ABT263 treatment in naturally aged mice

PRIMARY OUTCOME

rejuvenation of aged HSCs and MuSCs through senescent cell depletion

EXPECTED OUTCOMES

1. **Senescent cell clearance**: ABT263 will reduce SA-β-gal+ cells by 40-60% in liver, lung, and adipose tissue, and reduce p16INK4a+ cells in bone marrow by 35-50% compared to vehicle-treated aged mice. 2. **HSC functional rejuvenation**: In competitive repopulation assays, aged ABT263-treated donors will show 2.5-3.5-fold higher engraftment efficiency at 16 weeks compared to aged vehicle donors, reaching 60-75% of young donor engraftment levels. 3. **MuSC regeneration improvement**: Notexin-injured muscles in ABT263-treated mice will show 30-40% smaller central nucleation index at Day 7 and 25-35% larger mean fiber CSA at Day 14 compared to vehicle controls. 4. **SASP reduction**: Plasma IL-6 will decrease from ~85 pg/mL (aged baseline) to 40-55 pg/mL (ABT263) vs ~75 pg/mL (vehicle), representing 35-50% reduction. TNF-α similarly reduced by 30-45%. 5. **HSC lineage skewing correction**: Myeloid-biased LSK frequency will decrease from ~65% (aged vehicle) to 50-55% (ABT263), approaching young mouse levels of 40-45%. 6. **Physical function improvement**: Rotarod latency will increase from ~60s (aged vehicle) to 100-130s (ABT263) vs ~180s (young), and grip strength will improve by 25-40% in ABT263-treated mice. 7. **MuSC cell cycle re-entry**: Ki-67+ MuSC fraction will increase from ~25% (aged vehicle) to 40-50% (ABT263), indicating restored proliferative capacity comparable to young mice (~55-65%). ---

SUCCESS CRITERIA

- **Senescent cell clearance**: ABT263 reduces SA-β-gal+ cells by ≥35% in at least 3 of 5 tissues (liver, lung, adipose, muscle, bone marrow) compared to vehicle controls (two-tailed t-test, p<0.01). - **HSC engraftment**: Donor cell chimerism in peripheral blood at 16 weeks post-transplant reaches ≥50% for ABT263-treated aged donors, significantly different from vehicle (≤25%) with p<0.001 (Mann-Whitney U test) and effect size r≥0.7. - **MuSC regeneration**: Mean muscle fiber cross-sectional area in ABT263 group is ≥25% larger than vehicle group at Day 14 post-injury, with p<0.01 (ANOVA with Tukey post-hoc) and power ≥0.85. - **SASP suppression**: At least 2 of 3 measured cytokines (IL-6, TNF-α, CXCL1) show ≥30% reduction in ABT263 vs vehicle, with p<0.05 per cytokine (paired t-test or repeated measures ANOVA). - **Lineage balance restoration**: Myeloid:lymphoid ratio in bone marrow of ABT263-treated aged mice normalizes to within 1.5 standard deviations of young mouse mean. - **Physical function**: Combined z-score of rotarod + grip strength for ABT263-treated aged mice is ≥0.5 standard deviations above vehicle-treated aged mice, with p<0.05 (two-tailed). - **Safety threshold**: No significant difference in ALT, AST, BUN, or creatinine between ABT263 and vehicle groups (p>0.05), and platelet count remains within 80-120% of baseline to confirm acceptable thrombocytopenia risk.

