Oncolytic viruses are being investigated as therapeutic agents in cancer, yet their mechanisms of entry into tumor cells remain incompletely understood. We previously showed that SG33, a veterinary vaccinal strain derive...Oncolytic viruses are being investigated as therapeutic agents in cancer, yet their mechanisms of entry into tumor cells remain incompletely understood. We previously showed that SG33, a veterinary vaccinal strain derived from a pathogenic myxoma virus, displays oncolytic activity in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the entry pathways of SG33 into primary PDAC-derived cultures. We found that macropinocytosis, an endocytic process frequently upregulated in PDAC, contributes to SG33 uptake. Moreover, SG33 infection itself induced macropinocytosis in a subset of primary PDAC cultures. Mechanistic studies revealed that phosphatidylserine exposed on the viral envelope promotes SG33 internalization through apoptotic mimicry. In PDAC cultures lacking detectable macropinocytosis, SG33 employed clathrin-mediated endocytosis as an alternative entry route. These findings provide the first insights into the entry mechanisms of SG33 into PDAC-derived cells and indicate that this virus can utilize distinct endocytic pathways depending on the cellular context.
Adeno-associated virus (AAV) is widely regarded as a leading vector for gene therapy, underscored by clinical successes such as Luxturna and Zolgensma. However, efficient gene delivery to hard-to-transduce tissues-includ...Adeno-associated virus (AAV) is widely regarded as a leading vector for gene therapy, underscored by clinical successes such as Luxturna and Zolgensma. However, efficient gene delivery to hard-to-transduce tissues-including the retina, deep skeletal muscle, and the central nervous system-remains a significant challenge, limited by structural barriers, preexisting immunity, and dose-dependent toxicities. This review systematically outlines recent advances in overcoming these delivery bottlenecks. We delve into four key strategic areas: (i) capsid engineering (, rational design, directed evolution, and computational approaches) to enhance tropism and evade immune detection; (ii) innovative delivery routes (local, systemic, and physical/chemical methods) to improve vector bioavailability; (iii) modulation of intracellular trafficking to boost nuclear delivery; and (iv) immunomodulatory strategies to mitigate both innate and adaptive immune responses. We further highlight translational progress in neuromuscular and retinal diseases and discuss persistent challenges. Looking forward, we envision that the convergence of next-generation capsids, smart vector systems, and integrated delivery platforms will be critical to expand the therapeutic landscape of AAVs from rare monogenic disorders to broader clinical applications.
Type 1 regulatory T (Tr1) cells are CD4/FoxP3/CD49b/LAG-3 regulatory T cells that are induced in the periphery following chronic exposure to antigen in the presence of IL-10. Activated Tr1 cells secrete high quantities o...Type 1 regulatory T (Tr1) cells are CD4/FoxP3/CD49b/LAG-3 regulatory T cells that are induced in the periphery following chronic exposure to antigen in the presence of IL-10. Activated Tr1 cells secrete high quantities of anti-inflammatory cytokines, especially IL-10, and enforce robust bystander immune suppression, rendering them particularly attractive for adoptive cell therapy of autoimmune and autoinflammatory diseases. Here we introduce retroviral transduction of human CD4 T cells with membrane-attached IL-10 (memIL-10) or memIL-10/CAR as a new method for generating large cell populations of high purity, stability, and antigen specificity displaying the main phenotypic and functional characteristics of Tr1 cells.
