We are grateful for the commentary by Ángel Fernández-Flores on our concept of “desmosomal-type acantholysis”, a distinct histologic form of acantholysis associated with mutations in genes encoding desmosomal proteins [...]
Adeno-associated virus (AAV)-mediated gene transfer remains a promising strategy for cystic fibrosis (CF), but durability of expression and optimal dosing intervals are unresolved challenges. Here, we evaluated long-term gene transfer, transduction, and immunological responses following a single pulmonary administration of an AAV1 vector encoding Δ27-264 CFTR to juvenile rhesus macaques. Four animals received 1 × 101³ vector genomes via endotracheal spray. Two were untreated. All were analyzed 180 days post-delivery. Vector genomes were detected throughout the conducting airways and distal lung at levels ranging from ∼5 × 106 to 9 × 107 vg/µg genomic DNA. Vector genomes were also detected in extrapulmonary tissues, including liver, pancreas, spleen, heart, and kidney. Cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA expression was detected in all tissues examined and in several respiratory and extrapulmonary tissues, accompanied by increased CFTR protein expression in multiple tissues. Transgene expression was observed in airway basal cells and FOXI1-positive ionocytes, indicating targeting of key epithelial and progenitor cell populations. Despite persistence of vector genomes, immune activation was limited. Cytokine and chemokine expression profiles showed only sporadic, tissue-specific changes without evidence of coordinated inflammation. Neutralizing antibodies to AAV1 developed in all animals but did not correlate with capsid-specific T cell responses, which remained low and infrequent. These findings indicate sustained gene transfer and transduction for at least 180 days following a single AAV1 administration to the primate lung, with minimal immunopathology. The durability of expression supports the potential feasibility of extended dosing intervals and informs the development of repeatable AAV-based gene therapies for CF.
To date, prediction of manufacturing failure for CAR T cells is still lacking. In a retrospective multivariate analysis of our 368 manufactured CAR-T cell batches (tisagenlecleucel), we investigated potential factors that might be associated with manufacturing failure. In this letter to the editor, we provide a summary of our findings and outline recommendations for future advanced quality control strategies.
Accurate classification of Genetically Modified Organism (GMO) gene therapies remains a significant challenge to initiating clinical trials in the United Kingdom, often delaying patient access to innovative treatments. Current regulatory processes require separate GMO risk assessments at each National Health Service (NHS) trial location, leading to inconsistent approaches and duplication of effort. This article presents the development of a decision tree designed to streamline GMO classification for Gene Therapy Investigational Medicinal Products (GTIMPs). The proposed tool aims to address key challenges in trial setup by providing a structured and evidence-based approach to GMO classification, clarifying international misalignment in hazard classification and offering practical guidance for the in vivo and ex vivo GMO GTIMPs most encountered in clinical practice. By harmonizing decision-making and clarifying containment requirements, the adoption of the decision tree across NHS organizations could reduce variability, accelerate trial setup, and strengthen the United Kingdom's position as a competitive destination for GTIMP clinical trials.
Friedreich ataxia (FA) is a progressive neurodegenerative disorder caused by reduced expression of frataxin (FXN), a mitochondrial protein essential for iron-sulfur (Fe-S) cluster biogenesis. Although gene therapy strategies aimed at restoring FXN have shown promise, excessive expression can lead to mitochondrial dysfunction, emphasizing the importance of maintaining FXN within a physiological range. Here, we evaluated a gene therapy approach based on a human mini-frataxin construct (miniFXN7) incorporating an endogenous regulatory element to enable controlled FXN expression. The construct was delivered systemically using an AAV-PHP.eB vector in the Pvalb -cKO mouse model of FA. MiniFXN7 treatment resulted in widespread neuronal transduction and restoration of FXN expression toward a near-physiological range in the neuronal populations examined. Treated mice exhibited sustained improvements in motor coordination and proprioceptive function, including normalization of H-reflex responses. At the cellular level, miniFXN7 restored succinate dehydrogenase activity, a mitochondrial Fe-S enzyme, and was associated with partial normalization of mitochondrial morphology. In parallel, neuronal integrity was preserved and astrogliosis reduced across the cerebellum. These findings demonstrate that physiologically regulated FXN replacement is sufficient to achieve substantial functional rescue in FA, supporting a gene therapy strategy based on a transgene expression driven by endogenous regulatory elements.
