Dose-Window Characterisation, Evolutionary Timing, and Stochastic Simulation of the Staged Cavitation-Gated Editing Framework: From Principle to Protocol — A Quantitative and Critical Extension of the Acoustic-Genomic Interface Programme

Volume 1 of this series argued that focused ultrasound, used below the fractionation threshold, could act as a spatial actuator for transient genome editing in solid tumours, and that the resulting partial selective pressure must be timed against clonal evolution rather than applied once and abandoned. That argument was mechanistic. This volume converts it into a quantitative experimental and mathematical architecture that can be tested, and - more importantly - falsified. It defines the sub-ablative dose window in terms of peak negative pressure, pulse length, duty cycle and cumulative passive-cavitation-detection energy; couples the local editing-efficiency model to spatially resolved multi-omic readouts so that coverage is measured inside the mapped high-risk niche rather than averaged across a section; formalises evolutionary rescue under partial editing as a birth-death process with an explicit second-strike window; and specifies a multi-clone stochastic simulation of the full map-prime-edit-clear-strike sequence together with the sensitivity analysis that would identify its dominant parameters. The critical contribution of this volume is as important as the constructive one. The framework's central unverified assumption is that a usable separation exists, in perfused heterogeneous tumour, between the pressure at which membranes are transiently permeabilised and the pressure at which tissue fractionates irreversibly. Published threshold data come overwhelmingly from gels, ex vivo tissue and homogeneous models; the window may narrow or close under in vivo perfusion, stiffness gradients and bubble-nucleus heterogeneity. A second assumption, that passive cavitation detection can resolve and control that separation in real time across a tumour volume rather than at a single focal point, is likewise unproven. Both are stated here as go/no-go criteria with pre-specified thresholds, not as expectations. Speculative elements are flagged throughout, no new physics is introduced, and nothing in this paper is medical advice or a treatment recommendation; it is a research design intended for immunocompetent preclinical models.