Seven cases of hematological malignancy reported in recipients of Skysona™ (elivaldogene autotemcel) have reignited long-standing concerns about insertional mutagenesis in lentiviral vector (LV)-based gene therapy. Here,...Seven cases of hematological malignancy reported in recipients of Skysona™ (elivaldogene autotemcel) have reignited long-standing concerns about insertional mutagenesis in lentiviral vector (LV)-based gene therapy. Here, we dissect the molecular and clinical evidence underlying these events, place them in the broader context of over 300 patients treated with LV-modified hematopoietic stem and progenitor cells (HSPCs), and review the real-world safety record of LV-engineered chimeric antigen receptor T cells. We show that cancers associated with Skysona are mechanistically linked to the use of a potent viral MNDU3 promoter probably combined with intensive conditioning and growth-factor support, whereas LV products employing weak or physiological promoters continue to display an excellent safety profile. With event rates <0.6/100 patient-years, lower than those after autologous HSCT, the therapeutic index of approved LV-HSPC advanced therapy medicinal products remains favorable. Ongoing optimization of vector design, conditioning, and long-term surveillance, together with emerging genome-editing platforms, is expected to further mitigate residual risk.
Palacios-Alonso D, Bazan-Peregrino M, Zalacain M
… +10 more, Marrodán L, Mato-Berciano A, Gállego Pérez-Larraya J, Ausejo-Mauleon I, Patiño-García A, Zavala-Romero L, Cascalló M, Alemany R, González-Huarriz M, Alonso MM
Among solid pediatric tumors, brain tumors are the leading cause of cancer-related mortality. While survival rates have improved for certain pediatric brain tumor subtypes, the overall prognosis remains poor. Consequentl...Among solid pediatric tumors, brain tumors are the leading cause of cancer-related mortality. While survival rates have improved for certain pediatric brain tumor subtypes, the overall prognosis remains poor. Consequently, there is an urgent need for novel therapies that are not only effective but also less toxic. Oncolytic viruses have emerged as promising therapeutic agents due to their ability to selectively replicate in tumor cells while sparing healthy tissue and their potential to induce systemic antitumor immune responses. VCN-01 is a replication-competent oncolytic adenovirus whose efficacy has been demonstrated in clinical trials after systemic administration in combination with chemotherapy. Evidence of antitumor activity has also been obtained after intracranial administration in preclinical models of various brain tumors, including high-grade gliomas. However, before progressing to clinical trials for those indications, it is essential to assess the safety of its intracranial administration. In this study, we evaluated the toxicity and biodistribution of VCN-01 following intracranial injection in a Syrian hamster model. Two viral doses were tested: 1.5 × 10 and 1.5 × 10 viral particles (vp)/animal, corresponding to 5 and 50 times the starting clinical dose (10 vp/patient), respectively. Our toxicity analysis revealed a favorable safety profile, with no adverse effects observed following administration. Biodistribution studies demonstrated that VCN-01 primarily remained confined to the brain, with only minimal presence detected in peripheral tissues. The neutralizing antibody response against the virus was stronger in females than in males, correlating with a lower detection of vp in females compared with males. In conclusion, these findings support the safety of intracranial administration of VCN-01 and provide a strong rationale for its further development as a therapeutic option for patients with brain tumors.
Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment for hematological malignancies, yet translating this success to solid tumors remains challenging. Major obstacles include antigen heterogeneity,...Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment for hematological malignancies, yet translating this success to solid tumors remains challenging. Major obstacles include antigen heterogeneity, on-target off-tumor toxicity, limited infiltration and persistence, and the immunosuppressive tumor microenvironment (TME). The present review discusses recent engineering strategies designed to overcome these barriers. Innovations such as affinity-tuned and logic-gated CARs improve specificity and safety, while multi-antigen targeting helps address tumor heterogeneity by avoiding antigen escape. Gene-editing approaches enhance CAR T cell fitness by promoting memory phenotypes, metabolic resilience, and resistance to inhibitory signals imposed by the immunosuppressive TME. Additional modifications improve trafficking, enable extracellular matrix degradation, and reprogram CAR T cells to withstand the hostile conditions of the TME. Together, these advances reflect a growing shift toward rational CAR design and synthetic immunology, with the goal of achieving durable and safe responses in solid tumors. Early clinical trials show promise, and continued translational efforts will be key to unlocking the full therapeutic potential of CAR T cells in this setting.