Recombinant adeno-associated virus (rAAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, sev...Recombinant adeno-associated virus (rAAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, several DNA viruses are known to remodel host chromatin accessibility, for example, Herpes simplex virus type 1 (HSV-1) in mammalian cells and baculoviruses in insect systems. Here, we evaluated genome accessibility and nuclear organization of primary human fibroblast cells at 24 and 48 h after infection with wild-type AAV2, single-stranded (ss) and self-complementary rAAV2, as well as ssAAV2 and ssAAV-DJ in human lung carcinoma cells. Genome-wide assay for transposase-accessible chromatin using sequencing showed no detectable change in host chromatin accessibility at either time point. A DNase I digestion assay at five candidate loci supported this observation. Although host accessibility remained stable, immunofluorescence-based single-cell analyses uncovered modest changes in histone abundance, RNA polymerase II-associated signals, nuclear morphology, and the organization of liquid-liquid phase-separated nuclear compartments. These effects were generally mild in primary fibroblasts but showed greater dependence on vector type, cell cycle state, and transgene expression in carcinoma cells. Collectively, at the tested doses and times, AAV-based vectors did not remodel chromatin accessibility at scale and induced only small changes in polymerase-associated readouts and nuclear architecture. These data are consistent with limited nuclear perturbation under these conditions.
Developing gene therapies involving gene editing is a rapidly evolving field with large potential implications for improving health for both rare and common diseases. Ensuring that these technologies are developed safely...Developing gene therapies involving gene editing is a rapidly evolving field with large potential implications for improving health for both rare and common diseases. Ensuring that these technologies are developed safely, efficiently, and fairly is essential. To better understand the ethical considerations and regulatory requirements and challenges with gene therapies involving gene editing that may advance precision health. Through a multistakeholder workshop and subsequent engagements, multiple ethical and regulatory barriers to developing and implementing gene therapies involving gene editing were identified. Eight major themes emerged that warrant careful consideration, including (1) objectives (treatment, risk reduction, and enhancement) for the intervention; (2) competing interests of safety, equity, and desire for research efficiencies; (3) unique aspects of gene editing related to rare and ultrarare genetic conditions; (4) considerations in the pediatric population; (5) regulatory requirements and ethics oversight; (6) challenges with long-term follow-up and data sharing; and (7) communication. To promote the safe, efficient development of gene therapies involving gene editing that will reach their full potential, all stakeholders will have to undertake an unprecedented degree of collaboration. However, this will be essential to ensure that these interventions are effective, ethically sound, and patient-centered.
Niemann-Pick disease type C1 (NPC1) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants of the gene that encodes a protein essential for lysosomal cholesterol transport. A deficiency in NP...Niemann-Pick disease type C1 (NPC1) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants of the gene that encodes a protein essential for lysosomal cholesterol transport. A deficiency in NPC1 results in the accumulation of unesterified cholesterol and sphingolipids, leading to neurological, psychiatric, and hepatic manifestations from infancy to adulthood. The currently approved treatment is palliative. Although the efficacy of gene therapy has been demonstrated in murine models, reliable biomarkers for evaluating the treatment effects remain unknown. We evaluated adeno-associated virus (AAV) vector-mediated NPC1 gene therapy in homo-knockout () mice, focusing on blood-based biomarkers. An AAV vector carrying human under a cytomegalovirus promoter (AAV-) was administered intraperitoneally on days 6-8 after birth at varying vector doses and analyzed at multiple time points: 1.8 × 10 vector genomes/mouse analyzed at 7 weeks (Low/7w) and 1.0 × 10 vector genomes/mouse at 4 weeks (High/4w) and 9 weeks (High/9w). is expressed in the brain and liver, and a degree of neuronal cell survival is observed. High-dose AAV treatment improves body weight and rotarod performance. Plasma -palmitoyl--phosphocholine-serine (PPCS) and lysosphingomyelin (lyso-SM) levels were significantly elevated in mice. PPCS increased with disease progression but was significantly decreased after later points of high-dose AAV treatment (saline-treated mice: 12.88 ± 3.53 ng/mL, AAV-treated mice: 7.87 ± 1.67 ng/mL, = 0.0008). Lyso-SM and oxysterols showed limited changes after therapy. Vector genome analysis revealed higher and more sustained levels in the brain than in the liver, which is consistent with rapid hepatocyte proliferation-reducing vector persistence. These findings demonstrate that systemic AAV- therapy ameliorates motor and neurological deficits but has a limited impact on several cholesterol-related biomarkers. PPCS has been suggested as a sensitive biomarker of therapeutic response and warrants further evaluations in preclinical and clinical NPC1 gene therapy trials.
