Adeno-associated virus (AAV) vectors are pivotal in gene therapy for neurological disorders due to their ability to enable long-term gene expression in the central nervous system (CNS). However, transducing larger brains, such as those of non-human primates (NHPs), remains challenging, necessitating alternative delivery routes and optimized capsids. This study directly compares the transduction efficiency and biodistribution of the benchmark AAV9 and its engineered derivative, AAV-PHP.eB, following intracerebroventricular (i.c.v.) administration in juvenile Macaca nemestrina. Employing a neuron-specific promoter and nuclear-localized reporter, we systematically quantified transduction across cortical, subcortical, and spinal regions. AAV-PHP.eB demonstrated significantly higher transduction rates in cortical and spinal regions compared to AAV9, despite similar expression patterns. Both vectors exhibited limited subcortical penetration and significant peripheral leakage, highlighting key challenges in CNS targeting. This is the first study to quantitatively compare AAV-PHP.eB and AAV9 in NHPs, providing valuable insights into the advantages and limitations of engineered AAV capsids for CNS gene therapy. These findings lay a critical foundation for optimizing vector designs and delivery strategies to improve outcomes in clinical applications for neurodegenerative and neurodevelopmental disorders.
Scientific breakthroughs in the field of cell and gene therapy lay the groundwork for therapeutic innovation, but their translation and integration into routine care is delayed by systemic inefficiencies and limited cross-sector alignment. The Advanced Therapies model of Lund is a proactive and integrated approach designed to accelerate the development of advanced therapies from discovery to reimbursement, facilitating close collaboration among three main entities: university, hospital, and the iActors (a coalition of the innovation system, incubators, investors, and industry). Unlocking the full potential of therapeutic innovation requires inclusive and proactive engagement from all entities to accelerate development timelines, optimize resource use across the ecosystem, and ultimately deliver curative, cost-effective advanced therapies to patients. This will ensure broad patient access and establishment of optimal conditions for developing the next generation of cell and gene therapies, while also facilitating innovation and economic growth. To operationalize this model, we developed the Cell and Gene Therapy Navigator, a visualization instrument designed to track and optimize the development of advanced therapies within the model framework. This tool leverages three-dimensional visualization to provide a dynamic and comprehensive overview of progress of the advanced therapy development across the three crucial entities: university, hospital, and iActors. We are confident that working according to the model will minimize translational gaps and result in seamless good manufacturing practices (GMP) transfer, fewer re-works, faster time to first-in-human, and the creation of more viable biotech companies.
Eliminating hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) remains a major challenge, requiring innovative treatment strategies and drug candidates. Clinical studies reveal that wild-type HBV in the blood is often replaced by gradually rising mutant populations. This replacement reflects loss of the early cccDNA pool, then replenished predominantly through de novo infection. We proposed that blocking de novo infection is essential for cccDNA elimination and establishing a finite HBV treatment regimen. To achieve sustained inhibition of de novo cccDNA replenishment, we developed HBVZ10, a gene therapy candidate that utilizes an optimized adeno-associated virus (AAV) vector 8 to deliver human anti-HBs antibody genes into muscle cells for expanding endogenous anti-HBs antibody production capacity. HBVZ10 expression and therapeutic function were evaluated in uPA/SCID chimeric mice. HBVZ10 therapy achieved sustained antibody expression of ≥100,000 mIU/mL for at least 200 days following a single dose administration. Combining HBVZ10 with intracellular replication inhibitor entecavir resulted in >100-fold reductions in cccDNA within a few months, accompanied by progressive reductions in serum HBeAg and HBsAg to undetectable levels. These findings establish preclinical evidence of HBVZ10 as a novel gene therapy candidate and support a paradigm-shifting cccDNA elimination strategy.
Adeno-associated viruses (AAVs) are widely acknowledged as versatile vectors for gene therapy due to their non-pathogenic nature, inherent capacity for tissue-specific targeting, and their potential for customizable engineering. The N terminus of the AAV capsid protein VP1 plays a pivotal role in guiding AAV capsids into the cell nucleus. However, the precise dynamics of the interaction between the VP1 protein and host nuclear transport proteins, especially across diverse AAV serotypes, remain incompletely understood. AAV11 has emerged as a promising alternative for individuals with elevated antibody titers against AAV2, and in the field of neuroscience, it has demonstrated a strong capability for mapping and manipulating neural circuits, offering the potential for treating neurological and neurodegenerative disorders. In this study, we characterize the molecular interface between AAV11 VP1 and host importin-α (IMPα). Structural and biochemical analyses reveal that the basic regions BR1 and BR3 of the VP1 N-terminal domain engage IMPα in a bipartite nuclear localization signal (NLS)-like manner. These findings provide mechanistic insight into VP1-IMPα recognition and suggest a role for these interactions in AAV11 nuclear import. While direct functional evidence is pending, this work establishes the molecular basis for VP1-host protein binding and informs future capsid engineering.
