Molecular Therapy-Methods & Clinical Development最新文献

Despite the clinical success of redirected T cells in the setting of cancer adoptive cell immunotherapy, patients may exhibit resistance to treatment, resulting in uncontrolled disease and relapses. This phenomenon partly relies on impaired ex vivo-produced T cell metabolic fitness, including a decreased respiratory reserve, as well as a greater sensitivity to tumor-mediated metabolic stress. To improve the respiratory capacity of cultured T cells, we sought to target the nicotinamide adenine dinucleotide/sirtuine-1/reactive oxygen species (ROS) axis through supplementation of culture medium with resveratrol. Resveratrol-treated T cells display broader respiratory capacities, along with sustained ROS control ability. Strikingly, we reveal that the effect of resveratrol on T cells is restricted to cytomegalovirus (CMV)-exposed donors, a virus known to promote immune aging. Herein, CMV prior infection is associated with the influence of terminally differentiated T cells on the fate of companion T cell subsets. Moreover, beyond resveratrol's effect on redirected T cell metabolic features, it provides a functional anti-tumor advantage to these CMV-seropositive donor-derived T cells, in a third-generation CD123-specific chimeric antigen receptor-T cell in vitro model. This highlights the necessity to consider patient's intrinsic attributes, especially immune aging-related ones, when assessing new T cell production processes to improve clinical efficacy, pushing the limits of personalized medicine.

The lysosomal storage disease alpha-mannosidosis (AMD) is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions in the brain and other tissues. Animal models of lysosomal diseases have demonstrated the benefit of early treatment; however, many human diagnoses occur after patients are symptomatic. We demonstrate here partial correction of the globally distributed storage lesions by infusion of a high dose of adeno-associated virus 1-feline alpha-mannosidase into the cerebrospinal fluid via the cisterna magna in the gyrencephalic AMD cat brain at different ages, corresponding with different stages of disease progression. Significant improvements in clinical parameters were observed, and partial correction was documented pre mortem by non-invasive magnetic resonance spectroscopy and diffusion tensor imaging. Post mortem analysis demonstrated that higher levels of lysosomal alpha-mannosidase activity in animals treated at 12 weeks of age did not translate into increased correction of lysosomal storage lesions throughout the brain when compared with animals treated at earlier time points. These results further demonstrate the importance of early detection and treatment of a lysosomal storage disease to successful outcomes.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition (CRISPRi) using several epigenetic regulators successfully targets and represses DUX4 in FSHD myocytes with no adverse effects on the muscle transcriptome. However, for translatability, adeno-associated virus (AAV)-mediated gene therapies must include all components within a single vector that expresses at therapeutic levels in all target tissues. Thus, we have re-engineered our CRISPRi platform to an all-in-one (AIO) single-vector system. Key modifications were as follows: (1) optimizing our regulatory cassette for high activity in all skeletal muscles while maintaining low or no activity in FSHD-unaffected tissues and (2) minimizing epigenetic regulators to their essential functional repressor domains, together creating enough space to include a single guide RNA (sgRNA) expression cassette within the AAV packaging limit. Targeting these AIO cargos to DUX4 suppressed pathogenic gene expression in FSHD1 and FSHD2 myocytes. Importantly, AAV-mediated delivery of our best AIO cargo also repressed DUX4 in an FSHD xenograft mouse model. These therapeutic cassettes represent a clinically relevant CRISPRi platform for gene therapy of FSHD and a useful proof-of-concept platform for other skeletal muscle gene therapies.

