Human gene therapy最新文献

Adeno-associated virus (AAV)-based vectors are the most commonly used vectors for gene therapy. Wild-type AAV infections occur widely in humans and nonhuman primates (NHPs), and an accurate assessment of preexisting AAV antibodies is crucial for the efficient use of AAV-based gene therapies in preclinical and clinical studies. Cynomolgus macaques (Macaca fascicularis) are well-established preclinical large animal models for evaluating the efficacy and safety of AAV-mediated gene therapies intended for human use. We provide a retrospective evaluation comparing preexisting AAV-neutralizing or total antibody titers against serotypes AAV2, AAV5, AAV8, or AAV9 in cynomolgus macaque cohorts of Asian or Mauritian origin. We used an in vitro neutralizing antibody (NAB) assay to detect NAB titers or an in vitro Meso Scale Discovery-based assay for the quantification of total binding antibodies (TABs) in blood samples. Results were obtained to measure the serostatus of animals. In our analysis, the in vitro NAB assay revealed the lowest seroprevalence for AAV5 (13 ± 15% to 21 ± 6%) independent of origin. In the same assay, Asian animals were highly seropositive against AAV8, followed by AAV2 and AAV9 serotypes (88 ± 13%, 71 ± 10%, 69 ± 9%, respectively). Whereby, the prevalence of seropositivity was lower in animals of Mauritian origin with the highest seroprevalence for AAV9 (58 ± 7%), followed by AAV8 (53 ± 17%) and AAV2 (51 ± 20%) assessed by in vitro TAB assay. Notably, co-prevalences of antibody responses against AAV2, AAV8, and AAV9 serotypes resulted in 39.8% seropositivity (in vitro NAB assay) in NHPs of Asian and in about 32.6% (in vitro TAB assay) of Mauritian origin.

Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is an ultra-rare autosomal recessive disorder caused by mutations in SGCB, the gene that encodes for β-sarcoglycan (SGCB), a component of the dystrophin-associated protein complex that stabilizes muscle fibers during contractions. Bidridistrogene xeboparvovec is an investigational adeno-associated virus-mediated gene transfer therapy designed to deliver a codon-optimized, full-length human SGCB and induce targeted expression of functional human SGCB protein. Interim safety and efficacy data from a clinical trial in patients with LGDM2E/R4 aged 4-15 years (NCT03652259) support further clinical development of bidridistrogene xeboparvovec. However, less is known about the effects of this agent in patients with more advanced LGMD2E/R4, who on average are older and heavier, which prompted their inclusion in studies VOYAGENE (NCT05876780, phase 1) and EMERGENE (NCT06246513, phase 3). In the preclinical study presented here, we delivered bidridistrogene xeboparvovec (0.185 × 10¹³ vg/kg, 0.37 × 10¹³ vg/kg, 0.74 × 10¹³ vg/kg, 1.85 × 10¹³ vg/kg, or 7.4 × 10¹³ vg/kg) to Sgcb-/- mice aged 27-42 weeks (n = 4 per dose) with age-matched saline-treated Sgcb-/- and C57BL/6J mice used as controls. Approximately 12 weeks after administration, we observed SGCB expression and found evidence of reduction in muscle fibrosis, reduction in muscle damage, and restoration of muscle force. Overall, a dose-dependent increase in vector exposure across tissue types was observed, with a nonlinear, exposure-dependent increase in both SGCB expression and functional improvement that reached saturation at 7.4 × 10¹³ vg/kg. Pharmacokinetic and pharmacodynamic analyses demonstrated a robust relationship between vector biodistribution, SGCB expression, and muscle force, further supporting clinical development of bidridistrogene xeboparvovec at the highest dose (7.4 × 10¹³ vg/kg), across a broad LGMD2E/R4 population and regardless of disease progression.

Cell and gene therapies present unique challenges for long-term follow-up as they may lead to adverse events that could emerge years after treatment. Long-term follow-up helps identify potential delayed adverse events, such as oncogenesis or immunogenicity, which might not manifest immediately after treatment. Current regulatory guidelines emphasize a risk-based approach, recommending follow-up durations based on the therapy's mechanism of action between 5 and 15 years. To facilitate long-term monitoring, regulatory authorities recommend the establishment of long-term follow-up protocols, often involving patient registries and supported by real-world data sources to systematically capture and track data from treated patients. These long-term follow-ups are instrumental in both post-approval safety studies and reimbursement decisions, where payers may link payments to treatment outcomes. As the field of cell and gene therapy evolves, regulatory frameworks continue to adapt, balancing the need for comprehensive long-term follow-up with the feasibility of implementation to ensure that therapies are adequately monitored, ensuring patient safety and therapeutic effectiveness over time. However, maintaining patient engagement over extended periods, ensuring high-quality data collection, and addressing privacy concerns present significant challenges. Innovative solutions such as decentralized data collection, digital health technologies, and data linkage with electronic health records aim to alleviate patient burden and improve data reliability.