The variability in structure and stability of adeno-associated virus serotype 9 (AAV9) in response to changes in buffer composition, pH, and temperature was investigated using charge detection mass spectrometry (CDMS). AAV9 virus-like particles (VLPs) consisting of only viral protein (VP) 3 and wild-type AAV9 capsids (i.e., capsids containing varying stoichiometries of VP1, VP2, and VP3) showed differences in structure, indicating that these different VP stoichiometries and compositions may contribute substantially to conformational heterogeneity. Significant differences in AAV9 structure and stability were observed in ammonium acetate (AA) vs. phosphate buffered saline (PBS) solutions under some conditions. At 37°C under acidic conditions, AAV capsids fell apart in AA, whereas in PBS, capsids underwent structural compaction. Subsequent nuclease binding experiments indicated that partially extruded DNA was the likely origin of this structural compaction that occurred under different physical and chemical conditions. Results from one freeze-thaw cycle indicated that the capsids degraded by a similar mechanism to that in acidified solution. The structural complexity revealed by CDMS highlights the advantages of this biophysical characterization method in providing, for the first time, a holistic insight into the potential heterogeneous conformational transitions of AAV9 during purification, storage, and the natural infection process.
A challenge in gene editing for hematopoietic stem and progenitor cells (HSPCs) is achieving efficient editing while preserving long-term engraftment and clonal diversity. Tracking edited clones with high resolution is essential to understand the impact of editing on hematopoiesis. We developed a barcoded AAV6 donor template (BC-AAV) to precisely monitor the fate of edited HSPCs following transplantation. Our findings reveal that, despite initial barcode diversity in vitro, human hematopoiesis generated by edited HSPCs transplanted in immunodeficient mice is driven by a limited number of dominant clones. The engraftment of gene-edited cells follows an oligo/polyclonal pattern, indicating that editing does not alter clonal dynamics in this model. Using BC-AAV, we optimized a gene editing protocol for correcting the PKLR gene, responsible for pyruvate kinase deficiency, a rare disorder that causes severe anemia due to red blood energy imbalance. We implemented key improvements. GMP-grade StemSpan AOF medium and StemRegenin-1 increased clonal diversity while maintaining hematopoietic potential. NHEJ inhibitor AZD-7648, significantly boosted editing efficiency in vitro, and a shorter transduction period enhanced engraftment and clonal balance without compromising editing outcomes. This refined strategy for gene editing in human HSPCs optimizes both efficiency and long-term polyclonal dynamics and has important implications for clinical applications.
A scalable, serotype-agnostic, and fully chromatographic downstream platform was developed for the purification of adeno-associated virus (AAV) based on affinity chromatography (AC), anion-exchange chromatography (AEX), and multi-modal polishing chromatography (MMC). Quantitative proteomic profiling across each purification stage was performed using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to characterize residual host cell protein (HCP) retention for four AAV serotypes (AAV2, -5, -8, and -9) produced with suspension HEK293 cells. The downstream process showed cumulative vector genome (VG) yields ranging from 44.6 to 69.2% with full capsid enrichments ranging from 2.62- to 5.93-fold across all AAV samples. A total of 880, 99, and 21 HCPs were identified in all AAV samples after AC, AEX, and MMC, respectively. Vector-mediated mechanisms of retention are proposed (i.e., capsid or genome association) based on enrichment in protein- and nucleic acid-binding properties of difficult-to-remove HCPs, little clearance across multiple modes of separation, and divergence of HCP profiles for AAV-containing samples vs. null lysate material. The characterization of HCP retention described here advances the understanding of process-related impurity clearance in scalable AAV downstream systems, with implications for both purification process design and AAV interactions with host cell factors.