In gene therapy using adeno-associated virus (AAV) vectors, treatment-induced anti-AAV antibodies pose barriers for re-administration of the same or different AAV serotype vectors. We aimed to investigate whether the adm...In gene therapy using adeno-associated virus (AAV) vectors, treatment-induced anti-AAV antibodies pose barriers for re-administration of the same or different AAV serotype vectors. We aimed to investigate whether the administration of AAV5, AAV8, or AAV9 in Cynomolgus monkeys resulted in the formation of cross-reactive antibodies. To achieve this, we developed a Biacore SPR-based total binding antibody (TAb) assay to identify anti-AAV antibodies in monkey plasma and assess the cross-reactivity of these antibodies against AAV5, AAV8, or AAV9 vectors on a sensor chip. AAV5, AAV8, and AAV9 vectors were immobilized onto the surface of a CM5 sensor chip on Fc2, Fc3, and Fc4 flow cells, respectively, using amine coupling, while Fc1 served as a reference. Plasma samples flowed through four channels, followed by injecting anti-monkey IgG and IgM antibodies to determine the immunoglobulin (Ig) isotypes. We analyzed TAb against the AAV serotypes in the plasma using a Biacore-based TAb assay 29 days after administration to evaluate the anti-AAV antibody responses. The TAb detected by the Biacore-based assay showed cross-reactivity between antibodies against AAV8 and AAV9; however, there was minimal cross-reactivity between antibodies against AAV5 and those against AAV8 or AAV9. Both IgG and IgM TAb were detected at 29 days post-dosing, and the antibody profiles determined by both the Biacore and ELISA platforms were comparable. The Biacore assessment confirmed the absence of cross-reactivity of anti-AAV5 antibodies against AAV8 and AAV9 vectors, and vice versa. This absence of cross-reactive antibodies against a specific AAV serotype indicated the possibility of re-administering a different AAV serotype.
After many years of promising clinical results splashed with some serious adverse events, gene therapy has finally reached maturity, as demonstrated by the increasing number of medicinal products approved for commerciali...After many years of promising clinical results splashed with some serious adverse events, gene therapy has finally reached maturity, as demonstrated by the increasing number of medicinal products approved for commercialization by regulatory authorities. The approved products tackle monogenetic inherited diseases as well as cancer, include both and approaches, and comprise mostly gene additions but also a genome-edited product, demonstrating proof of concept for most gene therapy modalities. Uncertainties still remain, especially on their long-term safety and efficacy, which can only be solved with time. These successes should not lead to self-complacency but rather stimulate the development of necessary improvements concerning manufacturing or the safety and efficacy profile. Here, we review the different categories of gene therapy medicinal products and highlight potential areas for improvement. Products approved for commercialization are taken as the basis for the discussion, since information on their assessment is publicly available. New products and manufacturing approaches under development are also reviewed, with an emphasis on the regulatory challenges expected for some of them.
Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic disorders, cancers, and immune-related conditions through technologies such as viral vector delivery, genome editing, and gene...Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic disorders, cancers, and immune-related conditions through technologies such as viral vector delivery, genome editing, and genetically modified cell therapies. Despite significant advancements, the classification of gene therapy medicinal products (GTMPs) as genetically modified organisms (GMOs) under EU legislation imposes significant regulatory burdens, hindering early and timely patient access to such therapies. Current GMO regulations, originally designed for agricultural biotechnology, require environmental risk assessments (ERAs) and additional approvals, creating delays and increasing costs-with a particularly negative impact on early academic research. This article examines the scientific and regulatory discrepancies in classifying GTMPs as GMOs, arguing that replication-deficient vectors and non-persistent modified cells may not meet the criteria for GMOs. We highlight the negative impact of GMO requirements on clinical trial feasibility in Europe compared to the U.S., where a categorical exclusion from ERA applies to investigational medicinal products. Proposed solutions include adopting a risk-based regulatory model, harmonizing ERA processes under the revised EU Clinical Trials Regulation, and establishing exemptions for low-risk therapies. By aligning regulatory frameworks with scientific evidence, policymakers can accelerate the translation of gene therapies while maintaining safety standards, ultimately improving patient access to these transformative treatments.
Bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos) with their molecular cargo have therapeutic potential for pulmonary fibrosis (PF). This research was performed to uncover how microRNA-31-5p (miR-31-5p), ca...Bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos) with their molecular cargo have therapeutic potential for pulmonary fibrosis (PF). This research was performed to uncover how microRNA-31-5p (miR-31-5p), carried by BMSCs-Exos, affects PF via modulating IGFBP7. C57BL/6 mice were treated with bleomycin (BLM) to induce PF. Pulmonary function was tested, and fibrotic changes in the mouse lung tissues were examined. Levels of fibrosis-related inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, were tested. Mouse BMSCs were isolated and identified, and BMSCs-Exos were obtained by ultracentrifugation. Exosome morphology was observed by transmission electron microscopy, the surface markers were measured, and the expression levels of BMSCs-Exo marker proteins were assessed. The targeting relation between miR-31-5p and IGFBP7 was assessed, and the expression of both was tested. After modeling, mice exhibited decreased functional residual capacity, lung compliance, inspiratory capacity, vital capacity, total lung capacity, and forced vital capacity. After 14 days of BLM induction, thickening of the main tracheal wall, fibroblast accumulation, immune cell infiltration in lung interstitium, and increased collagen deposition were observed. Elevated levels of TNF-α, IL-1β, and IL-6 were also noted. BMSCs-Exos attenuated BLM-induced PF, and BMSCs-Exo-derived miR-31-5p ameliorated PF in mice. miR-31-5p was shown to target IGFBP7, diminishing both transcript and protein levels. IGFBP7 overexpression reversed the ameliorative impact of miR-31-5p on PF in mice. BMSCs-Exos ameliorate PF development by delivering miR-31-5p to repress IGFBP7.
Daniel HDJ, Farzana J, Kannangai R
… +11 more, Kumar S, Lakshmi KM, Jashal CB, Dhamne H, Lahoti V, Kanjwani D, Abraham A, Coleman K, Srivastava A, Srivastava A, Abraham AM
The natural tissue tropism of adeno-associated viruses (AAVs) is being widely exploited in their use as vectors for transgene delivery for gene therapy of human diseases. A major limitation of this approach is the preval...The natural tissue tropism of adeno-associated viruses (AAVs) is being widely exploited in their use as vectors for transgene delivery for gene therapy of human diseases. A major limitation of this approach is the prevalence of antibodies to AAV capsid antigens, which can neutralize the infused vector and thus affect expression of the transgene. There are limited data on the prevalence of such antibodies, total (TAb) and neutralizing (NAb), to determine the eligibility of patients for gene therapy using specific AAV vectors. Using whole capsid ELISA for TAb and transduction inhibition assay (mCherry-based flow cytometry method for AAV5 and luciferase-based assay for AAV8) for NAb, in this study, we have evaluated the seroprevalence of anti-AAV5 and anti-AAV8 antibodies in three groups: healthy individuals (AAV5 = 130, AAV8 = 75), individuals with hemophilia A (AAV5 = 62, AAV8 = 88), and individuals with hemophilia B (AAV5 = 42, AAV8 = 55). The TAb prevalence for AAV5 in the three groups was 77.7%, 90.3%, and 95.2%, respectively, and that for AAV8 was 89.3%, 93.2%, and 92.7%, respectively. The AAV5 NAb seropositivity in the three groups was 89.2%, 100%, and 100% and that against AAV8 was 45.3%, 46.6%, and 45.5%, respectively. To check endpoint titer for AAV5 NAb, 21 out of the 42 hemophilia B samples were screened, using a dilution of 1 in 80 and 1 in 160. Among the AAV5 hemophilia B samples with higher dilutions, 81% of the individuals had a titer of ≤80. Age-stratified AAV5 and 8 TAb and NAb seroprevalence showed high prevalence across all age groups in all three groups of samples screened. High positivity among AAV5 NAb at lower dilutions should be further evaluated for cross-reactivity.
