Adoptive transfer of gene-modified autologous T cells, for example, expressing chimeric antigen receptors (CARs), is a promising therapy option for a variety of malignant and nonmalignant diseases. Given their increased risk to develop certain cancer types, namely lymphoma, and the current progress with HIVenv-directed CARs, people living with HIV (PLWH) might particularly profit from CAR-T therapies. At the same time, clinical translation of novel advanced therapy medicinal products remains challenging, partly due to the elaborate ex vivo production under good manufacturing practice (GMP) conditions. This holds especially true for T-cell products from PLWH, which require special manufacturing conditions to suppress HIV replication. We aimed to exemplarily address this limitation in the context of CD19-CAR-T cells for HIV-positive cancer patients. To this end, we developed an integrated, GMP-compliant process for lentiviral CAR transfer and simultaneous knockout of the HIV coreceptor C-C chemokine receptor 5 (CCR5) by mRNA electroporation of our transcription activator-like effector nuclease (CCR5-Uco-hetTALEN) based on the CliniMACS Prodigy. We successfully performed three manufacturing runs to produce CCR5-edited CD19-CAR-T cells on a clinical scale, generating up to 3.9 × 10[9] viable T cells. Thirty-seven percent to 55% of the T cells in the final product expressed functional CD19-CARs, the overall CCR5 editing rates ranged from 42% to 54%. The third large-scale process was performed in the presence of the antiretroviral drug saquinavir to suppress potential viral replication during manufacturing of cells from HIV-positive patients. In summary, we have developed a GMP-compliant manufacturing process for gene-edited CAR-T cells under antiretroviral suppression, which might help to accelerate the translation of cellular immunotherapies, namely CAR-T cells for patients living with HIV in the future.

In recent years, gene therapy has emerged as a major frontier in biomedicine, with translation from basic research to clinical application accelerating worldwide. Clinical research in this field has also expanded substantially in China. Prior to the implementation of the new regulatory framework in 2026, investigator-initiated clinical research played an important role in early clinical exploration and technology validation, alongside product-oriented clinical trials conducted under the pharmaceutical regulatory pathway. However, under the previous framework, the pathways for translating research outcomes into clinical use and their interface with product development remained insufficiently defined. Against this background, this review systematically examines the evolution of China's regulatory framework for gene therapy clinical research and translation, with particular attention to the institutional changes introduced by the Regulation on the Administration of Clinical Research and Translational Application of New Biomedical Technology (the "818 Regulation"). Relevant regulatory approaches in the United States, the European Union, Japan, and South Korea are also examined for comparative context. The 818 Regulation provides a more clearly defined institutional framework for gene therapy clinical research and translation, while also introducing new practical requirements for research implementation. In particular, initiating institutions and clinical research institutions need to strengthen their professional implementation capacity and cross-departmental coordination. For innovative technologies at an early stage of development, where the ultimate translational direction may not yet be fully determined, greater attention should be given to the assessment of technological characteristics and prospective planning of translational pathways. Differences in data standards, quality-control requirements, and evidentiary expectations across translational pathways also place greater demands on evidence continuity and the future usability of research data. Accordingly, under the new regulatory environment, initiating institutions and clinical research institutions should strengthen internal governance and professional management, prospectively assess technological characteristics and intended translational objectives, appropriately plan research and translational pathways, and adopt high-standard data governance to improve the completeness, reliability, and traceability of research evidence. Such efforts would preserve opportunities for future data linkage and reuse, as well as the potential acceptability of data across different translational pathways. Effective institutional adaptation to the new framework will be critical to realizing the potential benefits of the 818 Regulation and improving the quality and efficiency of gene therapy clinical translation in China.

Biological cell and gene approaches have great potential to provide durable, disease-modifying therapies for, as yet, incurable neurological disorders. The central nervous system (CNS), unlike other organ systems, however, poses unique challenges for access for biological therapies. Access challenges include physical barriers such as the blood-brain barrier, technical barriers such as procedural considerations, and variability in response to biological therapies that are dependent on route of delivery. In this review, these challenges are discussed along with the practicalities of therapeutic access for biological therapies to the CNS by systemic intravenous (IV) delivery, intrathecal delivery, and direct intraparenchymal delivery in the context of epilepsy and with relevance to other neurological disorders. In the context of IV delivery, the practicalities of CNS target and off-target systemic effects are discussed. In the context of intrathecal delivery, the practicalities of procedural access, cerebrospinal fluid dynamics, and tissue penetrance are reviewed. Finally, in the context of direct intraparenchymal delivery, the technical aspects of therapy administration, local and systemic spread, and immune response are highlighted. Each delivery method provides a different balance of procedural, targeting, and inflammatory considerations, which may affect future therapeutic agent selection and trial design. This framework highlights that delivery-route selection should be individualized according to therapeutic modality, target anatomy, desired distribution, procedural risk, and clinical infrastructure.

