Genome-Wide Characterization of Recombinant AAV6 Vector Integration in Human CD34＋ Cells.

Identification of AAV Vector Integration Sites in Primary Human and Macaque Hepatocytes in vivo, and Analysis of Oncogenic Risk.

RAAV integration patterns in primary human hepatocytes xenografted into FRG mouse livers and in hepatocytes from cynomolgus macaques following systemic rAAV administration support a low oncogenic risk profile for the evaluated vector while reinforcing the value of direct human liver integration analyses to refine risk assessment and guide the development of safer gene therapy platforms.

Long-read target enrichment sequencing for rAAV integration site analysis in engineered clones with targeted viral insertion

Integration site analysis (ISA) plays a critical role in the genotoxicity assessment of recombinant adeno-associated viruses (rAAVs); however, ISA is challenged by the low integration rate, frequent vector genome rearrangements and the absence of well-characterized controls. Here, we generated 34 MCF10A-derived clones harboring recombinant adeno-associated viruses (rAAV) insertions at CRISPR-Cas9-induced double-strand breaks as cellular reference material and used these cell lines to evaluate long-read target enrichment sequencing (LR-TES) for rAAV ISA. LR-TES findings were complemented by droplet digital PCR quantification of vector copy number, long-range PCR analysis of on-target integration, and whole-genome sequencing on selected clones. Dilution experiments established a limit of detection of 1% under the tested conditions. LR-TES enabled genome-wide identification of vector-host junctions and structural characterization of integrated vector genomes, detecting targeted and non-targeted insertions, genomic deletions, and extensive vector restructuring across the clone panel. Only ∼29% of the integrated vector payload remained intact, while the observed truncations, rearrangements, or concatemers highlighted the structural complexity that ISA must resolve in rAAV settings. Together, the engineered clonal controls and optimized LR-TES provide reference material and a technical framework supporting rAAV ISA in safety evaluation, particularly when high structural resolution and specificity are needed to interpret predominant integration events.

Adeno-associated Virus (AAV) Synthetic Inverted Terminal Repeat Enhances Tissue Specific Transduction and Altered Vector Induced Stress Response

AAV Vectors in Regenerative Medicine and Cellular Reprogramming: Potential, Pitfalls, and Specificity Constraints

Evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing are synthesized.

Addressing the package: Cell-specific gene delivery using lentiviral vectors.

Lentiviral vectors have revolutionized gene therapy by efficient and stable transduction of dividing and non-dividing cells, their large packaging capacity, and their compatibility with pseudotyping to alter viral tropism. The vesicular stomatitis virus glycoprotein (VSV-G) is widely used as a viral envelope protein of choice to pseudotype lentiviral vector particles as it confers exceptional particle stability and a broad tropism, due to the ubiquitous nature of the low-density lipoprotein receptor (LDLR). While this broad tropism facilitates transduction of diverse cell types, it precludes accurate in vivo targeting of specific cell populations. Structural insights into VSV-G have made receptor-blinding possible and revealed sites amenable to mutation while preserving fusion capacity. Coupled with targeting moieties, VSV-G pseudotyped lentiviral particles are redirected towards cells expressing target antigens. Such targeted vectors open new possibilities for in vivo gene therapy across oncology, infectious diseases, transplantation medicine, and other diseases. Use of targeted vectors will make in vivo gene therapy more accessible than cost-intensive ex vivo gene therapies. Since targeted vectors will be available as 'off-the-shelf' drugs, they will also drastically reduce time-to-treatment. This review highlights advances in bioengineering to exploit the versatility of VSV-G-pseudotyped lentiviral vectors and explores their vast potential for targeted gene delivery.

A novel adeno-associated viral vector derived from human spleen isolate AAV.hu.S17

This study generated a novel recombinant AAV vector rAAV.hu.hu.S17, derived from the human spleen isolate AAV.hu.S17, and systematically evaluated its capsid features, in vitro transduction, and in vivo tissue tropism.