The first marketed gene therapy medicinal products based on adeno-associated virus (AAV-GTMP) show promise for the treatment of various diseases, including rare diseases with unmet medical needs. AAV is traditionally con...The first marketed gene therapy medicinal products based on adeno-associated virus (AAV-GTMP) show promise for the treatment of various diseases, including rare diseases with unmet medical needs. AAV is traditionally considered nonpathogenic to humans, is incapable of self-replication, and, after introduction into various cell types, remains primarily episomal. Several reports have examined the risks of AAV-GTMP, including the risks associated with unintended integration events of elements from the recombinant (r) AAV vector into the host genome. Such events can be one of the steps in the multistep process of tumor formation. To date, rAAV-gene therapy (GT) vectors have not been shown to induce tumors in humans or non-rodent species, and the potential for rAAV-mediated carcinogenicity in humans is still considered theoretical. Nevertheless, a critical review of publicly available scientific data on rAAV-related integration events and a contextualization of the numbers of AAV-GT vector DNA integrations with the absolute burdens of environmental, lifestyle and background tumorigenic genotoxicities is warranted. From a regulatory perspective, it is advisable to implement a long-term safety follow-up for patients who have undergone treatment with high doses of AAV-GT, in accordance with the risk-based approach that has been established for advanced therapy medicinal products.
Iwanowicz A, Boudi A, Seeley C
… +10 more, Sapp E, Miller R, Liu S, Chase K, Shing K, Batista AR, Siena-Esteves M, Aronin N, DiFiglia M, Kegel-Gleason KB
Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT low...Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant allele with expanded cytosine-adenine-guanine (CAG) can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with adeno-associated virus 9 (AAV9) bearing a cDNA for a zinc finger protein (ZFP), which preferentially targets mutant HTT (ZFP-HTT) to repress transcription. Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by ZFP-HTT. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including the subclasses sphingomyelin, ceramide, phosphatidylethanolamine and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride and certain phosphatidylcholine species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD.
The development of efficient and safe muscle-directed gene therapy is an unmet medical need. One of the bottlenecks in muscle-directed gene therapy is the high levels of muscle-targeted transcription required in these af...The development of efficient and safe muscle-directed gene therapy is an unmet medical need. One of the bottlenecks in muscle-directed gene therapy is the high levels of muscle-targeted transcription required in these afflicted target tissues. To circumvent this problem, novel transcriptional cis-regulatory elements (CREs) were identified by transcriptome-wide data-mining that led to a significant increase of transgene expression in skeletal muscle, heart, and diaphragm after adeno-associated viral vector 9 (AAV9) gene transfer. The expression achieved with this CRE arrays outperformed that obtained with several quintessential muscle-targeted promoters, such as the synthetic SPc5-12 and MHCK7 promoters, used in various muscle-targeted gene therapy clinical trials. Incorporation of these CRE arrays led up to a robust 20- to 30-fold increase in luciferase reporter gene expression when compared with the SPc5-12 and MHCK7 promoters. To validate their therapeutic efficacy, AAV9 vectors containing CREs and encoding α-glucosidase () were administered to -/- knockout mice that mimic Pompe disease (glycogen storage disease type II) in human subjects. The CRE arrays resulted in a significant 25-fold increase in GAA protein and mRNA expression in different skeletal muscles, leading to GAA activity levels comparable with those of wild-type mice. Subsequently, this led to a significant decrease in glycogen accumulation and a restoration of centronuclear localization similar to those of wild-type levels. Most importantly, long-term correction of skeletal muscle, diaphragm, and cardiac function was achieved in -/- knockout mice treated with the CRE-containing AAV9 vectors yielding normal phenotypes indistinguishable from wild type. This robust phenotypic correction was demonstrated based on grip and hanging tests, cardiac conductance assays as reflected by PR interval prolongation, and diaphragm contractility function tests. The current study has broad implications for improving outcomes of future clinical trials in Pompe patients and other genetic disorders that affect skeletal muscle, heart, and diaphragm.
