Search Results (269)

RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides—eteplirsen, golodirsen, viltolarsen, and casimersen—have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies—including base and prime editing—offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships. Full article

Adeno-associated viral (AAV) vectors have established themselves as a promising platform for genetic material delivery in clinical practice, evidenced by regulatory approval of multiple therapeutics. Despite proven therapeutic efficacy, safety concerns remain a critical limitation requiring systematic analysis. This review analyzes clinical data to identify mechanisms of toxicity, clinical risks, and strategies for their minimization in AAV gene therapy. The study examines dose-dependent toxicity, immune responses, and organ-specific burdens associated with systemic and local administration routes. Analysis reveals a clear correlation between systemic delivery efficacy and dose-dependent toxicity, with principal mechanisms including capsid-directed immune responses, hepatic burden, and complement system activation leading to thrombotic microangiopathy. Key determinants of the safety profile include pre-existing neutralizing antibodies, vector dose, serotype selection, and patient baseline conditions. Contemporary strategies for toxicity minimization are evolving from reactive management toward proactive risk mitigation, including prophylactic immunosuppressive regimens, vector engineering to alter tropism or reduce immunogenicity, and rigorous post-infusion monitoring. Integration of improved vector constructs, rational immunosuppressive regimens, and rigorous post-infusion surveillance has the potential to expand the therapeutic window of AAV-based gene therapy, achieving an optimal balance between efficacy and safety for a broader patient population. Full article

Background/Objectives: Recombinant adeno-associated virus (AAV) stands at the forefront of gene therapy development, requiring stable formulations to support the expanding therapeutic applications. The growing diversity of serotypes and engineered capsids often creates complex challenges for formulation development, thus demanding innovative formulation development strategies beyond traditional manual approaches to characterize a large formulation design space quickly to discover stable formulations. Methods: Here, we address this critical need through a high-throughput automation platform that dramatically enhances formulation development efficiency and capability through rapid formulation preparation and high-throughput AAV analytics. This system prepares 96 distinct formulations in 40 min and completes AAV compounding in 20 min per plate, with precise control of pH, buffer components, and AAV titers. Results: In a proof-of-concept formulation development study using AAV1, we screened 128 formulations across multiple buffer systems, pH ranges, and excipient combinations. This comprehensive approach successfully identified optimal stable high-titer AAV1 formulations (1.2 × 10¹⁴ vector genome (vg)/mL) that maintained stability under frozen, refrigerated, and room temperature storage conditions. Conclusions: Our study demonstrated that this automation platform combined with high-throughput AAV analytics significantly accelerates formulation development, conserves AAV material, and enables systematic exploration of broader formulation design space. It allows us to achieve identification of robust and stable AAV formulations within a timeframe unmatched by traditional formulation development approaches. Full article

Recent advances in gene therapy have highlighted the potential of CRISPR-based gene-editing systems combined with adeno-associated virus (AAV) vectors. However, the limited packaging capacity of AAV remains a significant challenge for the simultaneous expression of Cas effector proteins and guide RNAs within a single vector. To address this limitation, we developed a compact AAV vector that enables the co-expression of Acidaminococcus sp. Cas12a (AsCpf1) and CRISPR RNAs (crRNAs) using a single bidirectional promoter derived from the mouse H1 promoter. Our single bidirectional H1 promoter supported indel formation comparable to that achieved by dual-promoter systems and facilitated scalable genome editing with single-, dual-, and triple-target configurations. Genome editing was successfully accomplished both in vitro and in vivo following AAV delivery. This study shows that our engineered compact AAV vector platform is capable of simultaneously delivering AsCpf1 and multiplexed crRNAs. Full article

