In vivo chimeric antigen receptor (CAR)-T cell therapy - Nature Reviews Drug Discovery In vivo chimeric antigen receptor (CAR)-T cell engineering uses targeted delivery systems to generate CAR-T cells directly in patients, bypassing ex vivo manufacturing. This Review examines emerging v...

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Direct optogenetic activation of skeletal muscle offers an alternative to invasive electrical stimulation. Knapman et al. tested the ability of adeno-associated viral vectors (AAV9 and AAVMYO) to confer light sensitivity to upper airway muscles in a model of obstructive sleep apnea. The image shows robust opsin expression (orange) in axial sections from the anterior and middle regions of a rat tongue 3 weeks after intramuscular AAV9 delivery. Image credit: Fiona L. Knapman.

Check out the latest issue of #MolecularTherapy Methods & Clinical Development for reviews on #LNPs and #genetherapy redosing, plus research on #AAV manufacturing, #siRNA therapy, and #celltherapy. Read it here: http://dlvr.it/TN2q33
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Development and implementation of an LC-MS-based multi-attribute method for adeno-associated virus Powers and colleagues have developed an AAV MS-based multi-attribute method (MAM) to quantitate deamidation, an important quality attribute for AAV therapeutics. The method was validated according to ...

Development and implementation of an LC-MS-based multi-attribute method for adeno-associated virus #MTMCD www.cell.com/molecular-th...

Development of LC-MS methods for AAV capsid protein quantification and host cell protein profiling Kontogiannis and colleagues developed a mass spectrometry-based method for quantification of the capsid proteins of recombinant AAV9 and measurement of the mean capsid stoichiometry.

Development of LC-MS methods for AAV capsid protein quantification and host cell protein profiling #MTMCD www.cell.com/molecular-th...

Comparative analysis of cell-specific promoters in AAV9-mediated gene therapy targeting the central nervous system This study presents a comparative evaluation of ubiquitous and cell-specific promoters in the central nervous system and introduces a novel astrocyte-specific promoter. Using quantitative and imaging-based analyses, Bradbury and colleagues highlight promoter-dependent differences in distribution, intensity, and cell-type specificity. The toolkit described enables rational promoter selection for targeted gene therapy.

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Elevated tripeptidyl-peptidase 1 corrects multiple phenotypes of disease in a mouse model of juvenile neuronal ceroid lipofuscinosis Juvenile neuronal ceroid lipofuscinosis (JNCL) is an untreatable, fatal neurodegenerative lysosomal disease. Sleat and colleagues have shown that increased levels of tripeptidyl peptide 1 attenuates disease in a JNCL mouse model, suggesting that administering a recombinant form of this protease may be a viable treatment option for JNCL patients.

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CAR T cell engineering impacts antigen-independent activation and co-inhibition Schultheiß, Stücheli and colleagues compare viral and non-viral engineering of CAR T cells targeting a leukemia neoepitope. They reveal that manufacturing mode and CAR design significantly shape T cell phenotypes, activation states, and inflammatory responses, with implications for clinical translation, especially when using patient-derived T cells and non-viral methods.

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Design of Cationic Ionizable Lipids for the Delivery of Therapeutic Nucleic Acids Ionizable cationic lipids are a critical component of lipid nanoparticles (LNPs), enabling the clinical success of nucleic acid therapeutics through effective encapsulation, delivery, and release. As the field accelerates beyond first-generation RNA medicines, the rational design of next-generation ionizable lipids has become a key area of research. In this review, we outline key design principles that guide the development of efficacious and safe ionizable lipids for nucleic acid delivery. We highlight emerging structural motifs and discuss how these features contribute to improved potency, tolerability, and endosomal escape.

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Reduced rAAV Interference Enhances rcAAV Detection Sensitivity This study develops a CRISPR-based rcAAV detection method. SpCas9-HEK293 cells were co-transfected with rAAV and a sgRNA targeting polyA site. This method cleaves the rAAV genome, reliably identifying 3E2 vector genomes (vg) of rcAAV, a threshold undetectable by traditional methods, offering a valuable tool for quality control of rAAV products.