PROTOCOL

### Study Design - **Animal Model**: C57BL/6J mice, naturally aged (18-22 months), both sexes - **Drug**: ABT263 (S6381, Selleck Chemicals), 50 mg/kg dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, 45% sterile water - **Control**: Vehicle-only formulation - **Group Size**: n=12 per group (vehicle, ABT263), based on power analysis (α=0.05, β=0.2, expected effect size d=1.2) --- ### Phase 1: Baseline Assessment (Day -7) **Timepoint**: 7 days before treatment initiation - Age-matched young controls (3-4 months, n=8) and aged baseline cohort (n=24) undergo baseline phenotyping - **HSC harvest**: Flush femurs and tibias with PBS + 2% FBS; enrich for lineage-negative cells (Miltenyi Biotec Lineage Cell Depletion Kit) - **MuSC isolation**: Digest gastrocnemius muscles with collagenase II (800 U/mL) + dispase II (2.4 U/mL); enrich via Pax7-GFP reporter or anti-α7-integrin magnetic sorting - **Flow cytometry panels**: - HSC: LSK (Lin−Sca-1+c-Kit+), CD48/CD150, CD34/CD16/32 (myeloid bias) - MuSC: α7-integrin+CD34+, Pax7, Ki-67 - **Senescence markers**: SA-β-gal activity (C12FDG staining), p16INK4a reporter (Luciferase or qPCR: Cdkn2a) - **SASP multiplex**: IL-6, TNF-α, CXCL1, MMP-3 (Luminex or ELISA) - Record body weight, grip strength, rotarod performance --- ### Phase 2: ABT263 Treatment (Days 0, 2, 4) **Dosing regimen**: Oral gavage, 50 mg/kg, every other day × 3 doses - Randomize aged mice into vehicle (n=12) and ABT263 (n=12) groups - Administer via oral gavage (20G curved catheter) in morning - Monitor daily for adverse effects (weight loss >15%, hunched posture, decreased mobility) - Collect plasma at 2h and 6h post-dose on Day 0 for pharmacokinetics (LC-MS/MS) --- ### Phase 3: Acute Senescent Cell Clearance (Days 5-7) **Endpoint**: 24-72 hours after final dose **Senescent cell burden** (n=4 per group sacrificed): - **SA-β-gal tissue staining**: Liver, lung, adipose, muscle, bone marrow - **p16INK4a immunofluorescence**: Tissue sections co-stained with DAPI - **TUNEL assay**: Quantify apoptotic cells in senescent cell populations - **SASP plasma levels**: Compare pre- vs post-treatment **Off-target toxicity assessment**: - Complete blood count (CBC) via IDEXX ProCyte Dx - Liver function: ALT, AST, bilirubin (Beckman Coulter AU680) - Kidney function: BUN, creatinine - Histopathology: H&E staining of liver, kidney, lung, heart --- ### Phase 4: Early Regeneration Phase (Days 14-21) **Stem cell function assays** (n=8 per group): **HSC function**: - **Competitive repopulation assay**: Transplant 1×10^6 CD45.2 donor bone marrow cells (aged vehicle or ABT263) + 1×10^6 CD45.1 competitor cells into lethally irradiated (900 cGy) CD45.1 recipients - Analyze peripheral blood chimerism at 4, 8, 12, 16 weeks post-transplant (flow cytometry: CD45.2 vs CD45.1) - Lineage distribution: B220 (B cells), CD3 (T cells), Gr-1/Mac-1 (myeloid) - **Serial transplantation**: Collect bone marrow from primary recipients at 16 weeks; transplant into secondary recipients **MuSC function**: - **Notexin-induced muscle regeneration**: Inject 10 μL of 10 μM notexin into tibialis anterior; harvest at 7 and 14 days post-injury - **Muscle histology**: H&E staining to assess central nucleation (regenerating fibers), fiber cross-sectional area (CSA), fibrosis (Masson's trichrome) - **MuSC quantification**: Pax7+ cell density per 100 fibers - **Functional recovery**: Grip strength and running wheel activity at 7, 14, 21 days post-injury --- ### Phase 5: Long-Term Rejuvenation (Weeks 8-12) **Final endpoints** (remaining n=8 per group, plus young controls): **Hematopoietic parameters**: - Complete blood count with differential - Bone marrow cellularity (femur + tibia total nucleated cells) - HSC frequency and absolute numbers: LSK, LT-HSC (CD150+CD48−), ST-HSC (CD150−CD48−) - Myeloid:lymphoid ratio in bone marrow **MuSC parameters**: - MuSC yield per gram muscle (gastrocnemius, quadriceps) - Cell cycle analysis: Ki-67/DAPI flow cytometry - Mitochondrial function: Seahorse XF analyzer (OCR/ECAR in MuSC culture) - Telomere length: qFISH or Flow-FISH **Systemic markers**: - Inflammatory cytokines: IL-6, TNF-α, IL-1β, IFN-γ (Luminex) - Metabolic panels: fasting glucose, insulin (ELISA), cholesterol - Physical function: rotarod latency, hanging wire duration, stride length analysis --- ### Phase 6: Mechanistic Studies (Parallel, Weeks 4-8) **BH3 profiling** (Bio-techne or in-house): - Measure apoptotic threshold in aged vs young HSCs and MuSCs - Determine Mcl-1, Bcl-2, Bcl-xL expression (Western blot: Bcl-2, Bcl-xL, Mcl-1, NOXA, BIM) - Cyto-ID flow cytometry for autophagic flux **Senescent cell engraftment model** (validation): - Transplant DiR-labeled senescent fibroblasts (γ-irradiated, p16-high) into aged mice ± ABT263 - Track clearance via IVIS imaging at 24, 48, 72h post-transplant ---

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