In recent decades, medical and scientific advances have led to the development of new therapeutic approaches for Duchenne muscular dystrophy (DMD), including gene therapy (GT), which is currently being evaluated. Recruit...In recent decades, medical and scientific advances have led to the development of new therapeutic approaches for Duchenne muscular dystrophy (DMD), including gene therapy (GT), which is currently being evaluated. Recruiting enough children in clinical trials remains a challenge, depending on parental decisions. Numerous studies have already been carried out to understand these decision-making factors. To date, no study in Europe has been conducted among the various stakeholders lived experience in a DMD GT trial. Our qualitative study explored participants' perceptions using a social representation method and compared them. We recruited 42 participants, divided into 2 groups comprising 21 parents and 21 professionals participating in GNT-014, a DMD natural history study. Each participant was interviewed on four questions about clinical trials, GT, and the facilitators and barriers of the clinical trial pathway. A prototypical and categorical analysis was carried out using "Pointe-au-Sel" software to analyze the data quantitatively. This method highlights which perceptions are shared within the same group and brings out the most important and most frequently evoked terms. We exported the data as a superimposed scatterplot of the representations of both groups for each question. We obtained a total of 453 evocations for the parents' group and a total of 611 evocations for the professionals' group. For clinical trial and GT, are common to the core of both groups but are not at the same level of representation. Parents evoked as the main facilitator and what their child may undergo and become for barriers. For professionals, the facilitators and barriers are centered on the terms that can influence the proper conduct of the trial. These comparative results imply that the vision of the different stakeholders is not totally shared in trial participation. On the contrary, the term GT may also have an influence on professionals, including caregivers.
Several unexpected fatalities in patients who received adeno-associated virus (AAV)-based gene therapies have recently occurred. These tragic events have cast a pall over the entire sector with some stakeholders suggesti...Several unexpected fatalities in patients who received adeno-associated virus (AAV)-based gene therapies have recently occurred. These tragic events have cast a pall over the entire sector with some stakeholders suggesting that AAV is patently unsafe as a gene delivery platform and ought not to be pursued. This conclusion is not warranted.
The study of β-hemoglobinopathies and associated β-globin genes has revealed that genetic elements, such as the Locus Control Region (LCR) or the replication Initiation Region (IR) of the β-globin gene locus, are essenti...The study of β-hemoglobinopathies and associated β-globin genes has revealed that genetic elements, such as the Locus Control Region (LCR) or the replication Initiation Region (IR) of the β-globin gene locus, are essential for the regulation of β-globin genes replication and expression. The LCR at 5' of the β-globin genes plays major role in the intricate regulation of transcription of the "β-like globin genes" expression and in gene therapy protocols by viral gene transfer, ensuring globin gene expression independent from integration site and exerting a critical role in chromatin organization and boundary formation. The IR element, located at the 5' site of the gene promoter, functions as the initiation point for physiological, bidirectional DNA replication, both and within an episomal vector, and induces replication in positions that do not possess such capacity. It enhances plasmid replication, establishment, and transgene expression in the descendants of transfected human CD34+ cells during colony-forming cell assays. A third required genetic element is the promoter of the transgene(s). This is either the gene native promoter or the CD34+ cell-functional ubiquitous promoter spleen focus-forming virus. Both promoters, in studies, can direct accurate, efficient transcription from episomal, S/MAR-based vectors. Mutations in the gene native promoter as well as in LCR and IR lead to β-thalassemia. Another genetic element, the S/MAR, deriving from the 5' of the human β-interferon gene, ensures plasmid nonintegration and long-term nuclear retention in the prototype episomal vector pEPI-1 and derivative episomal vectors. Such S/MAR-based episomal vectors form the basis from which the genetic elements collectively- promoter, LCR, and IR-represent a comprehensive model for the design of efficient episomal vectors with efficient transcription, replication, and long-term nuclear retention of vector for gene therapy applications for the β-hemoglobinopathies within the context of strategy.
Antibody gene transfer offers a promising solution to the high cost and frequent administration of monoclonal antibodies (mAbs), enabling the body to produce its own drugs economically and sustainably. This review addres...Antibody gene transfer offers a promising solution to the high cost and frequent administration of monoclonal antibodies (mAbs), enabling the body to produce its own drugs economically and sustainably. This review addresses the challenges faced by antibody therapies, including economic and environmental impacts, as well as patient-related issues such as efficacy and tolerance. We propose that direct protein production, or autologous production, via plasmid DNA (pDNA) injection may address some of these challenges. This pDNA-based strategy provides a cost-effective alternative while maintaining flexibility and adaptability for various proteins, making it suitable for a wide range of pathological contexts. Additionally, gene therapy with plasmids could reduce the need for frequent injections, improving patient compliance. In this review, we provide an overview of the pioneering studies that introduced the use of pDNA for protein production. We focus on key factors for successful autologous production, such as plasmid design, vectorization, and methods of administration. Finally, we explore various applications where autologous production could serve as a promising alternative for therapeutic antibody treatments.