Molecular Therapy-Methods & Clinical Development最新文献

We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) with a quantitative variant allele frequency (VAF) down to 5% lower limit of detection at >300× genome coverage. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggest that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.

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.

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.

Administration of adeno-associated virus (AAV) gene therapies via blood or cerebrospinal fluid (CSF) in non-human primates (NHPs) can lead to degeneration of dorsal root ganglion (DRG) neurons and nerve fibers in the spinal cord and peripheral nerves. AAV cargo expression is implicated in AAV DRG toxicity, but the underlying mechanism(s) is unknown. Here, we performed a time course study of intra-cisterna magna (ICM) administration of an AAV9 variant encoding human survival of motor neuron 1 (hSMN1) to identify molecular and cellular changes preceding pathology. Increases in inflammatory gene modules, cerebrospinal fluid (CSF) cytokines, and immune cell infiltrates as early as day 5 prior to neuron and nerve fiber degeneration on days 15 and 29 suggested a role for the immune response in AAV-mediated toxicity. Prophylactic treatment with a glucocorticoid steroid dexamethasone and a calcineurin inhibitor tacrolimus diminished pathology in NHPs following administration of three AAV gene therapy vectors. Collectively, these data demonstrate a causal role for the immune response to AAV in AAV-mediated DRG and nerve fiber toxicity. The efficacy of immunosuppression with three different AAV cargos suggests broad utility across AAV vectors and provides a clinically feasible approach to mitigating this potential toxicity in patients.

Current therapeutic strategies for non-Hodgkin's lymphomas (NHLs) and chronic lymphocytic leukemia (CLL) are limited by systemic toxicity, acquired resistance, and tumor-mediated immune suppression. To address these challenges, we introduce a novel nano-liposomal (NP) delivery system that exploits the overexpressed, open-active conformation of lymphocyte function-associated antigen-1 (LFA-1) on malignant hematopoietic cells. This approach facilitates the selective delivery of Wiskott-Aldrich syndrome (WAS)-specific short interfering RNA (siRNA) to lymphoma and leukemia cells. Compared to conventional therapies, our LFA-1-targeted NP system provides a significant advantage by enhancing cell-specific delivery, thereby minimizing off-target effects and potentially reducing systemic toxicity. LFA-1-targeted nanoparticles facilitated the precise delivery of WAS siRNA, resulting in potent inhibition of cytoskeletal-mediated oncogenesis. In vitro assays using patient-derived NHL and CLL cells confirmed significant reductions in proliferation, migration, and invasive capacity. These findings were further validated in vivo, where the targeted delivery system effectively suppressed tumor growth in a human B-NHL xenograft model. This targeted delivery strategy offers a precision approach to gene silencing in hematologic malignancies, potentially improving therapeutic efficacy and reducing adverse effects compared to current non-selective treatments for hematological malignancies, including B-NHL and CLL.

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing therapy optimization. We assessed the potential of retinal organoids for optimizing CRISPR-Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant retinitis pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR-Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual adeno-associated virus (AAV) systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases.

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.

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.

Adeno-associated virus (AAV)-based gene therapies are emerging as transformative treatments for serious diseases; however, determining the optimal duration of nonclinical toxicity studies remains a key regulatory and scientific question. To address this, the EFPIA Gene Therapy Working Group surveyed 24 AAV gene therapy programs across 13 companies to assess current practices and the value of long-term (≥6 months) toxicity studies. Results showed that ≤3-month studies were sufficient to characterize the toxicology profile in 87.5% of programs that completed a toxicity assessment in a ≥6-month long-term chronic studies, with only one program identifying new toxicities in longer chronic studies with impact on clinical development. Common AAV-related toxicities, such as liver and dorsal root ganglia effects, were observed within the first 6 weeks post-administration. Longer studies were often driven by sponsor's perception based on internal experience or need to assess durability, rather than regulatory requirements. These findings aligned with regulatory reviews of approved AAV products (e.g., Zolgensma, Luxturna, Roctavian) that consistently demonstrated the adequacy of ≤3-month studies for approved and marketed products. The outcome of this survey supports a risk-based, science-driven approach to in vivo study duration, emphasizing that shorter-term studies are generally sufficient for identifying relevant toxicities associated with AAV-based gene therapies. Embracing this approach can reduce animal use, accelerate development timelines, and support harmonized regulatory expectations for AAV gene therapy products.

[This corrects the article DOI: 10.1016/j.omtm.2025.101607.].