Adeno-associated virus (AAV) gene transfer vectors mediate long-term expression in nondividing cells, an advantage for treating chronic disorders. However, current platforms lack a way to selectively shut down transgene expression if adverse effects arise. To create an "off switch," we hypothesized that incorporating unique artificial microRNA (amiRNA) target sequences into an AAV expression cassette would allow subsequent suppression of transgene expression using a second AAV vector encoding the cognate amiRNA. We introduced 22-nt sequences absent from human and mouse transcriptomes into the 3' untranslated region (UTR) of a therapeutic AAV cassette. To identify optimal amiRNAs, two tandem copies of each amiRNA were cloned into the 3'UTR of an mCherry reporter gene. In vitro assessment of six amiRNA/target pairs using a dual luciferase assay identified four amiRNAs that efficiently suppressed reporter expression. Cells cotransfected with target site 3 (TS3) and amiRNA-T3B showed the greatest reduction in luciferase activity (80%, p < 0.0001) and were selected for further study. The "off-switch" system was then evaluated using an AAV5 therapeutic vector expressing a recombinant humanized anti-IgE monoclonal antibody (AAV5-TBG-anti-IgE-TS3), designed for long-term suppression of allergen-induced reactions. Co-transfection of HEK293T cells with anti-IgE-TS3 and amiRNA-T3B significantly reduced anti-IgE mRNA and protein levels relative to a control amiRNA (p < 0.0001). In vivo testing in Balb/c mice (n = 5) involved intravenous administration of AAV5-anti-IgE-TS3 (3.2 × 1010 gc), followed 4 weeks later by an AAVrh.10 amiRNA vector (AAVrh.10-TBG-amiRNA-T3B; 1 × 1011 gc). Control mice receiving only the therapeutic vector expressed 18.4 ± 13.8 µg/mL serum anti-IgE at 10 weeks. In contrast, mice receiving the amiRNA "off" vector showed marked suppression of anti-IgE (0.3 ± 0.15 µg/mL, p < 0.0001). These findings provide proof-of-concept that AAV-delivered amiRNAs can selectively switch off transgene expression, offering a strategy to improve the safety of AAV-mediated gene therapies.
The integration of immunohistochemical biomarkers like PRAME (Preferentially Expressed Antigen in Melanoma) into the histomorphological assessment of melanocytic lesions is gaining prominence. While PRAME’s diagnostic value is widely recognized, its independent weight compared directly to classical morphology remains underexplored in large, challenging cohorts. This retrospective study quantifies the diagnostic utility of PRAME alongside traditional histomorphology in a high-risk cohort of 954 primary melanocytic lesions (335 nevi, 215 melanomas in situ, 404 invasive melanomas) where PRAME was clinically requested to resolve diagnostic ambiguity. Using Firth’s penalized likelihood regression, a baseline diagnostic model utilizing 13 histomorphological features was established and subsequently integrated with PRAME expression. The pure morphology baseline model demonstrated a high cross-validated area under the curve (AUC) of 0.969. As a standalone marker, PRAME achieved an AUC of 0.895, with a diffuse expression score of 4+ yielding peak specificity (94.3%) and moderate sensitivity (77.9%). Integrating PRAME into the morphological model significantly enhanced diagnostic accuracy (AUC: 0.980; p < 0.001), establishing PRAME as a dominant independent predictor of malignancy alongside core features like junctional atypia and upward melanocytes (Odds Ratio 19.08 for Score 4+). Analysis of discordant cases revealed that completely PRAME-negative melanomas were morphologically indistinguishable from typical PRAME-positive melanomas. Conversely, diffusely PRAME-positive (4+) nevi exhibited significantly higher rates of upward migrating melanocytes, constituting a critical diagnostic pitfall. In conclusion, while classic histomorphology remains the indispensable gold standard in dermatopathology, PRAME functions as a highly objective, reproducible tie-breaker. The synergistic integration of PRAME with morphological assessment effectively resolves diagnostic ambiguity and maximizes diagnostic confidence in challenging melanocytic lesions.
Common genetic variants of APOE are major risk factors for sporadic late-onset Alzheimer’s disease (AD). APOE has three common variants: APOE3, APOE4, and APOE2. Epidemiological, clinical, and experimental evidence demonstrate that APOE3 is associated with an average risk for AD, APOE4 is pathogenic and conveys a high risk, and APOE2 is protective and reduces risk. In prior mouse studies, we have demonstrated that (1) adeno-associated virus (AAV)-mediated central nervous system (CNS) gene transfer of APOE2 is highly protective against the toxic effects of APOE4 and (2) AAV-mediated CNS transfer of anti-APOE4 microRNA (miRNA) significantly suppresses the expression of APOE4. The ideal therapy for APOE4 homozygotes would both reduce CNS levels of APOE4 and substitute APOE4 with the addition of APOE2. We have developed a “silence-and-replace” therapy, where suppression of CNS APOE4 is achieved through AAV-mediated expression of miRNAs designed to silence the endogenous human APOE4 messenger RNA (mRNA), together with simultaneous replacement by an APOE2 coding sequence [APOE2(R)] resistant to those miRNAs. AAV vectors expressing APOE2(R) with and without the miRNAs were administered to the hippocampus of human APOE4 mice, and APOE levels at the mRNA and protein levels were the same for both vectors. Dideoxy Sanger sequencing and allele-specific real-time quantitative PCR were used to assess the CNS APOE2 to APOE4 ratio. As expected, both vectors with APOE2(R) increased the E2/E4 ratio relative to controls by 3.5 ± 0.6-fold for APOE2(R) and 6.3 ± 0.4-fold for APOE2(R) + miRNAs. We conclude that a single AAV vector can deliver a cassette with dual functionality: suppression of the toxic APOE4 variant and simultaneous delivery of the protective APOE2. This dual approach may provide a more potent gene therapy for APOE4-dependent AD than either strategy.