Pompe disease (PD) is a multisystemic progressive disease caused by acid-alpha glucosidase (GAA) deficiency. Patients display a spectrum of phenotypes ranging from the severe, rapidly progressive infantile-onset PD (IOPD) form to the slower progressing late-onset PD (LOPD). Enzyme replacement therapies (ERTs) are the only approved treatments; they decrease mortality in IOPD while maintaining or improving motor and respiratory function in LOPD. These therapies do not cross the blood-brain barrier (BBB) and the long-term survival of ERT-treated IOPD patients revealed underlying neurological disease. We used PD as a model to evaluate delivery of GAA across the BBB using anti-transferrin receptor (TfR) antibodies and show robust glycogen clearance and reduced neuroinflammation in the CNS of mice with PD. Importantly, anti-TfR-GAA treatment resulted in superior glycogen clearance in skeletal muscle compared with two approved ERTs. Glyco-chemical exchange saturation transfer/nuclear overhauser effect magnetic resonance imaging successfully estimated glycogen content in Pompe mouse brains, providing a non-invasive and translational method to evaluate brain-penetrant therapies for PD. Moreover, hexose tetrasaccharide was elevated in the cerebrospinal fluid of diseased mice and mirrored CNS glycogen levels, suggesting it may be a promising CNS biomarker. These data support the theory that the neurogenic and myogenic manifestations of PD can be effectively treated by anti-TfR-GAA.

AAV-based gene therapy represents an attractive treatment for hereditary peripheral neuropathies Charcot-Marie-Tooth diseases. We recently showed that AAV2/9 vector-expressing GFP, locally injected into mouse and rat sciatic nerves, transduced a large amount of myelinating Schwann cells (mSCs). The local delivery of a similar vector expressing a small hairpin RNA (shRNA) targeting PMP22 mRNA prevented CMT1A disease in a rat disease model. Here, we investigated the regional anesthesia standard perineural injection route in non-human primates using AAV2/9 expressing GFP and AAV2/9 expressing shRNA targeting human and monkey PMP22 mRNA. Injecting at multiple sites to cover the largest part of arm and leg nerves, we found that AAV2/9 easily crossed perineurium sheaths to transduce up to 90% of mSCs. Injections on 10 nerves in each animal did not generate any adverse effects. Injected nerves functioned properly, and fine motor dexterity remained unaffected. Enzymatic, metabolite, and blood cytometry were marginally affected, and no systemic inflammation was detected. Locally, we detected a slight to moderate perineural infiltration of monocytes only with vector-expressing GFP. Vector-expressing PMP22 shRNA decreased PMP22 expression in a dose-dependent manner and levels remained physiological. Perineural injections appear to be safe, well-tolerated, and efficient to deliver AAV2/9-based gene therapy vector to treat Charcot-Marie-Tooth diseases.

Assessment of the biodistribution and shedding is required for the development of viral vector-based vaccines. Vector copy number is commonly quantified using digital PCR (dPCR) or quantitative PCR (qPCR). However, the regulatory guidelines for bioanalytical PCR assays have not been released at present. To consider the future setting of acceptance criteria for method validation in a guideline, we aimed to develop and validate a method for quantifying a model adenoviral (Ad) vector vaccine (Ad-hCovHKU1S-2A-GFP) using dPCR and qPCR in biological matrices and assessed its biodistribution in mice. Primers and probes were designed for the region between the sequences of the Ad and HKU1S. Method development and validation were performed using dPCR and qPCR with 1 μg of mouse genomic DNA (gDNA)/reaction. The validation parameters and their acceptance criteria were pre-defined as possible values referred from the literature. Lower limits of quantitation were set as 12 copies and 48 copies/reaction for dPCR and qPCR, respectively. Both dPCR and qPCR met the pre-defined acceptance criteria for intra- and inter-run accuracy and precision. Cross-validation showed similar quantitative results using both dPCR and qPCR. The pre-defined acceptance criteria on dPCR and qPCR may be applicable to the biodistribution and shedding of viral vector-based vaccines.

Leukodystrophies form a group of rare genetic disorders characterized by the progressive degeneration of white matter in the brain, often presenting in childhood with life-threatening consequences. Current therapeutic options are limited, particularly after symptom onset, and delayed diagnosis exacerbates disease progression. Stem cell and gene therapies have emerged as promising strategies to address these challenges. Neural stem cells (NSCs) and hematopoietic stem cells (HSCs) offer potential for CNS repair through differentiation into neurons and/or glial cells. Human pluripotent stem cells (hPSCs) provide a valuable model for studying disease mechanisms, drug screening, and therapeutic development. Additionally, mesenchymal stem cells (MSCs) exhibit immunomodulatory and secretory properties that may confer therapeutic benefits. Advanced gene therapies, including stem cell-based gene therapy and adeno-associated virus (AAV)-mediated gene replacement therapy (GRT), hold promise for curative interventions. This review highlights recent advances and challenges in stem cell and gene therapies for leukodystrophies, proposing a strategic integration of these approaches to maximize therapeutic efficacy by addressing their respective strengths and limitations.