The adenovirus E3 region's immune-modulating genes (gp19K, Adenovirus Death Protein [ADP], E3B) are frequently modified in oncolytic adenoviruses (OAds) through deletion and transgene insertion like granulocyte-macrophage colony-stimulating factor (GM-CSF). However, the synergistic effects of dual-gene deletions on antitumor efficacy and transgene capacity remain unexplored. To address this, we constructed three E3-modified OAds including OAd5-delgp19K (delgp19K), M20 (delgp19K and ADP), M22-0 (delgp19K and E3B), and their GM-CSF-armed derivatives, systematically evaluating the impact of ADP and E3B deletions on viral replication, tumor cell lysis, immune modulation, and in vivo antitumor activity. Key findings revealed that gp19K/ADP deletion OAd prolonged intracellular viral replication, creating a "viral bomb" effect that delayed cell lysis, evading anti-adenovirus antibodies, sustained GM-CSF expression, and culminating in superior tumor suppression. Gp19K/E3B deletion OAd accelerated viral dissemination but triggered rapid antibody-mediated clearance in immunocompetent hosts, resulting in transient GM-CSF expression and diminished therapeutic persistence. In immunocompetent Syrian hamster Hap-T1 subcutaneous tumor models, gp19K/ADP deletion OAd demonstrated potent tumor inhibition, durable immune microenvironment remodeling, robust viral replication, and evading anti-adenovirus antibodies. These results underscore the critical role of coordinated gp19K/ADP deletion in optimizing viral replication, transgene expression, and immune evasion, providing a strategic framework for engineering next-generation OAds.

Hunter syndrome, also known as mucopolysaccharidosis type II, is an X-linked lysosomal storage disease caused by the deficiency of functional iduronate-2-sulfatase (I2S) enzyme, leading to the accumulation of lysosomal glycosaminoglycans (GAGs) affecting multiple organs. Two-thirds of patients have central nervous system (CNS) manifestations. The current standard of care, enzyme replacement therapy (ERT) via weekly intravenous delivery of recombinant human I2S (rhI2S), does not address the neuropathy in the CNS due to its inability to cross the blood-brain barrier (BBB). Next-generation ERTs consisting of systemically administered rhI2S linked to antibodies that target the transferrin receptor (TfR) have shown clinical efficacy in addressing CNS and peripheral manifestations of disease. We demonstrate here that systemic administration of recombinant AAV9 gene therapy vectors encoding human I2S fusion protein with a TfR1-targeted Variable Heavy chain domain of Heavy chain (VHH) nanobody at the N-terminus normalized brain and cerebrospinal fluid GAGs in symptomatic Ids knockout (Ids KO) mice. This ability to correct toxic substrate accumulation in the CNS was superior to gene therapy vectors expressing I2S with a C-terminal VHH tag or untagged I2S control. The VHH-I2S transgene product demonstrated a broader distribution in the brain parenchyma, coincident with a significant reduction of lysosomal-associated membrane protein 1 immunoreactivity, unlike untagged I2S and I2S-VHH transgene products. These data illuminate strategies to enhance AAV gene therapy vector design and leverage receptor-mediated transcytosis to strategize BBB-penetrating gene therapy for addressing the unmet medical needs of neuronopathic Hunter syndrome.

Gene therapy has become a widely accepted treatment for inherited or acquired genetic diseases. Lentiviral vectors are of particular interest because of their favorable biosafety profile and ability to introduce their therapeutic cargo into non-dividing cells. For clinical use, these viral vectors must be generated under conditions of good manufacturing practice in large quantities, which currently are provided via transient production. A solution for stable, robust, easy to scale, cost-effective, and predictable production of the therapeutic vectors is currently not available. Here, we describe the design, generation, and characterization of EL1-820, a packaging cell line for the stable production of lentiviral self-inactivating (SIN) vectors pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. EL1-820 enables the introduction of a lentiviral SIN-vector expression cassette via Flp-recombinase-mediated cassette exchange (RMCE) into a predefined locus selected for optimal vector production, with expression units designed to improve reliability. EL1-820-based producer clones generated similar titers (1 × 10⁷ TU/mL) from a targeted, single-copy integration of a lenti-GFP or a lenti-chimeric antigen receptor transfer vector as transient production. In initial scale-up experiments, multiple harvests from bioreactors could be achieved, resulting in titers of around 8-9 × 10⁷ TU/mL after tangential flow filtration and a total yield of about 2.3 × 10¹¹ TU. In conclusion, RMCE-based introduction of the transfer construct allows stable, defined, predictable, and safe vector production suitable for clinical applications.

Although the safety of retroviral vector (RV) gene therapy has been improved over the last years, insertional mutagenesis is still a risk factor, as seen in some of the clinical trials targeting hematopoietic stem cells. This highlights the necessity of appropriate preclinical genotoxicity assays. Our group previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) to evaluate the risk of side effects by integrating vectors. In this study, murine hematopoietic stem and progenitor cells are transduced with RVs, and genotoxicity can be detected by a proliferation advantage under limiting dilution conditions (IVIM) or the activation of genes associated with oncogenesis and stem cell-like properties (SAGA). A limitation of SAGA is the costly microarray technology. In this study, we present the digital droplet-based SAGA-Quantification (SAGA-Q) as a cost-efficient and faster alternative. Murine samples transduced with known mutagenic vector designs consistently showed upregulation of genotoxicity predictor genes. Based on a training set of 140 IVIM samples (including untransduced controls and samples transduced with long terminal repeat-driven γRV, SIN-LV.SF, SIN-LV.EFS, SIN-LV.PGK.RAG2, SIN-LV.MND.RAG1, and SIN-LV.MND.RAG2), we used random forest prediction for reliable and fast identification of genotoxic vector designs. The relevance of the predictor genes for the immortalization process was further highlighted by an elevated expression in immortalized clones. By simplifying SAGA to SAGA-Q, we aim to increase the accessibility of genotoxicity assessment and, thus, support the safer translation of gene therapy products to clinical trials.

Genome and RNA editing modalities have revolutionized precision gene therapy, offering a safer alternative to traditional gene replacement approaches. Alpha-1 antitrypsin deficiency (AATD) is a compelling model for precision medicine because the disease mechanism is well defined-mutations in a single gene are responsible for both liver and lung pathology. In this review, we summarize the current preclinical and clinical efforts for AATD, with an emphasis on genome and RNA editing strategies.