Fig. 1. Overview of AAV library generation, in vivo evolution, and validation.

(A) Workflow comprising nine consecutive steps: (i) creation and production of two AAV capsid libraries by DNA family shuffling of multiple cap genes; (ii) in vivo selection/cycling via systemic injection into mice, followed by vector genome amplification from on-target tissues (diaphragm, heart, and skeletal muscle), subcloning and production of secondary libraries for repeated injection; (iii and iv) barcoding and stratification in mice; (v) rational P1 peptide transfer into selected shuffled capsids; (vi) barcoding of the ensuing bioengineered capsids plus benchmarks; (vii) validation of the muscle specificity in WT mice; (viii) combination of the two best capsids (AAVMYO2 and AAVMYO3) with two myotropic promoters; and (ix) application in two mouse models of human muscle diseases. (B) Sequence analysis of shuffled AAV libraries. Top: Parental libraries composed of AAV1, -6, -8, and -9 (library A) or AAV1, -6, -8, -9, and po.1 (library B). Shown are representative examples. Each row depicts one clone. (C) Composition of the secondary barcoded library. Shown are log₂(FC) values relative to the mean per barcoded AAV variant in the library. The two dotted lines mark a twofold change. Negative values indicate underrepresentation of a variant, and positive values imply overrepresentation. FC, fold change. (D) Viral DNA distribution of the library in multiple organs. Shown are vector genomes (individual mice and averages) per diploid genome (vg/dg; means + SD) from three C57BL/6J mice (each mouse is one dot, injected with 1 × 10¹² vg, euthanized 1 week later). Vector genomes (EYFP probe) were normalized to RPP30 as a housekeeper. Gastrocn., gastrocnemius; Quadriceps, quadriceps femoris.