Six Core Pillars of AAV Gene Therapy Development: Technical, Immunological, and Regulatory Challenges with Strategic Solutions.

Adeno-associated virus (AAV) vectors have become a leading vehicle for in vivo gene therapy, characterized by their ability to maintain transgene expression in non-dividing cells and a relatively good safety profile. In 2025, AAV-based gene therapy advanced with the US Food and Drug Administration (FDA) approval of Itvisma to treat spinal muscular atrophy (SMA) in patients aged two years and older. Despite these advances, broader clinical applications remain limited by the interplay among biological, immunological, manufacturing, and regulatory challenges. This minireview highlights the fundamentals and contemporary progress in addressing developmental issues such as capsid tropism, pre-existing immunity, payload capacity, manufacturing scalability, dose-dependent toxicity, and regulatory strategy. The latest emerging solutions, including directed evolution, machine learning-aided design, and new purification strategies, are also addressed. The article serves as a roadmap for researchers seeking to develop the next generation of AAV-based genomic medicines by synthesizing these complex impediments.