Recombinant adeno-associated virus (rAAV) vectors are widely used for gene delivery but show limited efficiency in immune cells, including T lymphocytes and natural killer (NK) cells. To overcome this barrier, we have developed a CD7-targeted rAAV vector (CD7-AAV6/9) featuring a nanobody-fused hybrid capsid derived from a rationally selected chimeric combination of AAV6 and AAV9. CD7-AAV6/9 enables efficient and selective transduction of immortalized and primary human T and NK cells in vitro and in vivo in a humanized mouse model, achieves high production titers, and exhibits markedly reduced off-target transduction compared with wild-type serotypes. Incorporation of a human gene-derived intron into the vector genome to overcome host-mediated transcriptional repression enables robust transgene expression in human CD7[+] T lymphocyte and NK cell populations. Together, our findings establish an integrated capsid-genome design framework for targeting human T and NK cells, notoriously challenging immune cell populations for gene therapy, and provide a versatile platform readily adaptable to alternative surface markers and therapeutic payloads.

Duchenne muscular dystrophy (DMD) is a devastating X-linked disorder caused by out-of-frame mutations in the DMD gene, most commonly large deletions or duplications, as well as nonsense and splice site mutations, that result in the absence of functional dystrophin protein. These mutations lead to progressive skeletal and cardiac muscle failure. In particular, exon 52 of the DMD gene represents a mutational hotspot in DMD patients. Deletion of exon 52 (DMDΔ52) disrupts the reading frame, leading to premature termination of translation. Here, we used a dual recombinant adeno-associated virus (rAAV) system employing an intein-mediated split strategy to deliver ABE8e using the PAM-less SpRY Cas9 nickase for adenine base editing of splice acceptor sites (SAS) in the dystrophin gene. Targeting the SAS of exon 51 or exon 53 in a DMDΔ52 background aims to induce exon skipping, thereby restoring the open reading frame, in effect converting the severe DMD phenotype into a milder Becker muscular dystrophy (BMD)-like phenotype. We systematically screened sgRNAs in porcine kidney fibroblasts and human embryonic kidney cells (HEK293T), identifying a guide RNA targeting the SAS of exon 53 as the most effective candidate with high on-target editing efficacies and minimal bystander editing. In human DMDΔ52 iPSC-derived cardiomyocytes cultivated as 2D monolayers, high editing efficiencies were achieved with the optimized 1:2 ratio of N-Terminus to C-Terminus. Functional assessment in 3D engineered heart patches revealed a trend toward normalization of the arrhythmic DMD phenotype with an increase in the effective refractory period (ERP) and a reduction in arrhythmic load compared with untreated DMD patches, despite lower editing efficacy in the 3D setting. These findings suggest that even modest levels of base editing-mediated exon skipping may ameliorate the DMD cardiac phenotype toward a BMD-like state, supporting the translational potential of this dual rAAV base editing approach for DMD cardiomyopathy.

Advancing the development of specific gene therapies for rare genetic eye disorders is a major challenge in modern translational vision research. It requires integrated clinical, molecular, mechanistic, and regulatory expertise, yet these aspects are often addressed in isolation. Combining complementary skills, knowledge, and expertise within an integrated, highly interactive research framework has the capacity to drive and advance therapeutic development. Moreover, early-stage involvement of patients and targeted clinical observation, alongside molecular and therapeutic research, potentially enable clinical trial readiness and support the translation of preclinical therapeutic innovations into clinical care. In this Perspective article, we present the InsightRP2 framework, an integrated translational strategy that incorporates clinical data, artificial intelligence-supported imaging analysis, experimental disease modeling, and adeno-associated virus design with the aim to facilitate the development of a targeted gene therapy for RP2-associated retinitis pigmentosa.

Natural killer (NK) cells lead a rapid and potent innate immune response following exposure to infectious agents, but their activity may also contribute to chronic inflammation and augmented disease pathogenesis. Previous reports have shown that Janus kinase 3 inhibitors or anti-IL-15 monoclonal antibodies potently deplete NK cells in nonhuman primates (NHPs). However, they also disrupt T cell homeostasis, making it difficult to use these reagents to delineate the nonredundant role of NK cells in NHPs. To induce the specific depletion of NK cells in vivo, we designed chimeric antigen receptor (CAR) T cells targeting either a broad (CD16[+]) or narrow (NKG2A/C[+]) proportion of NK-inclusive innate immune subsets in rhesus macaques (RM). Ex vivo screening assays identified anti-CD16 and anti-NKG2A/C CAR constructs that potently and specifically depleted CD16[+] and NKG2A/C[+] cells, respectively, in the presence of autologous peripheral blood mononuclear cells (PBMCs). Adoptive transfer of autologous anti-NKG2A/C CARs was associated with a profound depletion of NK cells in blood and tissues of RM. NK cell depletion occurred despite modest anti-NKG2A/C CAR T cell expansion and persistence. No target cell depletion was observed following anti-CD16 CAR T-cell infusion, potentially due to the CAR T-cell dose and/or infusion frequency, animal-specific factors, or limitations of the CAR construct. Collectively, our pioneering study expands the application of CAR T cells as a basic science tool and further advances the NHP model for studying NK cell activity in disease contexts, such as infectious diseases and allotransplantation.