Tissue fibrosis is a pathological feature of many diseases including muscular dystrophies such as Duchenne muscular dystrophy (DMD). Fibrosis may limit the effectiveness of gene therapy in muscle impacting on viral dosing but direct evidence is lacking. Strategies to reduce skeletal muscle fibrosis are limited. The fibrosis Fap gene is over-expressed in the skeletal muscles of a severe mouse model of DMD, suggesting that cells expressing membrane fibroblast activation protein (FAP) could be targeted by chimeric antigen receptor (CAR)-T cells. Two consecutive administrations of FAP-specific CAR-T cells in the severe DMD model reduced collagen deposits and fibrotic biomarkers and also reduced the number of FAP-positive cells in muscle. Single cell transcriptomics revealed that FAP-CAR-T cells triggered cellular interactions with otherwise inactive muscle resident macrophages and depleted specific subsets of FAP-highly-expressing fibro-adipogenic progenitor cells, pointing to their importance in the fibrosis process. Reducing fibrosis with FAP-CAR-T cells enhanced adeno-associated virus (AAV) microdystrophin gene transfer in the model by increasing vector copies, demonstrating that fibrosis is a restriction factor for AAV gene delivery in skeletal muscle. These results provide novel insights into therapeutic strategies for DMD or other fibrotic diseases.

Mucopolysaccharidosis type I (MPS-I) is a rare pediatric disease caused by mutations in the α-L-iduronidase (IDUA) gene encoding for a lysosomal enzyme involved in glycosaminoglycan metabolism. While newborns with the severe Hurler variant are usually asymptomatic at birth, progressive disease manifestations emerge early in life. Since previous studies on lentiviral vector gene therapy (GT) in Hurler patients have demonstrated superior metabolic correction and early beneficial clinical effects, we investigated whether applying this GT approach during the neonatal period could be effective in preventing disease pathology before it becomes irreversible. Thus, newborn MPS-I mice were transplanted with affected bone marrow-derived progenitor cells transduced with an IDUA-encoding lentiviral vector. Treated animals displayed increased IDUA levels, significantly reducing substrate accumulation in analyzed organs, indicating metabolic correction. Skeletal manifestations, typically resistant to conventional therapies, showed improvements at radiographic and histological levels post-treatment. Additionally, a decrease in brain cortex vacuolization and inflammation suggested neurological amelioration. Overall, this study provides a proof of principle demonstrating the effectiveness of neonatal ex vivo GT in MPS-I mice and supports its potential for further optimization at the pre-clinical level.

B cells have immense potential as gene therapy carriers because of their ability to secrete therapeutic proteins and mediate immunological memory and tolerogenic functions. However, efficient gene delivery into primary B cells remains a challenge, as DNA electroporation frequently induces significant cell death. Here, we demonstrate that primary murine B cells undergo apoptosis and pyroptosis after DNA electroporation, which is driven primarily by activation of the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway in response to cytoplasmic double-stranded DNA (dsDNA). To overcome this, we pretreated B cells with a caspase inhibitor, which increased cell viability and improved DNA delivery and knock-in efficiency. Notably, compared with the inhibition of either apoptosis or pyroptosis alone, treatment with the pan-caspase inhibitor Boc-D-FMK resulted in a more significant improvement in cell viability and DNA electroporation efficiency. This approach significantly increased the expression and secretion of human erythropoietin (hEPO) both in vitro and in vivo. These findings not only elucidate the mechanisms underlying DNA-electroporation-induced cell death in B cells but also establish a strategy to optimize B-cell-based gene therapies and therapeutic protein production.