Mucopolysaccharidosis type I-Hurler (MPS I-H) is a severe lysosomal storage disorder caused by α-L-iduronidase (IDUA) deficiency, leading to glycosaminoglycan (GAG) accumulation and progressive multisystem dysfunction, i...Mucopolysaccharidosis type I-Hurler (MPS I-H) is a severe lysosomal storage disorder caused by α-L-iduronidase (IDUA) deficiency, leading to glycosaminoglycan (GAG) accumulation and progressive multisystem dysfunction, including the central nervous system (CNS). Early hematopoietic stem cell transplantation remains the primary disease-modifying intervention for MPS I-H despite its clinical challenges. Conventional enzyme replacement therapy (ERT), which has limited CNS efficacy due to blood-brain barrier (BBB) restrictions, is being addressed by the development of novel BBB-penetrating ERT platforms. Recently, mRNA therapy has become a promising treatment option for rare genetic diseases by enabling the supplementation of defective or deficient proteins to ameliorate the disease phenotype. To evaluate mRNA therapy for MPS I-H, we screened ionizable lipids to develop lipid nanoparticle systems optimized for hepatic delivery, which were subsequently used to encapsulate mRNA encoding human IDUA (hIDUA) fused to a validated BBB-penetrating melanotransferrin peptide (MTfp). This formulation was intravenously administered to neonatal MPS I-H mice at a dose of 1.0 mg/kg every 2 weeks for 4 months. The treatment significantly increased IDUA enzyme activity in both the serum and liver. Concurrently, it reduced GAG accumulation in the urine, peripheral tissues, and partial CNS regions (olfactory bulbs, hippocampus, and cerebellum). Furthermore, the treatment significantly improved cardiac and skeletal development, as assessed by echocardiography and micro-computed tomography, respectively. Notably, it also enhanced cognitive function, as evidenced by the improved performance in the Delayed-Matching-to-Place dry maze test. Our findings demonstrate that LNP-MTfp-hIDUA treatment effectively ameliorates key pathological features and promotes neurodevelopment in MPS I-H, establishing a promising and safe therapeutic strategy for this disorder.
Adeno-associated virus (AAV) vectors are promising platforms used in a growing number of approved gene therapy (GT) products across diverse therapeutic areas. Due to the potential safety and efficacy concerns associated...Adeno-associated virus (AAV) vectors are promising platforms used in a growing number of approved gene therapy (GT) products across diverse therapeutic areas. Due to the potential safety and efficacy concerns associated with AAV-based immune responses, patients with pre-existing anti-AAV antibodies (Abs) are routinely excluded from GT trials to prevent treatment of patients who are hypothesized to have potentially higher risk and/or little or no benefit. However, the exclusion of seropositive patients without prior GT exposure is not based on data-driven Ab titer cut-offs, and diagnostics to identify levels of pre-existing immunity have not been standardized, precluding data generation that would substantiate or reject this hypothesis. There are also significant gaps in clinical data comparing the impact of pre-existing immunity with treatment-induced immune responses for a variety of disease states. This review aims to address these gaps by examining the impact of pre-existing anti-AAV Abs on the safety and efficacy of approved and failed GT products and ongoing clinical trials. Together, these data suggest that pre-existing immunity may not be the principal determinant of GT success. Therefore, to expand the number of patients eligible for treatment, novel AAV GTs should be optimized to mitigate against the effects of anti-AAV Abs and avoid the need for exclusionary screening; in turn, including both seronegative and seropositive patients in clinical trials will enable characterization of the clinical relevance of anti-AAV Abs. Strategies to prevent an undesirable immune response to GT, including immunosuppressive regimens and modifications to GT manufacturing, design and delivery, are presented for consideration in future GT trials.
