Navigating Quality, Safety, and Analytical Testing Frameworks for Viral Vector–Based Gene Editing Therapies

Therapeutic CRISPR/Cas9 delivery approaches: strengths, limitations, and a roadmap for clinical translation

Advances in gene transfer technologies: comparing viral and non-viral vectors for therapeutic applications

Standardisation in the Analytical Characterization of Adeno-Associated Virus (AAV) Vectors.

Adeno-associated virus (AAV) has become a leading vector for in vivo gene therapy, with eight products currently holding marketing authorization. As the field rapidly evolves, the need for robust analytical methods to characterize critical quality attributes (CQAs)-including capsid titer, genome titer, capsid content (empty/full ratio), identity, and purity-continues to grow. Reference Standard Materials (RSMs) play a pivotal role by providing well-characterized, standardized AAV batches that serve as universal benchmarks. RSMs facilitate the validation of emerging analytical technologies, ensure the accuracy and reproducibility of routine assays, and enable inter-laboratory comparability. However, developing universal AAV RSMs is fundamentally constrained by the complex biology, diversity of serotypes, vector genomes, and engineered capsid variants, necessitating serotype-specific and application-specific standards. Recent advances, including the release of pharmacopeial AAV8 reference standards characterized by multiple orthogonal methods, represent meaningful progress toward measurement harmonisation. This review addresses the critical need for RSMs in AAV gene therapy, evaluates the currently available pharmacopeial and commercial standards, and outlines practical strategies for in-house RSM development. Establishing robust, serotype-specific AAV RSMs and harmonised standard operating protocols (SOPs) are essential for advancing AAV gene therapy and ensuring accuracy, reproducibility, and safety across research, development, and clinical manufacturing.

Delivery Systems for Therapeutic Genome Editing: Challenges, Innovations, and Future Perspectives

Therapeutic genome editing has advanced rapidly with the development of diverse programmable nucleases, from zinc‐finger nucleases and transcription activator‐like effector nucleases to clustered regularly interspaced short palindromic repeats (CRISPR)‐based systems such as base and prime editors. Despite these breakthroughs, clinical translation remains constrained by the challenge of achieving safe, efficient, and tissue‐specific delivery. Viral vectors, particularly adeno‐associated viruses, have enabled durable editing in selected organs but are limited by their restricted cargo capacity, immunogenicity, and complex manufacturing. Nonviral platforms, most notably ionizable lipid nanoparticles, have demonstrated remarkable efficacy for hepatic targets, with clinical trials reporting up to 93% protein knockdown after a single dose. An expanding set of emerging modalities, including virus‐mimicking nanosystems, cell‐derived extracellular vesicles, cell‐penetrating peptides, and intelligent‐responsive multifunctional scaffolds, further enriches the delivery toolbox by supporting transient expression and programmable targeting across diverse editors and tissues. Parallel advances in high‐throughput barcoded screening and machine learning are accelerating vector optimization, while rational chemical modification of payloads improves in vivo stability and specificity. This review provides a comprehensive overview of current and emerging delivery systems for genome editing, highlighting key innovations, unresolved challenges, and interdisciplinary strategies poised to unlock broader therapeutic potential.

Advanced CRISPR-Cas Genome Engineering Coupled with Nanotechnology-Based Drug Delivery for Treating Genetic and Infectious Diseases

Background CRISPR-Cas genome editing offers curative potential for monogenic disorders and persistent infections, but its clinical translation is hindered by delivery inefficiency, off-target effects, and immunogenicity. Nanocarrier platforms address these barriers by enabling targeted, transient intracellular delivery of CRISPR components. Methods This PRISMA 2020–guided systematic review and meta-analysis synthesized data from 127 preclinical studies and 14 Phase I/II clinical trials (2018–2025) evaluating CRISPR-nanocarrier systems. Results Pooled analysis revealed a median on-target editing efficiency of 52.4% (95% CI: 48.1–56.7). Ionizable lipid nanoparticles (LNPs) demonstrated superior hepatic delivery, while engineered extracellular vesicles (EVs) enabled extrahepatic tropism. Safety profiles were highly favorable: off-target edits remained consistently <0.1%, chromosomal aberrations were negligible (98.4% compliance), and pathogen loads decreased by 3.12 log₁₀. Functional protein restoration yielded a large pooled effect size (SMD: 2.84). Adverse events were primarily limited to transient cytokine elevation and mild, manageable hepatotoxicity. Carrier architecture and ribonucleoprotein (RNP) cargo format emerged as significant predictors of editing success. Conclusion CRISPR-nanocarrier systems represent a highly viable precision medicine platform for achieving durable, potentially curative outcomes. Accelerating clinical deployment requires prioritizing standardized GMP manufacturing, longterm genomic surveillance, scalable access frameworks, and the continued optimization of stimuli-responsive carriers and high-fidelity editors.

Biophysical considerations for designing viruses, lipid nanoparticles and virus-like particles for CRISPR-based genome editing.

CRISPR-based genome editing has opened new pathways towards precision medicine, but its success depends on more than just molecular engineering. Cargo and carrier dynamics are profoundly influenced by the underlying biophysical properties of cells and vectors. Consequently, this domain is moving beyond simple "lock and key" approaches, and towards disease-customised fits. In this review, we examine how biophysical properties of viral vectors, lipid nanoparticles, and hybrid virus-like particles influence editor delivery performance. We examine parameters such as size, cargo capacity, charge, shape, stiffness, membrane composition, internalisation strategies, tropism, endosomal escape, protein corona formation, and immune recognition as key drivers of intelligent, modular engineering. Finally, we explore how the notion of carrier systems shifts in diseased states like solid tumour cancers, autoimmune psoriasis of the skin, and the autosomal monogenic cystic fibrosis, where altered biophysical landscapes demand adaptable, context-informed genome editing solutions.