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In Vivo Delivery of a Fluorogenic Antisense Oligonucleotide into Human Leukocytes in a Humanized Mouse Model System Using a fluorogenic delivery system originally developed for peptides and oligonucleotides in vitro, the data in this paper demonstrate delivery without toxicity of an antisense oligonucleotide targeting β-actin messenger RNA into human leukocytes in the blood, bone marrow, liver, spleen, brain, and gut of a humanized mouse model.

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Genome-replicating HC-AdV – a novel high-capacity adenoviral vector class featuring enhanced in situ payload expression Plückthun and colleagues present a novel genome-replicating high-capacity adenoviral vector that enhances in situ transgene expression without inducing cytotoxicity. This new adenoviral vector class combines large payload capacity (≥ 22 kb) with self-induced and self-sustained genome replication, offering a promising tool for improved gene therapies and vaccination strategies.

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Variation of VP2 stoichiometry and deamidation of VP1 during production and their impacts on the transduction efficiency of AAV vectors Uchiyama and colleagues demonstrated, based on the repeated productions of adeno-associated virus vector using HEK293 suspension cell line, that the stoichiometric ratio of viral protein 2 and deamidation ratios of Asn57 and Asn94 in viral protein 1 could be potential critical quality attributes (pCQAs) in terms of gene transduction.

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Landscape of Ex vivo Gene Therapies: Technological Trends and Future Prospects This study provides the current landscape and future directions of ex vivo gene therapy. Using a strategic foresight methodology a database was built with extensive data treatment (preparation, curation, enrichment and verification) of ∼1,500 products exploring cell types and sources, genetic modifications, vectors, molecular targets, indications, and development stages.

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Morphological changes induced by buffer exchange during preparation of mRNA-lipid nanoparticles occur in a step-wise manner as pH is elevated Manufacturing of LNPs involves entrapment of nucleic acids at pH 4 and then a buffer exchange to pH 7.4. During buffer exchange, particles fuse and undergo morphological changes. Pouton and colleagues showed using physicochemical techniques that a series of changes occur in a step-wise manner as the pH is increased.

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pH-dependent DNA degradation pathways for adeno-associated virus gene therapy Adeno-associated virus (AAV) degradation reduces gene therapy efficacy, but mechanisms remain unclear. Researchers developed a capillary gel electrophoresis assay to quantify encapsidated DNA degradation, identifying acid-driven chemical breakdown and base-induced ejection. DNA degrades faster inside capsids, impacting AAV potency. Ensuring genome stability is crucial for manufacturing and efficacy.

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Functional, sustained recovery of hearing in Otoferlin-deficient mice using DB-OTO, a hair cell-specific AAV based dual vector gene therapy DB-OTO, a hair cell-specific AAV-based dual-vector gene therapy, was developed to provide hearing for OTOF-related deafness. A synthetic Myosin15 promoter was engineered to restrict the transgene expression to hair cells. Safe and sustained recovery of hearing in Otoferlin-deficient mice supported the initiation of an ongoing Phase I/II registrational clinical trial.

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Directed evolution of liver-detargeted AAV vectors for systemic gene delivery to skeletal muscle and heart To improve the safety of AAV gene therapy by reducing hepatotoxicity risks, AAV capsids with reduced liver and enhanced muscle transduction were identified using targeted mutagenesis, serotype screening, and directed evolution approaches. The capsid structures were elucidated by Cryo-EM and capsid activity was characterized in mice and non-human primates

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Effects of CRISPR-Cas9-mediated FOXP3 knockout on CAR T-cell potency Peter and colleagues demonstrate that FOXP3, traditionally known for its role in regulatory T cells, limits effector function in CAR T cells. Using CRISPR-Cas9 knockout, they reveal FOXP3 as a potential modulator of therapeutic T-cell activity, offering a strategy to improve CAR T-cell efficacy in disease settings.

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Durable tissue-specific transgene expression in newborn mice following intraperitoneal delivery of non-cytotoxic herpes simplex virus-based vectors Miyagawa and colleagues explore the distribution and transgene expression of non-cytotoxic, replication-defective herpes simplex virus vectors following intraperitoneal delivery to newborn mice. The results demonstrate tissue-specific, durable transgene expression, suggesting the potential of these vectors for the treatment of skin and neuromuscular diseases by systemic gene therapy.