Gene therapy is reshaping the therapeutic paradigm in hemophilia by enabling sustained endogenous clotting factor production after a single administration. This approach moves disease management beyond lifelong replacement therapy. While clinical trials have demonstrated marked reductions in bleeding rates and treatment burden, real-world implementation has revealed emerging complexities. These include interindividual variability in transgene expression reflected by a progressive reduction in circulating FVIII or FIX activity over time, uncertainty regarding the long-term durability of expression, immune-mediated constraints, and episodes of transaminase elevation. This review addresses a critical transition point in the field: the shift from proof-of-concept efficacy toward integration of gene therapy into long-term hemophilia care. We examine determinants of therapeutic stability, host–vector immune interactions, and mechanisms underlying loss or fluctuation of expression, with emphasis on monitoring strategies and post-therapy management pathways. Immunogenic processes affecting vector transduction, hepatocellular responses, and transgene persistence are discussed alongside current approaches to immune modulation. This review uniquely focuses on post-gene therapy clinical integration rather than vector design or trial outcomes. Beyond direct factor correction, evolving therapeutic concepts targeting coagulation rebalancing and immune regulation are considered within a systems-based framework. Psychosocial adaptation and patient-reported outcomes are also explored, underscoring that therapeutic success extends beyond hemostatic control. In aggregate, these perspectives position gene therapy not as a singular curative event but as a component of an evolving, biologically integrated management strategy. Long-term follow-up translational research (LTFU) and coordinated global efforts will be essential to optimize durability, safety, and equitable access. Full article

Pompe disease (PD) is a neuromuscular autosomal recessive disorder caused by mutation in the GAA gene, which encodes acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing glycogen to glucose. Deficiency of this enzyme leads to pathological accumulation of glycogen in almost all tissues of the body, with the most pronounced effects in cardiac and skeletal muscle, as well as in the central nervous system. Two major clinical forms of PD are recognized: infantile-onset PD, characterized by almost complete absence of GAA activity and severe cardiomyopathy and neurological abnormalities, and late-onset PD, which primarily presents with impairment of respiratory and motor function. Since 2006, enzyme replacement therapy with recombinant GAA has been used to treat PD, improving survival and quality of life. However, this approach has several limitations: the need for lifelong infusions, the risk of immune responses, and the inability of the enzyme to cross the blood–brain barrier, which is particularly critical for infantile-onset PD. Consequently, alternative strategies are being developed, including gene therapy using adeno-associated virus vectors for GAA delivery to target tissues; these approaches are currently in phase I/II clinical trials. Transplantation of genetically modified hematopoietic stem cells also represents a promising therapeutic strategy, offering a single-intervention treatment with long-lasting effects. This review discusses the molecular mechanisms of PD, current and emerging disease models, and therapeutic approaches, which together open prospects for the development of potentially one-time curative treatments, despite persistent challenges such as immunogenicity and the need for long-term efficacy monitoring. Full article

Gene therapy using recombinant adeno-associated virus (rAAV) vectors has emerged as a transformative therapeutic modality for genetic disorders, demonstrating high transduction efficiency and a generally favorable safety profile during pre-clinical development. However, serious adverse events, including thrombotic microangiopathy, acute respiratory distress syndrome, hepatotoxicity, myocarditis, cytokine storm, and hemophagocytic lymphohistiocytosis, have been observed across multiple gene therapy clinical trials. Significant efforts have been made to understand the toxicities that cause these adverse events and clinical care for patients receiving gene therapies has evolved to mitigate their effects. These toxicities arise from a complex interplay between the innate and adaptive immune responses directed against the viral capsid and transgene products and are often compounded by pre-existing anti-AAV immunity. Immunomodulatory strategies have been developed to combat these responses to improve the long-term success of gene therapies, and this review provides clinicians managing gene therapy patients with an overview of mechanisms underlying AAV-associated immunotoxicities and a discussion of syndromes and mitigation strategies that have been reported in the clinical care of patients. Full article

Vascular endothelial growth factor (VEGF)-driven pathological angiogenesis constitutes a primary driver of neovascular diseases, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). Although anti-VEGF agents demonstrate clinical efficacy, their limited intraocular half-life mandates repeated intravitreal injections, thereby highlighting the imperative for long-term therapeutic strategies. In the present study, we assessed the anti-angiogenic potential of retinal organoid-derived microRNAs (miRNA) delivered via an engineered adeno-associated virus vector. Human umbilical vein endothelial cells (HUVEC) were transduced with AAV2.7m8 vectors to overexpress three candidate miRNA (miR-26a, miR-122, and let-7a), followed by VEGF stimulation to evaluate downstream signaling pathways and angiogenic responses. AAV2.7m8-mediated transduction of HUVEC demonstrated high efficiency without inducing detectable cytotoxicity. Overexpression of these miRNA markedly attenuated VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Functional assays demonstrated suppression of endothelial cell proliferation and cell cycle progression, with miR-122-5p additionally inhibiting migration. All three miRNA substantially inhibited capillary-like tube formation. In aggregate, these results affirm that AAV2.7m8-mediated delivery of retinal organoid-derived miRNA —namely miR-26a-5p, miR-122-5p, and let-7a-5p—markedly suppresses VEGF-induced angiogenic signaling cascades and endothelial cell activation in vitro, thereby establishing their viability as a sustained therapeutic approach for pathological retinal neovascularization. Full article