Lentiviral vectors are widely used in both biological research and therapeutic applications. Accurate and reliable quantification is essential for their effective use, yet existing methods are often technically demanding...Lentiviral vectors are widely used in both biological research and therapeutic applications. Accurate and reliable quantification is essential for their effective use, yet existing methods are often technically demanding and time-consuming. Here, we describe a simple and robust approach to measure lentiviral titers by quantifying viral genomic RNA with nucleic acid-binding dyes. We first evaluated several newly developed commercially available fluorescent dyes for their ability to detect RNA and DNA, identifying SYBR Green II and SYBR Gold as the most sensitive for RNA, with detection limits in the low-nanogram range. We further observed that SYBR Green II readily stained nucleic acids in live cells, whereas SYBR Gold was only partially membrane-permeable. Based on these findings, we developed a quantification method combining SYBR Green II with benzonase to selectively detect encapsidated lentiviral RNA. This assay yielded results consistent with those obtained by enzyme-linked immunosorbent assays. Importantly, this SYBR Green II-based method is rapid (<60 min), reliable, and cost-effective, making it a practical tool for titering lentiviral vectors.
Rahmanian M, Khoshandam M, Mousazadeh M
… +7 more, Yang P, Soltaninejad H, Karami Dehkordi P, Sadeghizadeh M, Hedayati Goudarzi MT, Azimi AH, Sheykhhasan M
Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9 (CRISPR/Cas9) technology has become a revolutionary tool in medicine, offering substantial potential for treating a wide range of dise...Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9 (CRISPR/Cas9) technology has become a revolutionary tool in medicine, offering substantial potential for treating a wide range of diseases, including hematological disorders, cancers, genetic conditions, and ophthalmological diseases. This systematic review evaluates the efficacy, safety, and applicability of CRISPR/Cas9 in clinical trials. A comprehensive search of the PubMed, Scopus, Web of Science, and Cochrane databases was conducted. All studies, up to November 2024, meeting the eligibility criteria assessing the application of CRISPR for the treatment of diseases were included. A quality assessment of the included studies was conducted using the Cochrane risk of bias tool. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement for systematic reviews and meta-analyses was followed, and a total of 17 studies were included. This systematic review of CRISPR/Cas9 technology focused on its effectiveness and safety across various diseases. In nonmalignant hematological disorders, CRISPR successfully treated β-thalassemia and sickle cell disease, resulting in high transfusion independence and the elimination of disease crises. In malignant hematological disorders, B-cell acute lymphoblastic leukemia, CRISPR-engineered chimeric antigen receptor T (CAR-T) cells achieved an 83.3% complete remission rate. Furthermore, CRISPR-based CAR-T cells showed promising results in B-cell non-Hodgkin's lymphoma. In oncology, lung cancer and other solid tumors are among the diseases that have been safely engineered using CRISPR gene editing technology. For genetic disorders, CRISPR improved vision in retinal degeneration and reduced symptoms in hereditary angioedema and transthyretin amyloidosis with mild side effects. The results demonstrated CRISPR's potential across a wide range of conditions. In conclusion, the findings underscore the potential role of CRISPR/Cas9 technology across a wide range of diseases. However, challenges remain, including optimizing delivery systems, minimizing off-target effects, addressing immunogenicity concerns, and ethical considerations.
Recombinant adeno-associated virus (rAAV) vectors have demonstrated their versatile utility for gene therapy to deliver successful treatment for several diseases of unmet medical needs. The polytropic nature of AAV perm...Recombinant adeno-associated virus (rAAV) vectors have demonstrated their versatile utility for gene therapy to deliver successful treatment for several diseases of unmet medical needs. The polytropic nature of AAV permitted targeting diverse tissue types to support various diseases; however, it also resulted in vector transduction of non-target organs, including the gonads of males and females, which subsequently confer an additional investigation to derisk any potential germ cell transduction and/or germline transmission following rAAV administration. While a plethora of data is available for male gonad assessment, primarily due to the feasibility of longitudinal semen collection, there is a paucity of data on oocyte transduction by rAAV due to challenges with oocyte collection. In this brief report, we share polymerase chain reaction and hybridization data on oocyte transduction in a female cynomolgus macaque 4 weeks following systemic intravenous administration of an engineered rAAV and discuss the significance and potential mitigation strategies of such findings in the process of AAV-based gene therapy product development.