Gene therapy achieved through genetic manipulation and transfer of hematopoietic stem and progenitor cells (HSPC) is becoming an increasingly attractive option for personalized medicine. In the current clinical scenarios, use is restricted to overcoming genetic disorders, but it is envisioned that more diverse applications will be developed in the future. Immunotherapy of malignant disease already employs non-HSPC-based gene therapies, and there is a body of evidence that HSPC-mediated gene therapy has potential as a powerful immunotherapeutic for some diseases of immune dysregulation such as autoimmune disease and allergies. Administration of HSPC gene-engineered to encode antigen(s) induces antigen-specific immune tolerance in animal models. To translate this technology, we sought to identify whether human dendritic cells (DC) and other antigen-presenting cell populations derived from lentivirally transduced HSPC would express, process, and present antigens of interest. Lentiviral vectors were generated that encoded preproinsulin, a relevant autoantigen implicated in type 1 diabetes, or a control protein carrying a viral epitope along with reporter genes under transcriptional control of ubiquitous or antigen-presenting cell-directed promoters. In vitro, DC were differentiated from transduced HSPC and tested in antigen presentation assays using Jurkat lines carrying relevant type 1 diabetes-associated T cell receptors. In vivo, transduced HSPC were used to generate humanized mice, and the development of a range of antigen-presenting cells and their expression of lentivirus-encoded genes were determined. Antigen-presenting cell progeny of transduced HSPC expressed antigens and reporter genes both in vitro and in vivo, and in vitro-generated DC presented lentivirally encoded antigens. The studies characterize human chimerism and therapeutic gene expression in a new model and provide support for the use of HSPC-based gene therapies for immune dysregulation in humans.

Total elimination of replication-competent human immunodeficiency virus type 1 (HIV-1) remains a major clinical challenge, in part due to random integration of the proviral DNA into host cell chromosomes, which enables lifelong persistence and production of progeny. Although antiretroviral therapies (ARTs) suppress viral replication, they cannot eliminate integrated proviral DNA, which remains a fundamental obstacle to achieving a cure. To overcome this problem, we developed a combinatorial clustered regularly interspaced short palindromic repeats-Cas9 gene editing strategy to disrupt viral replication and inactivate host factors essential for HIV-1 entry and spread. This approach targets C-C chemokine receptor type 5 (CCR5), a chemokine receptor central to HIV-1 host cell entry, and mannosyl-oligosaccharide glucosidase (MOGS), a key enzyme in glycoprotein processing that modifies the HIV-1 envelope glycoprotein gp120, facilitating receptor engagement, viral entry, and morphogenesis of infectious virion. We demonstrate that our strategy, which includes editing of the integrated proviral DNA, in concert with two cellular genes whose products facilitate viral entry, results in robust suppression of viral replication in vitro and in ex vivo-infected cells. Using transmission electron microscopy, HIV-1 p24 ELISA, and GFP-based viral infection assays, we show that the combination knockout of CCR5, MOGS, and viral sequences profoundly reduces HIV-1 replication in an ex vivo cellular model, that is, HIV-1-infected peripheral blood mononuclear human cells, thus offering a pathway to launch further preclinical studies.

This retrospective follow-on study reviewed 25 consecutive patients with genetically confirmed choroideremia who were treated with subretinal AAV2-CAG-REP1 in dose-escalation gene therapy clinical trials conducted at Oxford University Hospitals NHS Foundation Trust and the University Eye Hospital Tübingen. Six patients in Oxford received 1010 vector genomes in 100 µL, while a further 19 patients across both centers received 1011 vector genomes in 100 µL. Although the primary safety outcomes of both investigator-led academic studies have previously been reported, retinal imaging changes were observed during follow-up, prompting a more detailed characterization of these findings. Twelve of 25 treated eyes developed imaging features consistent with perivascular astrocytic gliosis during follow-up, representing 48% of the cohort. All affected eyes had received the higher dose, accounting for 12 of 19 eyes treated with 1011 vector genomes. No patients receiving the lower dose (1.0 × 1010 vector genomes) developed this phenotype. The lesions followed the major vascular arcades toward the optic disc, and optical coherence tomography demonstrated inner retinal thickening with a fine, granular hyperreflective texture, without intraretinal cysts. Longitudinal imaging showed no progressive expansion of the involved regions. Clinically, the findings were silent: no patient reported uveitic symptoms, ocular examinations were largely quiet, and no peripheral cellular immune responses were detected in patients with available assays. Perivascular astrocytic gliosis therefore appears to be a common, clinically silent, nonprogressive, and noninflammatory retinal response after subretinal AAV2-CAG-REP1 gene therapy for choroideremia at a dose of 1011 vector genomes, which is generally accepted as the therapeutic human dose for targeting photoreceptors. We hypothesize that local REP1 overexpression in perivascular astrocytes suppresses FOXO3-mediated apoptosis and engages EGFR/STAT3 signaling, driving benign perivascular astrocytic accumulation analogous to retinal astrocytic hamartomas seen in phacomatoses. Gene therapy involving ubiquitous promoters may have unexpected effects, and while benign, may not be predicted.

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.