Over the past ∼25 years, structural studies of adeno-associated virus (AAV) capsids have greatly aided our understanding of the biology of these single-stranded DNA viruses and provided insights into their utilization as gene therapy vectors for the treatment of various human diseases. Recent advances in cryo-electron microscopy have yielded a library of currently ∼150 high-resolution AAV capsid structures, with more than 50% determined in the last 5 years alone. Comparative analyses of capsids from primate and nonprimate origins have revealed both conserved architectural elements, such as the canonical jelly-roll fold, and critical surface variations that affect receptor interaction, antibody recognition, and intracellular trafficking. These structures, as well as those in complex with glycan and proteinaceous receptors, purification agents, and antibodies, have been instrumental in rational capsid engineering, guiding the design of new variants with enhanced transduction efficiency, tissue specificity, and reduced detection by pre-existing neutralizing antibodies. This review summarizes the available AAV capsid structures to date, highlighting landmark discoveries over the years, and offers perspectives on how structural biology will continue to drive innovation in AAV gene therapy.
Gene therapies based on adeno-associated virus vectors hold strong potential for the treatment of central nervous system disorders. However, systemic delivery is limited by the blood-brain barrier, off-target effects, immune responses, and vector loss. Direct intraparenchymal injections can bypass these barriers by targeting specific brain regions, but broader vector distribution is essential to reduce the need for multiple injections and to achieve widespread transgene expression. The brain biodistribution of adeno-associated virus serotype 5 is partially mediated by its interaction with sialic acid. Here, we describe a modified serotype 5 variant, AAV5neo, carrying a single amino acid substitution that alters its sialic acid-binding properties. In cultured cells, AAV5neo exhibits transduction that is independent of sialic acid and is not inhibited by N-acetylneuraminic acid, a form of sialic acid highly abundant in the brain. Following direct striatal injection in mice, minipigs, and non-human primates, AAV5neo consistently demonstrated enhanced transduction efficiency, broader distribution to cortical and deep brain regions, and achieved comparable transgene expression at approximately 10-fold lower doses relative to the parental serotype. These findings highlight AAV5neo as a potent and efficient vector candidate for localized gene therapy applications targeting the central nervous system.
Recombinant AAV (rAAV) vectors are a leading viral vector for gene therapy. Viral genome (Vg) titer is the primary method to determine potency of rAAV and dosing in preclinical/clinical studies. However, the rAAV genome comprises a heterogeneous population. These particles not only contain the intact genome but also include numerous truncated species, which likely lack functionality and may induce adverse effects. Consequently, the Vg titer does not accurately reflect the integrity of the rAAV genome. Currently, there is no reliable quantitative method available. In this study, we demonstrate that there is a disconnect between Vg titer and the activity of rAAV by using multiple vectors and high-throughput imaging assays. Importantly, we have developed a novel, high-throughput RNA-DNA hybrid capture-multiplex meso scale discovery (MSD) method for characterizing the integrity of the rAAV genome. This method quantifies the intact versus truncated genomes of both the plus and minus strands individually with high sensitivity and specificity. The integrity data generated by our novel method exhibits a strong correlation with the activity of the rAAV. We anticipate that our new method will significantly improve preclinical/clinical studies, enhance vector design, and increase delivery efficiency. Furthermore, this method can be used to characterize and quantitate RNA and DNA in various fields.
Adoptive T cell therapy, particularly T cell receptor-engineered T (TCR-T) cell therapy, holds promise for cancer treatment in solid tumors and hematological malignancies. Conventional lentiviral TCR-T cell therapies face insertional mutagenesis risks, while CRISPR-mediated T cell receptor α constant (TRAC) locus targeting suffers from suboptimal knockin efficiency and chromosome 14 loss. To address these challenges, we introduced a non-viral strategy combining CRISPR-Cas9 electroporation with methotrexate (MTX) metabolic selection. Primary human T cells were engineered to integrate a CMV-pp65-specific TCR and an MTX-resistant dihydrofolate reductase (DHFR)-FS cassette into the TRAC locus via homology-directed repair (HDR). Systematic optimization of electroporation timing, buffer systems, and HDR enhancers achieved initial TCR integration efficiency of ∼20%. Subsequent 6-day MTX treatment enriched engineered cells to ∼70% purity while selectively depleting unedited and chromosomally aberrant clones. Fluorescence in situ hybridization revealed that MTX enrichment reduced CRISPR-associated chromosome 14 loss comparable to unedited T cells. Functionally, TRAC-TCR-T cells exhibited enhanced interferon (IFN)-γ/tumor necrosis factor alpha (TNF-α) secretion and reduced exhaustion markers versus lentiviral counterparts, while maintaining equivalent tumor clearance efficacy in vitro and in xenograft models. In conclusion, this integrated platform mitigates viral vector risks, alleviates concerns of CRISPR-associated genomic instability, and provides a good manufacturing practice (GMP)-compatible approach that may facilitate the development of safer adoptive TCR-T immunotherapies.