The adeno-associated virus (AAV) capsid variant known as "true type" (AAV-TT) is a capsid with enhanced central nervous system tropism and efficient retrograde transport across brain circuits. This property enables AAV-T...The adeno-associated virus (AAV) capsid variant known as "true type" (AAV-TT) is a capsid with enhanced central nervous system tropism and efficient retrograde transport across brain circuits. This property enables AAV-TT to transduce neurons in multiple regions projecting to the site of injection, as previously shown in rodent and sheep models. Such spreading offers the potential for broad therapeutic vector distribution to functionally connected brain areas via a single intraparenchymal administration. To assess the clinical potential of AAV-TT, we delivered a green fluorescent protein (GFP)-expressing AAV-TT vector into the left commissural putamen of two young adult . For comparison, two additional animals received AAV9-GFP. Four weeks post-injection, animals were euthanized, and whole-brain transduction patterns were analyzed using a dedicated Aiforia deep learning-based image analysis algorithm to quantify GFP+ neurons. We observed GFP expression in structures known to innervate the injection site, consistent with retrograde transport. Both AAV-TT and AAV9 showed similar overall distribution patterns, but AAV-TT demonstrated modestly enhanced neuronal transduction efficiency compared with AAV9. This study provides the first direct comparison of AAV-TT and AAV9 in the adult nonhuman primate brain and positions AAV-TT as a promising tool for disease modeling and therapeutic interventions in neurodegenerative disorders targeting anatomically distributed neural circuits.
Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective don...Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective donor template for homology-directed repair because they can avoid integrating into the host genome. However, only a few AAV serotypes can efficiently transduce human primary T cells at low multiplicities of infection (MOIs) with high packaging efficiency. To address this problem, variants derived from an AAV2 peptide library were screened in Jurkat cells and later validated in primary T cells. A high-ranking sequence identified outside the VR-VIII region, NNSKLTV, was discovered after three rounds of selection and was named Tot3. Tot3 demonstrated transduction efficiency similar to AAV2, but at a 27-fold lower MOI. In addition, Tot3 exhibited greater packaging efficiency and reduced thermal stability. Simultaneously, programmed cell death protein 1 (PD-1) knockout and CAR overexpression were achieved in human primary T cells using Tot3, with knockout and knock-in efficiencies reaching up to 70% and 55%, respectively. These CAR-T cells demonstrated significantly enhanced antitumor activity and increased survival times in a mouse model of diffuse B cell lymphoma.
Mutations in the () gene are one of the leading causes of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size o...Mutations in the () gene are one of the leading causes of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the coding sequence (∼9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-directional and Site-specific Transgene Assembly by Recombination (Uni-STAR). The system consists of three AAV constructs carrying discrete segments flanked by engineered, noncompatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. , subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete gene and provide a foundation for future therapeutic development targeting -associated retinal degenerations.