Studies have shown that IL-10 has therapeutic potential for inflammatory diseases. However, it is challenging to use IL-10 as a therapeutic drug because it also possesses pro-inflammatory functions. To reduce these pro-inflammatory effects of IL-10, we have designed three IL-10 mutants using structure-based computational design technology. We demonstrated that these mutants exhibited significantly lower activity in IL-10-responsive cell lines than wild-type IL-10. Using recombinant adeno-associated virus (rAAV8) vectors expressing wild-type or mutant IL-10 molecules, we performed gene therapy experiments in IL-10 KO mice. The results showed that our vectors mediated high levels of transgene expression. Importantly, IL-10 gene therapy increased body weight gain, reduced colon injury, and prevented the development of inflammatory bowel disease (IBD). Moreover, IL-10 mutant gene therapy elicited significantly lower stimulation of CD8 T and NK cells compared with the wild-type IL-10 group. In summary, our IL-10 mutants provide a protective effect comparable to wild-type IL-10 in the IL-10 KO mouse model, suggesting that they may potentially have reduced pro-inflammatory function. While rigorous investigations of safety and efficacy in different disease models will be required, these results indicate the therapeutic potential of IL-10 mutant gene therapy for inflammatory diseases such as IBD. Full article

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction. Full article

Background/Objectives: Recombinant adeno-associated virus (rAAV) has become a leading vector in gene therapy. However, manufacturing limitations may result in replication-competent AAV (rcAAV) contamination of clinical rAAV products, posing safety risks. Rigorous testing is therefore essential, and the use of accurately calibrated rcAAV reference standard materials is critical for ensuring assay stability and reliability. A disadvantage of the widely used Tissue Culture Infectious Dose 50 (TCID₅₀) assay is its high variability. This study introduces an optimized TCID₅₀ assay for the precise quantification of infectious rcAAV particles. Methods: We developed a TCID₅₀ assay tailored to rep2-based rcAAV, optimizing key aspects such as viral infection conditions, qPCR reaction systems, and standard curve preparation. We employed an innovative strategy to prepare the standard curve using serial dilutions of rcAAV in cell lysate, ensuring alignment with the test sample matrices. Results: The rcAAV-derived standard curve demonstrated exceptional linearity (R² > 0.99), sensitivity (LOQ ≈ 38 copies), and reproducibility, enabling robust endpoint qPCR analysis. The optimized assay significantly improved the precision of the TCID₅₀ assay, as an inter-assay coefficient of variation (CV) of 11.4% was achieved. Conclusions: This refined TCID₅₀ assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing. Full article

