Molecular Therapy-Methods & Clinical Development最新文献

This report describes the distribution and transgene expression of two non-cytotoxic, replication-defective (rd) herpes simplex virus (HSV) vectors, JΔNI7 and JΔNI8, following intraperitoneal delivery to newborn mice. The two vectors are functionally defective for all immediate-early genes, and JΔNI8 is further deleted for the UL41 endonuclease (vhs). Both vectors were engineered to express a red luciferase gene from the LAT locus to track vector distribution and gene expression in vivo. A comparison of reporter gene activities under the control of four different promoters in JΔNI7 showed that the strongest expression was achieved with the CAG promoter. Distribution analysis at 1 week post-injection showed transgene expression in multiple tissues, but at 4 weeks, high-level expression was limited to the spinal cord, skin, and muscles. JΔNI8 showed rapid clearance of vector DNA in most tissues, suggesting a role for the vhs gene in vector stability. Compared with wild-type KOS strain injections, JΔNI7-based non-cytotoxic rdHSV did not induce substantial CD45⁺ immune-cell infiltration or tissue destruction, suggesting that our rdHSV vectors are safe. Taken together, these results demonstrate tissue-specific, durable transgene expression following systemic delivery of rdHSV vectors, suggesting their potential for systemic gene therapy for newborns with skin or neuromuscular diseases.

Testing for binding or neutralizing antibodies (NAbs) to adeno-associated virus (AAV) is part of the laboratory assessment of people with hemophilia considering AAV-based gene therapy. We evaluated the natural history of NAb titers to AAV serotype 5 (AAV5) in adult males ≥18 years old with hemophilia B (factor IX ≤ 2%) during the lead-in period of a phase 3 trial prior to the infusion of etranacogene dezaparvovec to characterize NAb in addition to immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-AAV5 binding antibody changes over time. At screening, 48% (32/67) of enrolled participants had detectable NAbs (NAb+) with a median titer of 58 (range: 9-3,440). Participant-specific lead-in periods differed and included discontinuers (median duration: 240 days; range: 1-360). The median intra-participant coefficient of variation of NAb titer over time was 25% (range: 2%-154%). NAb seropositivity was associated with older age (p = 0.0065). For participants with detectable anti-AAV5 NAbs and IgG, there was a high correlation of titers at each visit (median r = 0.96; range: 0.92-0.99). IgM anti-AAV5 antibodies were detectable in only 9% of participants, and seroconversion was infrequent. In conclusion, AAV5 NAb test results were consistent over 6 months, which informs the timing of NAb screening when considering gene therapy for hemophilia B.

Glycogen storage disease type IIIa (GSD IIIa) affects multiple tissues, including liver, heart, and skeletal muscles. We recently reported that an adeno-associated virus serotype 9 vector expressing pullulanase, a bacterial glycogen debranching enzyme, driven by an immunotolerizing dual promoter (AAV9-Dual-Pull), effectively decreased pullulanase-induced cytotoxic T lymphocyte response and corrected disease abnormalities in all major affected tissues in GSD IIIa mice. To reduce effective vector dosages for transgene delivery to skeletal muscles, we packaged the AAV-Dual-Pull vector into two muscle-tropic MyoAAV capsids, MyoAAV4A and MyoAAV4E. Six weeks after administration of the same dose vector (1 × 10¹³ vg/kg), both the MyoAAV vectors demonstrated remarkably greater transduction efficiency and glycogen clearance efficacy in the cardiac and skeletal muscles than the AAV9 vector, accompanied by the improvement of muscle function, reversal of liver abnormalities, and normalization of the disease biomarker, Glc4 in the urine. Furthermore, treatment with the MyoAAV4A-Dual-Pull vector at a 10-fold lower dose (1×10¹² vg/kg) achieved significantly better therapeutic outcomes in the skeletal muscles than the AAV9-Dual-Pull vector at a high dose (1×10¹³ vg/kg). Validation in human liver chimeric mice revealed that the MyoAAV vectors and the AAV9 vectors had a similar efficiency in transducing human hepatocytes, indicating increased translatability for clinical applications.

Autoimmune diseases are chronic conditions where the immune system mistakenly attacks healthy tissues, leading to potentially debilitating symptoms that require lifelong management. There are no cures for autoimmune diseases, and new treatments are urgently needed to improve patient outcomes. This review delves into the compelling advancements and ongoing challenges in harnessing mRNA-lipid nanoparticles (LNPs) as innovative therapies for autoimmune diseases. mRNA-LNPs enable a range of therapeutic approaches to combat autoimmune diseases, including targeted immune cell modulation, tissue regeneration, antigen-specific tolerizing immunotherapy, and in vivo chimeric antigen T cell therapies. To successfully advance this promising class of therapies to the clinic, key challenges must be addressed, such as mitigating unwanted inflammation caused by LNPs, overcoming biological barriers to delivery, and ensuring the long-term safety of mRNA-LNPs specifically in autoimmune contexts. Through their modular design, flexible application, and potential for cost-effective production, mRNA-LNP therapies offer exciting clinical potential to transform the management of autoimmune diseases.