The recent evolution of oncolytic virotherapy has yielded viral platforms with enhanced tumor tropism and expanded engineering flexibility, thereby enabling not only direct oncolysis but also deliberate promotion of anti...The recent evolution of oncolytic virotherapy has yielded viral platforms with enhanced tumor tropism and expanded engineering flexibility, thereby enabling not only direct oncolysis but also deliberate promotion of antitumor immune responses. Here, we systematically explore multiple insertion sites for calreticulin (CALR) within an oncolytic adenovirus, identifying the most optimal variant that exposes robust CALR on tumor cell membrane and functionally motivates macrophages in addition to directly mediating tumor cell lysis. Mechanistically, this variant incorporates a precise deletion within the domain (920-946 bp), enabling selective replication in retinoblastoma-deficient tumor cells. Furthermore, the region has been replaced with an exogenous Cytomegalovirus (CMV) promoter to achieve precise regulation of CALR overexpression and diminish systemic toxicity. Functionally, by bulk-RNA-Seq, we demonstrate that oncolytic adenovirus (oAd)-CMV-CALR transfection induces endoplasmic reticulum stress, as evidenced by upregulation of phosphorylated eIF2α, which facilitates the translocation of CALR to the plasma membrane. Via coculture assays, we validate that oAd-CMV-CALR transfection enhances the phagocytic capacity of M2 macrophages and promotes their repolarization toward an M1-like phenotype. These findings are further validated in patient-derived ovarian cancer spheroids, underscoring the translational potential of our approach. , oAd-CMV-CALR suppresses Hepa1-6 xenograft growth, boosts CD8 T-cell infiltration, and exhibits favorable safety. Collectively, our findings highlight oAd-CMV-CALR as a potential therapeutic approach to modulate the tumor microenvironment and improve cancer immunotherapy outcomes.
Shieh PB, Proud C, Diamond T
… +17 more, Chapin CA, Ahmad J, Salama AD, Bönnemann CG, Soslow J, Byrne BJ, Veerapandiyan A, Brandsema JF, Matesanz S, Samelson-Jones BJ, Wilkins BJ, Gerber M, Godwin Wild KE, Mason S, Asher D, McDonald CM, Mendell JR
Hum Gene Ther
· 2026 Mar · PMID 41681118
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Acute liver injury (ALI) is a recognized adverse event with adeno-associated virus (AAV)-based gene therapies, including delandistrogene moxeparvovec, an AAVrh74-based gene therapy for Duchenne muscular dystrophy. Progre...Acute liver injury (ALI) is a recognized adverse event with adeno-associated virus (AAV)-based gene therapies, including delandistrogene moxeparvovec, an AAVrh74-based gene therapy for Duchenne muscular dystrophy. Progression of ALI to acute liver failure (ALF) is rare. In clinical trials, ALF was defined as an international normalized ratio ≥1.5 with encephalopathy and/or ascites, occurring <26 weeks since product/treatment exposure, and in the absence of identified preexisting liver disease as assessed by the treating investigator. The two cases presented here represent the only known instances of ALF following delandistrogene moxeparvovec to date, both resulting in fatal outcomes. Both cases occurred in nonambulatory patients (ages 15 and 16 years). Both patients exhibited abrupt elevations in aminotransferases from baseline approximately 4 weeks after delandistrogene moxeparvovec administration. Over the subsequent weeks and despite interventions, aminotransferases, including gamma-glutamyl transferase (GGT), declined concurrently with a continued rise in total bilirubin, consistent with severe hepatocellular injury and a harbinger of ALF. An interdisciplinary expert panel was convened and concluded that no single biomarker or clinical feature consistently predicted the rapid progression of severe ALI to ALF. Advisors recommended basing treatment intervention for severe ALI on the rate and magnitude of changes from baseline in liver biomarkers ( 2-3× increase in alanine aminotransferase within ≤1 week). For these severe cases, most advisors recommended intravenous methylprednisolone as the initial treatment whereas typical ALI may be managed with oral corticosteroid adjustment or may resolve with time, as seen in the clinical trial experience. Advisors hypothesized that delandistrogene moxeparvovec-related ALI is T-cell-mediated and discussed the potential of additional immunosuppression. Advisors also recommended enhancing baseline evaluation for hepatic comorbidities with lipid profiling, transient elastography, and abdominal ultrasound to further study potential risk factors for ALI progressing to ALF. Finally, there was strong support for generating additional real-world evidence and conducting prospective clinical trials to inform clinical management of ALI in practice.