Recombinant AAV gene therapies deliver durable clinical benefit but face immune-mediated challenges that vary by vector, dose, route, and patient. Gulve and colleagues synthesize the clinical manifestations, temporal patterns, and mechanisms of rAAV immunogenicity, highlighting risk assessment and emerging mitigation strategies to support safer, more effective gene therapy development.

Infusion-related reactions (IRRs) can occur with AAV gene therapy. IRRs were observed in two participants who received AAV8 gene therapy and in one who received AAV9 gene therapy. AAV gene therapy IRRs may be rate-related, possibly driven by complement activation. In our studies, a slow, staged infusion mitigated additional IRRs.

Transduction efficiency is a critical determinant of CAR T-cell manufacturing quality. Analysis of 204 clinical CAR T-cell products revealed that transduction efficiency is shaped primarily by manufacturing workflows and protocol-dependent starting material composition. Higher transduction efficiency was associated with early memory-like cellular states, providing insights into optimizing CAR T-cell.

Automated ÄKTA-connected zonal-rotor CsCl ultracentrifugation enables high-resolution AAV polishing at 1.65-L scale. The workflow enriched full capsids and resolved empty and intermediate populations in AAV8-X and AAV9-Y. The AAV8-X intermediate fraction retained about a half of qPCR signal of equimolar full capsid but produced minimal mRNA expression potency.

Inoue and colleagues developed Digenome-Detect, a data analysis tool that enables highly accurate and sensitive assessment of off-target genome editing using Digenome-seq, the simplest cell-free assay. This advance improves genome-wide identification of off-target mutation events and thereby helps ensure the safety of genome-editing therapeutics.

Maeda and colleagues established of a 293-based cell line stably maintaining the HSV genome. This cell line enables efficiently producing HSV amplicon vectors without purification or transfection of the HSV genome. This system offers a more practical platform for HSV-based gene delivery, overcoming several limitations associated with conventional methods.

He and colleagues developed a transient, non-gene-editing platform for off-the-shelf allogeneic TCR-T therapy. By conferring tacrolimus resistance characteristics to IL-2/4/7 expanded TCR-T cells that display reduced alloreactivity, He et al. provided a proof-of-concept for an allogeneic TCR-T therapy approach that addresses both manufacturability, and unwanted graft and host alloreactivity concerns.

Todo and colleagues describe the development of IL-12-expressing oncolytic HSV-1 derived from the clinically validated G47Δ platform for malignant melanoma. Intratumoral treatment controls both injected and distant tumors and shows enhanced efficacy with PD-1 blockade. Favorable preclinical safety supports clinical translation and has enabled the ongoing investigator-initiated clinical trial.

In 2012, I had the distinct honor of being part of one of the trailblazing teams leading first-in-human/first-in-child chimeric antigen receptor (CAR) T cell trials in B cell acute lymphoblastic leukemia (B-ALL). Little did I know then that we were only at the tip of the iceberg for use of immunotherapy in pediatric oncology. While the multiple CAR T cell approvals for B cell malignancies, including in pediatric B-ALL, reflect the curative potential in relapsed/refractory disease, what has additionally transpired is a cascade of novel immune-effector cell therapy-based approaches, each iteratively building upon the prior to further extend the therapeutic index of immunotherapy.

Nguyen and colleagues engineered bispecific TCEs targeting p53R273H neoantigens presented by HLA-C*01:02. Machine learning-guided optimization (Boltz-2/EvotProGrad) yielded TCE01Cr3 (Kd = 2.51 nM), a small, high-affinity, specific TCE with mouse serum stability (t1/2 ≈ 40 h), and monotherapy efficacy (> 80% tumor elimination in 3D models) without off-target toxicity or chemotherapy.

Zhang and colleagues engineered the poly(A) tail as a programmable regulatory element, showing that embedding cell-type-specific microRNA target sites directly within it confers robust, position-dependent silencing in off-target tissues while preserving activity in target cells. This strategy offers a new modular tool to enhance mRNA therapeutic safety and precision.

Kis and colleagues report an integrated sequential-batch IVT–oligo-dT process that links RNA synthesis and affinity capture through a shared buffer, enabling direct crude-IVT loading and flowthrough recycling. The workflow improves cap-analog utilization and raw-material efficiency while preserving functional mRNA expression across five cycles.

COL17A1 nonsense mutations generate premature termination codons, reducing collagen XVII through truncated protein production and/or nonsense-mediated decay (NMD), and causing junctional epidermolysis bullosa. Translational readthrough drug offers a potential approach to nonsense mutations suppression. Different COL17A1 nonsense mutations showed distinct responses to readthrough drugs and NMD inhibitors, supporting personalized therapeutic strategies.

Small extracellular vesicles from differentiating human skeletal muscle cells are enriched in miR-193b-3p, which contributes to myogenic responses through AP2M1 regulation. These findings establish a miR-193b-3p–AP2M1 axis connecting extracellular vesicles to muscle regeneration and highlight its potential role in regulating myogenic processes.

CRISPR hybrid guides containing RNA and DNA nucleotides (chRDNA) enhance Cas nuclease specificity by reducing off-target editing. Intravenous administration of LNP-encapsulated Cas12a mRNA with a chRDNA guide in mice led to dose-responsive hepatic genome editing without detectable off-target editing and near elimination of the targeted plasma protein for one year.

Immunization with a nucleoside-modified mRNA-LNP vaccine encoding the conserved malaria antigen E140 induced durable humoral immunity in mice and generated invasion-blocking antibodies against Plasmodium vivax. The results highlight E140 as a promising candidate for next-generation malaria vaccines.

Et1Ψ-modified mRNA shows UUU-dependent translational sensitivity, but targeted UUU-to-UUC recoding restores protein expression. With this sequence constraint addressed, Et1Ψ reduces early innate immune activation while preserving vaccine-induced humoral, cellular, and neutralizing responses, thereby establishing codon–modification compatibility as a practical design principle for mRNA therapeutics.

Lipid nanoparticles incorporating DOTAP and DOPE enable serial mRNA transfection of primary human T cells with significantly improved viable cell yields compared to standard electroporation. This platform supports iterative functional modifications across multiple rounds of delivery while preserving cell viability, facilitating the manufacturing of complex, multi-payload cell therapies.

Tang and colleagues systematically review the structural biology, synthetic engineering, and immunological consequences of the mRNA 5' cap. By analyzing how Cap-0 through Cap-2 methylation patterns differentially affect translation and immune recognition, this work highlights the conceptual advances and translational potential of cap engineering for next-generation vaccines.

TTN truncating variants cause dilated cardiomyopathy and may be amenable to therapeutic upregulation. Disrupting TTN upstream open reading frames (uORFs) increased luciferase reporter expression, however disrupting endogenous uORFs in hiPSC-derived cardiomyocytes did not increase titin protein. This highlights the importance of validating regulatory mechanisms in disease-relevant cellular contexts.