Background: Effective tuberculosis vaccines capable of inducing durable pulmonary immunity remain an unmet need. Mucosal vaccination strategies and rational antigen selection are increasingly recognized as critical for improving protection against aerosol Mycobacterium tuberculosis (Mtb) infection. Objective: The objective of this study was to establish an intranasal recombinant adeno-associated virus (rAAV) platform and evaluate SapM (Rv3310) as a mucosal TB vaccine antigen in mice. Methods: We established and optimized an rAAV production and purification platform suitable for intranasal immunization and applied it to deliver Mtb antigen SapM. Immunogenicity was assessed by lung mucosal T-cell responses (CD69/CD103) and IFN-γ production in the lungs and spleen after mycobacterial antigen stimulation. Protective efficacy was evaluated after aerosol H37Rv challenge by quantifying pulmonary bacterial burden and lung pathology compared with vector controls and BCG. Results: rAAV6-SapM was successfully produced and efficiently transduced antigen-presenting cells without inducing phenotypic maturation. Intranasal immunization in mice induced mucosal T-cell responses in the lungs and increased expression of tissue residency-related markers (CD69 and CD103). It also elicited a Th1-biased cellular immune response characterized by enhanced IFN-γ production in both the lungs and spleen in response to mycobacterial antigen stimulation. Upon aerosol challenge with virulent Mtb H37Rv, rAAV6-SapM-immunized mice exhibited a significant reduction in pulmonary bacterial burden and attenuated lung pathology compared with vector-immunized controls. Conclusions: These findings provide proof-of-concept evidence that intranasal delivery of an AAV-based vaccine encoding SapM can induce antigen-responsive Th1 immunity and confer significant protection against early pulmonary TB, supporting further exploration of SapM as a vaccine antigen and AAV-based mucosal gene vaccination as a platform for TB vaccine development. Full article

Background/Objectives: Recombinant adeno-associated virus (AAV) vectors have revolutionized gene therapy for monogenic diseases such as Duchenne muscular dystrophy (DMD). However, high systemic doses required for muscle transduction cause a spectrum of toxicities ranging from transient hepatic inflammation to fatal multi-organ failure leading to death. These adverse events have reshaped the risk–benefit considerations for gene therapy in DMD. Methods: We conducted a narrative review describing complications associated with AAV-mediated gene therapies in the DMD field. PubMed and Clinicaltrials databases were used to search for peer-reviewed manuscripts published between 1987 and 2025. Publicly available abstracts and press releases were also used to describe AAV-mediated adverse events that have been discovered. Priority was given to large prospective cohorts, meta-analyses, and high-impact publications. Results: We outlined the mechanistic basis of AAV toxicity—spanning innate and adaptive immune activation, vector–host interactions, transgene overexpression, and host vulnerability—and discussed their therapeutic implications for DMD. We also highlighted ongoing strategies for vector re-design, immune modulation, patient selection, and regulatory adaptation, aiming to improve efficacy with safety in the next generation of muscular dystrophy gene therapies. Conclusions: Patient safety remains the number one priority in the AAV-mediated gene therapies field. Achieving long-term benefits requires continued optimization of existing vectors, implementation of strict criteria for patient selection, and regulation of immune responses, with close collaboration and transparent dialog among scientists, clinicians, and regulatory agencies, informed by both successful cases as well as tragic deaths reported in the fields of neuromuscular diseases. Full article

Recent years have seen rapid progress in biological treatments for genetic diseases, as well as conditions like type 1 diabetes that lack an obvious genetic component. The authors sought to explain why this progress has emerged at this particular moment. The best way to illustrate this is by showcasing a wide range of therapies targeting diverse diseases. This progress has been driven by technological advances in genetically modified CAR-T and CAR-NK cells (e.g., using CRISPR or transgenes), which have led to significant improvements in cancer therapy. A key trend now is the emergence of “off-the-shelf” approaches aimed at generating cellular therapies compatible with a range of recipients by mitigating alloreactivity and immune rejection. Different diseases impose distinct biological and logistical limitations; thus, treatment of each patient requires an appropriate strategy. Emerging advances include the modification of therapeutic cells, either ex vivo or in vivo. Current options for transgene delivery mainly comprise lipid nanoparticles (LNPs), adeno-associated virus (AAV) vectors, and lentiviral vectors. Researchers also focus on selecting suitable promoters for specific expression in selected cell types. Altogether, these advances have led to remarkable progress in treating various diseases in recent years. This publication discusses the development of biological therapies, with particular emphasis on cell and gene therapies, illustrated by viable examples across various disorders. It covers implemented solutions for several types of cancer, as well as selected hereditary diseases and syndromes, including Huntington’s disease, carbamoyl phosphate synthetase 1 (CPS1) deficiency, hemiplegia, epidermolysis bullosa, chronic granulomatous disease, and congenital deafness. Emerging applications in heart diseases and diabetes are also summarized, along with therapeutic strategies involving tRNA gene editing. Although numerous strategies exist, only the most representative, practical, and up-to-date examples are emphasized. Full article

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools. Full article