Active HSV-1 or HSV-2 lesions (vesicles, ulcers, crusts) on clinical examination at screening
• CMV or EBV IgM positive
• Increased bleeding risk, including platelet count <100 ×10⁹/L, active anticoagulation (unless washout possible), or known coagulation disorders
• Severe physical functional limitation (CFS>6)
• Prior exposure to any AAV gene therapy product (any AAV serotype)
• Prior exposure to any investigational drug within 90 days
• Participation in another clinical trial within 90 days
• Known hypersensitivity to investigational product components or immunosuppressive agents (e.g., prednisone, rapamycin)
• Life expectancy <6 months
• Any condition that, in the Investigator's judgment, may pose undue risk, interfere with study outcomes, or impair study participation

Criteria are shown verbatim from the registry (in English). Final eligibility is always assessed by the study center.

Healthy volunteers: Yes

Study design

Allocation

Non-randomized

Model

Sequential

Masking

Open label

Primary purpose

Prevention

Study locations

Honduras · 1 center

• GARM — Coxen Hole

Publications

• Suoranta T, Laham-Karam N, Yla-Herttuala S. Strategies to improve safety profile of AAV vectors. Front Mol Med. 2022 Nov 1;2:1054069. doi: 10.3389/fmmed.2022.1054069. eCollection 2022. PMID 39086961
• Tang R, Harasymowicz NS, Wu CL, Collins KH, Choi YR, Oswald SJ, Guilak F. Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet-induced obesity. Sci Adv. 2020 May 8;6(19):eaaz7492. doi: 10.1126/sciadv.aaz7492. eCollection 2020 May. PMID 32426485
• Mendell JR, Sahenk Z, Al-Zaidy S, Rodino-Klapac LR, Lowes LP, Alfano LN, Berry K, Miller N, Yalvac M, Dvorchik I, Moore-Clingenpeel M, Flanigan KM, Church K, Shontz K, Curry C, Lewis S, McColly M, Hogan MJ, Kaspar BK. Follistatin Gene Therapy for Sporadic Inclusion Body Myositis Improves Functional Outcomes. Mol Ther. 2017 Apr 5;25(4):870-879. doi: 10.1016/j.ymthe.2017.02.015. Epub 2017 Mar 6. PMID 28279643
• Nance ME, Shi R, Hakim CH, Wasala NB, Yue Y, Pan X, Zhang T, Robinson CA, Duan SX, Yao G, Yang NN, Chen SJ, Wagner KR, Gersbach CA, Duan D. AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice. Mol Ther. 2019 Sep 4;27(9):1568-1585. doi: 10.1016/j.ymthe.2019.06.012. Epub 2019 Jul 3. PMID 31327755
• Al-Zaidy SA, Sahenk Z, Rodino-Klapac LR, Kaspar B, Mendell JR. Follistatin Gene Therapy Improves Ambulation in Becker Muscular Dystrophy. J Neuromuscul Dis. 2015 Sep 2;2(3):185-192. doi: 10.3233/JND-150083. PMID 27858738
• Deev R, Plaksa I, Bozo I, Isaev A. Results of an International Postmarketing Surveillance Study of pl-VEGF165 Safety and Efficacy in 210 Patients with Peripheral Arterial Disease. Am J Cardiovasc Drugs. 2017 Jun;17(3):235-242. doi: 10.1007/s40256-016-0210-3. PMID 28050885
• Deev R, Plaksa I, Bozo I, Mzhavanadze N, Suchkov I, Chervyakov Y, Staroverov I, Kalinin R, Isaev A. Results of 5-year follow-up study in patients with peripheral artery disease treated with PL-VEGF165 for intermittent claudication. Ther Adv Cardiovasc Dis. 2018 Sep;12(9):237-246. doi: 10.1177/1753944718786926. Epub 2018 Jul 11. PMID 29996720
• Kota J, Handy CR, Haidet AM, Montgomery CL, Eagle A, Rodino-Klapac LR, Tucker D, Shilling CJ, Therlfall WR, Walker CM, Weisbrode SE, Janssen PM, Clark KR, Sahenk Z, Mendell JR, Kaspar BK. Follistatin gene delivery enhances muscle growth and strength in nonhuman primates. Sci Transl Med. 2009 Nov 11;1(6):6ra15. doi: 10.1126/scitranslmed.3000112. PMID 20368179

Identifiers

NCT: NCT07443826 · UBL-CALMAF-001

Primary sources (government registries)

• ClinicalTrials.gov (US NIH)

View this study on ClinicalTrials.gov ↗