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Simple, Specific, Rapid and Pharmacopoeia-Compliant qPCR-Based Approach for the Detection of Mycoplasma in Biopharmaceuticals Dos Santos and colleagues have developed a pharmacopoeia-compliant, mycoplasma-specific qPCR assay that uses two primers and two probes as a minimum requirement. The development involved selecting a target mycoplasma genomic sequence, designing primers for genomic DNA amplification, and designing hydrolysis probes specific to amplicons derived from the mycoplasma genome.

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Adeno-Associated Viral Vector Resource for the RNA-Targeting Cas13d: A Comparison of High-Fidelity Variants, DjCas13d and hfCas13d Ploski and colleagues created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d. They found that both hfCas13d and DjCas13d exhibit generally similar levels of RNA knockdown potential, with DjCas13d sometimes outperforming hfCas13d. However, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d.

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Natural history of preexisting AAV5 antibodies in adults with hemophilia B during the lead-in of the etranacogene dezaparvovec phase 3 study Adeno-associated virus serotype 5 neutralizing and IgG antibody levels were correlated and remained stable over 6 months prior to dosing in the HOPE-B trial of etranacogene dezaparvovec for hemophilia B, providing valuable guidance on timing for antibody screening when considering gene therapy

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Harnessing mRNA-lipid nanoparticles as innovative therapies for autoimmune diseases This review describes the development of RNA-lipid nanoparticle therapies for autoimmune disease. It highlights the importance of created targeted, non-inflammatory lipid nanoparticles and explores their potential applications in cellular therapies and tolerizing vaccines.

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Polymeric nanoparticles delivery of AMPK activator 991 prevents its toxicity and improves muscle homeostasis in Duchenne Muscular Dystrophy Duchenne Muscular Dystrophy (DMD) is a degenerative muscle disease associated with chronic inflammation and fibrosis. Juban and colleagues show that the encapsulation of the AMPK activator 991 into PLGA nanoparticles prevents its adverse effects and allows its delivery in muscle macrophages, improving muscle homeostasis in a DMD mouse model.

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AAV-mediated peripheral single chain variable fragments’ administration to reduce cerebral tau in adult P301S transgenic mice: mono- vs combination therapy Katel and colleagues show that peripheral vectorized scFvMC1 (in monotherapy) reduces pathological tau species in tau transgenic mice more efficiently than in combination with scFv-PHF1. The authors observed improved motor and behavioral functions together with increased brain glucose metabolism in scFv-MC1-treated mice

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Development of LC-MS Methods for AAV Capsid Protein Quantification and Host Cell Protein Profiling Kontogiannis and colleagues developed a mass spectrometry-based method for quantification of the capsid proteins of recombinant AAV9 and measurement of the mean capsid stoichiometry.

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HLA-A2 CAR/IL-2-CISC Engineered Treg display robust in vitro and in vivo antigen-specific regulatory function Subhash Tripathi and colleagues generated A2CAR-engineered Tregs (EngTreg) co-expressing A2CAR, FOXP3 and an inducible IL-2 signaling complex. A2CAR EngTreg exhibited robust suppressive activity, low cytotoxicity, and superior efficacy in a xenogeneic GvHD model, highlighting the potential for targeted immunotherapy in transplantation and autoimmune diseases.

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Development and Implementation of a LC-MS Based Multi-Attribute Method for Adeno-Associated Virus Powers and colleagues have developed an AAV MS-based multi-attribute method (MAM) to quantitate deamidation, an important quality attribute for AAV therapeutics. The method was validated according to according to ICH Q2(R2) and implemented for routine testing of AAV for development and stability purposes.

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GroβT rapidly and reliably mobilizes primitive hematopoietic stem and progenitor cells in nonhuman primates Radtke, Kiem and colleagues show that highly efficient and reliable mobilization of hematopoietic stem cells (HSCs) is achieved with AMD3100/GroβT. Bone marrow and blood displayed rapid recovery and robust multilineage long-term engraftment of gene-modified HSCs, offering a G-CSF-free regimen for sickle cell disease (SCD).

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AAV Hamartin Gene Therapy in a Stochastic, Cerebral Mouse Model of Tuberous Sclerosis Type 1 Systemic AAV9-mediated hamartin delivery reversed key neuropathological defects and extended survival in Tsc1-deficient mice, supporting its therapeutic potential in tuberous sclerosis complex type 1.

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