Adoptive immunotherapies have emerged as a promising strategy in the treatment of hematological malignancies. To date, seven chimeric antigen receptor (CAR)-T cell products have obtained market authorization in Europe for B-cell malignancies, where they have revolutionized treatment for eligible patients. In addition, natural killer (NK) cells and γδ T cells are of particular interest for cell-based therapies due to their strong intrinsic cytotoxicity and favorable safety profile. Addressing challenges facing the clinical translation of NK cell therapies was one of the issues discussed in the NK & ILC Symposium that took place in Freiburg, Germany from March 11 to 13, 2026, as the annual meeting of the NK & ILC study group of the German Society for Immunology (DGfI). Topics ranging from basic immunological research to technological innovations and clinical trial results were discussed during the 3-day conference, spanning over 40 presentations and 100 posters. A highlight of the conference was a workshop featuring short presentations and a panel discussion that focused specifically on the current challenges and future prospects of the clinical implementation of NK cell therapies; the findings of this workshop are summarized in this opinion paper.
Targeted in vivo transduction, entailing direct administration of viral vector preparations to patients, is the next big step in gene therapy. To redirect lentiviral vector (LV) particles selectively to desired cell type(s), different glycoproteins have been engineered to alter their tropism, including the glycoprotein G from Vesicular Stomatitis Virus (VSV-G) as the most commonly employed tropism-defining protein for LV particles. For detargeting from its natural receptor, the low-density lipoprotein receptor (LDLR), a VSV-G variant with two blinding substitutions, K47Q and R354A, is commonly used (VSV-G.pub). We provide first evidence for insufficient blinding of VSV-G.pub in human and murine cell lines and primary cells. In silico modeling pointed toward only slightly reduced LDLR binding affinity of VSV-G.pub. To lower the affinity further, we generated three novel VSV-G variants based on charge-reversing substitutions in two, four, or six key residues. These variants achieved a more stringent blinding compared with VSV-G.pub in the tested cell lines and primary cells. Codisplay of a CD4 binder enabled lentiviral particles pseudotyped with the novel variants to selectively transduce CD4-expressing cells. In summary, we present improved VSV-G variants with a better on/off-target ratio as attractive tools for in vivo gene therapy applications.
Helper-dependent adenoviral vectors (HDAdVs), which lack all viral coding sequences, enable the delivery of up to 35 kilobases (kb) of therapeutic DNA into target cells. This large packaging capacity facilitates the transfer of complex, large, or even multiple transgenes. Despite the technical complexity of their production, HDAdVs have been extensively evaluated in numerous preclinical studies, and several clinical trials have recently been initiated. This review provides a comprehensive, state-of-the-art overview of recent advances in the HDAdVs technology. It summarizes key strategies for vector development, including capsid engineering and improvements in production methodologies. Furthermore, it reviews preclinical in vivo studies with a focus on vector design, target indications, and therapeutic outcomes. Most preclinical applications have focused on targeting the liver and hematopoietic stem cells; however, additional organ systems and vaccination approaches based on HDAdVs are also being actively explored. In addition to preclinical progress, three recently initiated clinical trials using HDAdVs targeting knee osteoarthritis, chronic granulomatous disease, and solid tumors are highlighted. Nevertheless, several challenges must be addressed to enable broader clinical translation of HDAdVs. These include the complexity of large-scale manufacturing, achieving efficient and specific vector targeting, and overcoming host immune responses. In conclusion, HDAdV-based vector systems hold considerable promise for the treatment of a wide range of diseases. Continued technological advancements, including the development of next-generation adenoviral platforms and the integration of precision gene therapy approaches, are likely to further enhance their potential as versatile biotherapeutic tools.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has revolutionized genetic medicine by enabling precise genome editing for therapeutic benefit. CRISPR nucleases are programmed to target genomic sites with sequence complementarity to the spacer region of an associated guide RNA. However, these nucleases may target genomic loci with sequences similar to the target site, which can lead to unintended disruption of off-target genes. The risks associated with these off-target editing events are critical to assess as CRISPR-based in vivo editing systems advance to clinical development. Nonhuman primates (NHPs) are common model species for evaluating the human safety of many therapeutic modalities, but their relevance for evaluating human CRISPR off-target activity has yet to be determined. In this study, 1,220,908 Cas12a and 6,159,066 Cas9 spacer sequences targeting human genes were designed, and off-target editing sites were computationally predicted in humans and five common NHP species. Of the 7,413 Cas12a and 570,754 Cas9 spacers meeting defined on- and off-target inclusion criteria, only 14-21% of Cas12a and 7-15% of Cas9 human off-targets per spacer are recapitulated in the genomes of NHPs commonly used in preclinical studies. These results highlight the limitations of NHPs for the study of human CRISPR spacer specificity and contextualize human risk informed by these studies.