Muscular dystrophies, such as Duchenne muscular dystrophy (DMD), are caused by permanent muscle injuries leading to chronic inflammation, with macrophages harboring an altered inflammatory profile contributing to fibrosis through the secretion of transforming growth factor β1 (TGF-β1). We previously showed that AMP-activated protein kinase (AMPK) activation reduces TGF-β1 secretion by macrophages and improves muscle homeostasis and muscle force in a DMD mouse model. However, direct AMPK activators like compound 991 show strong adverse effects in vivo. To overcome this toxicity, we encapsulated 991 into biodegradable polymeric poly(lactic-co-glycolic) acid (PLGA) nanoparticles for in vivo delivery. We show that 991-loaded PLGA nanoparticles retained drug activity on fibrotic macrophages in vitro and in vivo. In the D2-mdx DMD mouse model, intravenously injected PLGA nanoparticles reached macrophages in gastrocnemius and diaphragm muscles, two severely affected muscles in this model, but not in heart and quadriceps. Chronic intravenous injections of 991-loaded PLGA nanoparticles decreased inflammation in both gastrocnemius and diaphragm, which was associated with TGF-β1 level and fibrosis reduction and increase in myofiber size and muscle mass in the gastrocnemius, without toxicity. These results demonstrate that nanomedicine is an efficient strategy to deliver AMPK activators in vivo to target inflammation and improve the dystrophic muscle phenotype in the gastrocnemius.

Chimeric antigen (Ag) receptor-expressing T regulatory cells (CAR-Treg) offer therapeutic potential for treating autoimmunity, allograft rejection, and graft-versus-host disease (GvHD). HLA-A∗02 (A2) CAR (A2CAR)-expressing natural Treg have shown efficacy in preclinical models and are being evaluated in phase 1/2 trials. In the current study, we utilized homology-directed-repair (HDR)-based gene editing to generate A2CAR-expressing engineered Treg (EngTreg). HDR at the FOXP3 locus in bulk CD4⁺ T cells was used to enforce stable co-expression of endogenous FOXP3 and a chemically inducible interleukin (IL)-2 signaling complex (CISC or IL-2 CISC). A2CAR expression was achieved by lentiviral transduction or via dual-HDR editing targeting A2CAR to the TRAC locus. A2CAR⁺ CISC⁺ EngTreg (A2CAR EngTreg) products displayed a Treg immunophenotype, low secretion of pro-inflammatory cytokines in response to stimulation, and low cytotoxicity toward A2⁺ target cells in vitro. In a xenogeneic GvHD model driven by human A2⁺ peripheral blood mononuclear cells, A2CAR EngTreg showed superior therapeutic efficacy compared with polyclonal EngTreg. Further, in vivo activation of the IL-2 CISC improved efficacy at limiting doses of A2CAR EngTreg. Together, these findings demonstrate efficient generation of Ag-specific EngTreg utilizing CAR as the targeting moiety and efficacy of A2CAR EngTreg in preclinical models, suggesting potential therapeutic benefit for CAR-expressing EngTreg in transplantation and autoimmune diseases.

Autologous hematopoietic stem cell (HSC) gene therapy has gone through remarkable advancements in recent years, especially for the treatment of sickle cell disease (SCD). However, the collection of HSCs from SCD patients requires unique considerations, as granulocyte colony-stimulating factor (G-CSF)-mediated mobilization is contraindicated, and plerixafor-only mobilization is highly variable. Consequently, alternative mobilization regimens that are safe for SCD patients and generate better cell yields are desirable for SCD HSC gene therapy. Here, we evaluated a combination of plerixafor (AMD3100, a CXCR4 antagonist) with GroβT (MGTA-145/GroβT, a CXCR2 agonist) against the current gold-standard G-CSF for HSC gene therapy in nonhuman primates (NHPs) for HSC mobilization, leukapheresis, ex vivo gene editing to reactivate fetal hemoglobin, and transplantation. AMD3100/GroβT rapidly and reliably mobilized phenotypically primitive HSCs within hours even in a G-CSF non-responder. Average CD34/CD90 frequency in the blood and yields after enrichment were comparable in both mobilization regimens. Rapid recovery and robust multilineage long-term engraftment of gene-modified HSCs was achieved in the bone marrow and blood of animals. In summary, AMD3100/GroβT allows highly efficient and reliable mobilization of HSCs, providing a G-CSF-free regimen specifically for SCD but also any other hematological disease or disorder treatable with HSC gene therapy.

[This corrects the article DOI: 10.1016/j.omtm.2025.101504.].

Accurate quantification and characterization of recombinant adeno-associated virus (rAAV) capsid proteins are critical for evaluating product quality and safety, ensuring batch consistency, and informing process development of their manufacture. The capsid consists of three proteins derived from the same gene, and while the mean capsid stoichiometry is nominally 1:1:10 (VP1:VP2:VP3), capsids with different stoichiometries exist. Recent studies show that variations in the capsid stoichiometry can impact vector infectivity. Here, a mass spectrometry (MS)-based method was developed to quantify VP1, VP2, and VP3 in rAAV9 capsids and determine stoichiometry. Additionally, the methodology delivers precise measurement of total capsid content and provides a greater depth of information than traditional ELISA capsid titer measurements. The method could be further refined as a reference method to standardize measurements and assign values to reference materials. Host cell proteins consistent with other findings reported in the literature were also identified and reported. The consistent detection of these host cell proteins across different studies highlights their potential relevance to gene therapy products and the importance of their monitoring. Our report exhibits the utility of MS for precise rAAV characterization and presents the first approach to using MS for the standardized measurement of rAAV across different drug products.