(Molecular Therapy 33, 4091–4112; September 2025)

This review presents the evolution of gene-editing technologies, with particular emphasis on the CRISPR-Cas system and its expanding applications from genetics and biotechnology to medicine. Furthermore, advanced gene editing approaches provide an overview of emerging trends.

Gu and colleagues demonstrate that plasmids expressing transcripts from various Pol III promoters localize to discrete foci in the nucleus that are distinct from Pol II transcription and processing domains. However, when both Pol II and a Pol III promoter-transcript cassettes are on the same plasmid, this localization is disrupted, accompanied by a profound reduction in expression of both cassettes.

NUT carcinoma depends on the BRD4::NUTM1 fusion, which current inhibitors can target but without durable clinical efficacy. This study demonstrates that CRISPR/Cas9 can directly disrupt this fusion, suppressing cancer cell growth. Therefore, cancer genome editing could be employed as an alternative and effective strategy for targeting fusion-driven solid cancers.

Affinity-enhanced TCR-engineered T cells show increased off-target reactivity, posing toxicity risks. CD8α-negative cells expressing these TCRs have reduced off-target reactivity and thus enhanced specificity. These findings suggest a pathway to improvements for safer, more effective TCR therapies.

Gee and colleagues successfully employed AAV-based gene supplementation with precise regulation of target cells and transgene expression to restore hearing in a mouse model harboring an Mpzl2 East Asian founder variant.

This review offers critical insights and highlights key challenges in establishing varicella-zoster virus (VZV)- and cytomegalovirus (CMV)-based cancer viroimmunotherapy platforms. The potential tumor cross-reactivity of the T cell and natural killer (NK) cell responses that are activated and amplified by VZV and CMV is also described.

CAR-engineered innate immune cells represent a promising new frontier for solid tumor treatment, overcoming limitations of CAR-T therapies such as toxicity and poor tumor infiltration. This review highlights their biological advantages and clinical potential, paving the way for innovative, off-the-shelf cancer immunotherapies.

An intuitive and comprehensive comparison of protein and nucleic acid recombination for the dual AAV strategy was performed in a mouse model of DFNB9 deafness.

This study applies Prime Editing to correct Dilated Cardiomyopathy-causing mutations in cardiomyocytes. PE4-targeting of the RBM20P633L-mutation achieved 34.8 % T-to-C editing and improved the cell’s molecular phenotype. This strategy paves the way for an in-vivo strategy to treat RBM20-mediated DCM and other inherited cardiac diseases.

Gao and colleagues investigated the pan-cancer neural regulation pattern and revealed the clinical applications for patient stratification and immunotherapy.

Yoshioka and colleagues developed mRNA vaccine expressing virus-like particles of enterovirus D68 (EV-D68), a respiratory virus emerging as a cause of polio-like paralysis. This vaccine induced antibodies against EV-D68 in the blood and respiratory tract and showed protective effects against viral challenge. This platform might help control future enterovirus epidemics.

Combes and colleagues present an ASO-based switch that controls synthetic mRNA translation in response to short trigger RNA. ASO length, chemistry, and conjugation influence suppression and activation. Cholesterol- and dendrimer-linked PMOs enhance uptake and triggerability in cells, supporting a protein-free strategy for conditional mRNA translation with therapeutic potential.

This study introduces a point-of-care stem cell engineering strategy in which hematopoietic stem cells are genetically targeted within gene therapy foam that is mixed with freshly isolated bone marrow aspirate concentrate and then directly injected back into the marrow. This method could lower cost and infrastructure for hematopoietic-targeted genetic therapies.

Cryopreservation represents a practical approach in manufacturing and productization of cell-based therapies. Mei and colleagues' study shows that the administration of cryopreserved MSCs containing DMSO, at a level that is equivalent to a concentration of 0.98 g/L in blood volume, is well tolerated in septic animals and in immunocompromised rats.

There is a critical need for novel interventions that can slow the progression of kidney disease in diabetes, particularly in Type 1 diabetes. Here, Foster and colleagues show that gene therapy is a viable option for targeted delivery in the kidney in diabetes, using adeno-associated virus to deliver a growth factor (VEGFC).

Huang and colleagues reported a series of novel modification patterns that significantly enhance siRNA efficacy. This enabled the identification of a potent siRNA targeting PCSK9, which demonstrated favorable preclinical efficacy and safety profiles, highlighting its potential for dyslipidemia treatment. The study provides valuable insights for developing efficient siRNA modification patterns.

Williams and colleagues used charge detection mass spectrometry to investigate the structures and stability of adeno-associated virus, serotype 9 (AAV9). AAV9 had multiple conformers that depended both on buffer composition and temperature. Partial extrusion of DNA at physiological pH with structural compaction provides insights into natural infection processes.

CD-MS can be used for AAV analysis from harvest to drug substance. Harvest material maintains consistent empty/partial/full ratios as affinity purified sample. Process impurities were measured concurrently and are consistent with industry standards. CD-MS is highly reproducible and effectively tracks the production process, providing quantitative results at each stage.

Wilson, Asokan, and colleagues expand the toolkit for kidney gene transfer by testing the novel adeno-associated virus AAV.cc47. They found enhanced gene delivery to the kidney both in human kidney organoids and mice in proximal tubule cells. The also expanded the gene delivery capacity of the vectors using dual AAVs.

This study compares two human tolerogenic dendritic cells (tolDCs), named ATDC and DC-10, to evaluate how their generations affect their phenotype, metabolism, and function. Results suggest that DC-10 induce tolerance through immune regulation whereas ATDC do it through metabolic adaptation. These results provide new insights for tolDC application in cell-based-therapies.

PAD4, elevated in DKD patients and models, promotes renal injury. Loss-of-function PADI4 variants associate with higher eGFR. In diabetic mice, PAD4 deficiency or inhibition reduced tubulointerstitial injury by decreasing macrophage infiltration. Mechanistically, PAD4 interacts with p65 to induce Cmklr1 expression, enhancing macrophage migration, crucial in DKD injury.

Classical adenine base editors (ABEs) induce widespread RNA off-target edits, contributing to potential toxicity. The mCherry-based INOsine Sensor (MINOS) enables rapid and effective evaluation of RNA A-to-I editing, facilitating high-throughput optimization of ABEs. Through these efforts, engineered ABEs were generated, exhibiting therapeutic potential in both cellular and animal models.

This study reports that exosomes produced by IL4-polarized THP-1 human macrophages (THP1-IL4-exo) can serve to reverse cardiac functional decline in mice with heart failure caused by diet-induced myocardial infarction (MI). Ng and colleagues identify Type 1 Interferon signaling as a key pathway attenuated by THP1-IL4-exo, responsible for conferring cardioprotection post-MI.

Langerhans Cell-Targeted Delivery System (LC-TDS) enables highly specific antigen delivery to skin Langerhans cells, boosting antigen-specific T cell responses. Intradermal or topical LC-TDS immunization efficiently activates adaptive immunity and achieves pronounced dose-sparing, supporting LC-TDS as a powerful and flexible platform for next-generation vaccines and immunotherapies.