{
  "protocol_schema": "nmd-vcell-dmd-minimum-perturbome/1.0",
  "object_id": "PROTOCOL:v1.0.0-database-resource:DMD-MIN-PERTURBOME:1.0-DRAFT",
  "resource_release": "v1.0.0-database-resource",
  "interface_build": "EA-20260729-15",
  "lifecycle": {
    "state": "DRAFT",
    "immutable": false,
    "registered_at": null,
    "experiment_started": false,
    "measured_outcome_count": 0
  },
  "research_question": "Across DMD and matched-control human myogenic systems, which perturbations change a predeclared functional phenotype without unacceptable viability or differentiation cost?",
  "selection_frame": {
    "perturbation_slot_count": 24,
    "strata": [
      {
        "stratum": "muscle_context_candidates",
        "slots": 6
      },
      {
        "stratum": "high_uncertainty_candidates",
        "slots": 4
      },
      {
        "stratum": "mechanism_diversity_candidates",
        "slots": 4
      },
      {
        "stratum": "known_myogenesis_or_dmd_controls",
        "slots": 4
      },
      {
        "stratum": "random_or_negative_controls",
        "slots": 4
      },
      {
        "stratum": "toxicity_controls",
        "slots": 2
      }
    ],
    "assignment_status": "SLOTS_DEFINED_GENES_NOT_ASSIGNED_UNTIL_SELECTION_RULES_ARE_FROZEN",
    "ranking_policy": "The panel must not contain only high-scoring candidates."
  },
  "experimental_axes": {
    "disease_contexts": [
      "DMD-derived human myogenic system",
      "matched healthy or isogenic-corrected system"
    ],
    "cell_states": [
      "proliferating myoblast",
      "early differentiation",
      "fusion",
      "maturing myotube"
    ],
    "perturbation_modalities": [
      "CRISPRi, CRISPRa, KO or siRNA selected per target and question",
      "two independent reagents where technically feasible"
    ],
    "time_windows": [
      {
        "window": "6–12 h",
        "role": "early molecular and signalling response",
        "status": "PLANNING_DEFAULT_REQUIRES_ASSAY_CALIBRATION"
      },
      {
        "window": "24–48 h",
        "role": "regulatory program and cell-state transition",
        "status": "PLANNING_DEFAULT_REQUIRES_ASSAY_CALIBRATION"
      },
      {
        "window": "4–7 d",
        "role": "differentiation and functional phenotype",
        "status": "PLANNING_DEFAULT_REQUIRES_ASSAY_CALIBRATION"
      }
    ],
    "blocking_factors": [
      "donor or isogenic pair",
      "differentiation batch",
      "plate",
      "reagent"
    ]
  },
  "endpoints": {
    "target_engagement": [
      "mRNA",
      "protein where a validated assay exists",
      "editing or knockdown efficiency"
    ],
    "candidate_primary_functional_endpoint": "Fusion index at an assay-calibrated late timepoint; must be confirmed or replaced before registration.",
    "muscle_differentiation": [
      "differentiation index",
      "fusion index",
      "nuclei per myotube",
      "myotube length, diameter and area",
      "MYOG, MYH3 and mature myosin markers"
    ],
    "dmd_function": [
      "membrane integrity",
      "contraction",
      "calcium handling",
      "mitochondrial function",
      "oxidative stress",
      "injury susceptibility",
      "regeneration response"
    ],
    "safety_cost": [
      "viability",
      "proliferation",
      "differentiation blockade",
      "global stress",
      "essentiality",
      "off-target phenotype"
    ]
  },
  "primary_estimand": "The DMD-versus-matched-control difference in perturbation effect on the frozen primary functional endpoint, with reagent-specific and donor-specific estimates retained.",
  "replication_rules": {
    "reagent": "Two qualified reagents must agree in direction; discordance is inconclusive, not averaged away.",
    "donor": "A favourable effect must not be attributable to one donor or one isogenic pair.",
    "batch": "A future batch is held out prospectively and evaluated without refitting."
  },
  "result_archetypes": [
    "robust_responder",
    "dmd_specific_responder",
    "donor_variable",
    "state_specific",
    "toxic_responder",
    "null_with_equivalence_margin",
    "discordant",
    "qc_failure",
    "inconclusive"
  ],
  "hard_gates_before_start": [
    "freeze the 24 slot assignments and selection rules",
    "freeze one primary endpoint and numeric minimally important effect",
    "freeze negligible-effect margin",
    "freeze sample size or pilot variance rule",
    "freeze target-engagement, viability and imaging QC thresholds",
    "confirm biospecimen, institutional and laboratory approvals",
    "register the immutable Study and Prediction objects before outcome access"
  ],
  "expansion_stages": [
    {
      "stage": "A",
      "scope": "24-slot prospective pilot",
      "purpose": "close the registration-to-outcome loop",
      "status": "DRAFT"
    },
    {
      "stage": "B",
      "scope": "96–192 target high-content perturbation screen",
      "purpose": "build a disease-background phenotype matrix",
      "status": "LOCKED_UNTIL_STAGE_A_REPLICATION_AND_ASSAY_QC"
    },
    {
      "stage": "C",
      "scope": "24–48 deep perturbations",
      "purpose": "paired imaging, RNA/protein and selected multimodal mechanism studies",
      "status": "LOCKED_UNTIL_STAGE_B_SELECTION"
    }
  ],
  "boundary": "This is a draft experiment-registration contract. It contains no assigned 24-gene panel, no experiment, no outcome and no efficacy claim."
}