Proliferative nodules (PNs) are benign, well-limited melanocytic proliferations that can occur within congenital nevi, particularly larger ones. Although they may mimic melanoma clinically and histologically, PNs are characterized by a monomorphic, well-defined cell population with peripheral blending with the adjacent nevus cells, and a lack of severe atypias, numerous mitoses (in most instances), necrosis, or inflammation. They generally present at birth or early childhood, and even with cytological atypia, they do not undergo malignant transformation. The risk of malignancy associated with a large/giant congenital nevus is low but increases with size and the presence of multiple satellite lesions. Diagnostic tools, including immunohistochemistry and, in selected cases, molecular techniques such as CGH-array or RNA-seq, can help differentiate atypical PNs from melanoma. Awareness of this entity and its diverse histological features is crucial to avoid over-diagnosis of malignancy and unnecessary interventions. Here we report a case of atypical PNs in a giant congenital nevus and discuss the literature.
Receptor engagement plays a key role in the cellular uptake, intracellular trafficking, and overall transduction efficiency of adeno-associated virus (AAV) vectors. Although heparan sulfate proteoglycan (HSPG) and α5ß1 integrin-binding motifs of the AAV serotype 2 (AAV2) capsid were mapped, it has remained incompletely understood how loss of these interactions affects AAV vector performance. Hence, we generated capsid variants harboring mutations at capsid residues responsible for HSPG binding (AAV2ΔHSPG), α5ß1 integrin binding (AAV2ΔIntegrin), or both (AAV2ΔHSPGΔIntegrin), and investigated the mutants systematically ex vivo and in vivo. While neither production nor packaging efficiency was affected, variants revealed distinct physicochemical alterations, including altered electrophoretic mobility and thermal stability. Ex vivo, loss of HSPG binding completely abolished transgene expression across various cell lines, whereas ablation of α5ß1 integrin binding lowered transduction efficiency. While cellular uptake was reduced for AAV2ΔIntegrin and almost eliminated for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin, mutants nevertheless reached the nuclear compartment, albeit with lower efficiency compared with AAV2. Strikingly, in vivo performance diverged sharply from ex vivo findings as we observed a substantially enhanced transduction in multiple non-hepatic tissues for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin in C57BL/6N albino and BALB/c mice, as well as a strong liver detargeting, while AAV2ΔIntegrin was non-infectious in vivo. These data uncover a fundamental dichotomy between ex vivo and in vivo determinants of AAV2 transduction and identify receptor-binding ablation, especially integrin binding, as a potential alternative to detarget AAV2 vectors for next-generation capsid engineering and tissue-specific retargeting.
Oncolytic virotherapy harnesses genetically engineered viruses to selectively infect and lyse tumor cells while stimulating antitumor immunity through pro-inflammatory microenvironment activation. Celyvir represents a pioneering cell-based oncolytic therapy combining mesenchymal stromal cells (MSCs) as carriers of the human oncolytic adenovirus ICOVIR-5. Developed over two decades through collaborative efforts, Celyvir leverages the tumor-homing capacity of MSCs to enhance systemic delivery and therapeutic efficacy of oncolytic virotherapy against solid tumors. Preclinical and clinical studies, including compassionate use programs and phase I trials, have demonstrated Celyvir’s safety, feasibility, and potential efficacy. Mechanistic insights reveal that MSCs with a low pro-inflammatory profile and patients’ baseline immune competence correlate with improved responses. The evolution from autologous (Celyvir) to allogeneic MSC-based delivery (AloCelyvir) aims to overcome manufacturing delays and optimize clinical outcomes. This review summarizes the conceptual foundations, preclinical models, translational milestones, and ongoing clinical trials of Celyvir, illustrating its trajectory from bench to bedside as an improved oncolytic virotherapy modality.
