Molecular Therapy-Methods & Clinical Development最新文献

Multiple sclerosis (MS) is a neurological disease characterized by demyelinating lesions in the CNS. This study investigated whether a minimally invasive adeno-associated virus (AAV) vector (AAV.PHP.eB) can direct transgene expression in CNS cell types relevant to MS, including astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), microglia, and neurons in experimental autoimmune encephalitis, a widely used MS model. In vivo bioluminescence imaging and histological analysis following AAV.PHP.eB-mediated gene delivery in healthy mice using the ubiquitous CAG promoter and five neural promoters (MBP, Sox10, hSyn1, gfa2, and gfaABC1D) revealed long-term, robust, and cell-type-specific activity across the brain and spinal cord. AAV.PHP.eB is capable of traversing the blood-brain barrier in experimental autoimmune encephalitis (EAE) and directs sustained and cell-type-specific transgene expression for the MBP, Sox10, hSyn1, and gfaABC1D promoters. The MBP and Sox10 promoters directed transgene expression in oligodendroglia around and within inflammatory demyelinating lesions, whereas the gfaABC1D promoter directs transgene expression in gray and white matter astrocytes and hSyn1 in neurons. The neural promoters were minimally active in the periphery, with the exception of gfa2. This methodological study is a first step toward the development of minimally invasive gene therapy to promote myelin repair and/or suppress inflammation in MS.

Ribonucleic acid (RNA)-mediated gene regulation is being widely investigated in preclinical and clinical studies owing to its immense potential for treating a broad spectrum of medical conditions. Because unmodified RNA molecules are rapidly degraded and cleared from circulation, carriers such as lipid nanoparticles (LNPs) are used to protect them, deliver them to target tissues, and facilitate their cellular entry and endosomal escape. However, most LNPs are trafficked to the liver upon intravenous administration, so new approaches are being explored to facilitate extrahepatic delivery. Recent studies suggest that modifying RNA-loaded LNPs with cell-derived phospholipid membranes can alter their biodistribution, cellular entry, and gene regulation potency, resulting in improved therapeutic outcomes. This review discusses the status of membrane-modified LNPs for RNA delivery, highlights key design criteria for these systems, and provides perspectives on the path toward clinical implementation. With further development, these exciting tools could enable RNA-based therapies to realize their full clinical potential.

In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A^∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

Congenital leptin deficiency is a monogenic disease originated from adipose tissue, causing hyperphagia, severe obesity, and hyperinsulinemia. Moreover, most forms of lipodystrophy syndromes exhibit leptin deficiency. Leptin replacement therapy with leptin protein analog requires frequent injection for life and is extremely expensive. We previously reported that a single intraperitoneal injection of an adipose-targeting Rec2-leptin adeno-associated virus (AAV) vector normalized metabolic syndromes in the leptin deficient ob/ob mice. Here, we conducted a dose-deescalating study with four doses (2E10, 1E10, 5E9, and 1E9 viral genome per mouse) in ob/ob mice and extended in vivo monitoring to 27 weeks post dosing. Rec2-leptin at all doses normalized excessive weight gain, hyperphagia, obesity, low core temperature, glucose intolerance, and hyperinsulinemia in the ob/ob mice. Transgene expression was restricted in the targeting visceral adipose tissues and sustained throughout the 27-weeks study. Rec2-leptin at the lowest dose 1E9 viral genome (vg)/mouse restored the circulating leptin level to 18% of the normal level of wild-type (WT) mice. Rec2-leptin at all doses reversed liver steatosis and pancreatic islet hyperplasia. Behavioral assessment, serum chemistry, and histopathology noted no significant adverse effects attributed to Rec2-leptin. This study demonstrates that Rec2-leptin is safe and highly efficacious, supporting further development for genetic or acquired leptin deficiency.

Safe and efficient nucleic acid delivery to targeted cell populations remains a challenge in the fields of cell and gene therapy. Toward this end, we attempted to utilize the "DogTag-DogCatcher" system to target adenoviral vectors. "DogTag" is a short peptide that forms a spontaneous isopeptide bond upon mixing with its partner protein, "DogCatcher." We genetically incorporated the DogTag peptide into the protein responsible for initial binding of the virus to its target cell, the fiber. This allowed permanent linking of DogCatcher-fused single-domain or single-chain antibodies at the fiber. This modification allowed simple, effective, and exclusive targeting of the vector to cells bound by the linked antibody. This enhanced gene transfer into primary B and T cells by up to 60-fold in vitro and 2- to 3-fold in vivo in mice without other alterations to vector tropism. Although the system's in vivo performance is currently suboptimal and additional engineering is needed prior to further use, these studies form the basis of a novel method for targeting adenovirus that can be combined with additional well-characterized adenovirus modifications toward applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

The ability to deliver a therapeutic sequence to a specific cell type in the human brain would make possible innumerable therapeutic options for some of our most challenging diseases; however, studies on adeno-associated virus (AAV) vector tropism have generally relied on animal models with limited translational utility. For this reason, establishing the tropism of common adeno-associated virus (AAV) vectors in living human brain tissue serves as an important baseline for further optimization, as well as a determination of human brain cell types transduced by clinically approved gene therapy vectors AAV2 and AAV9. We have adapted an ex vivo organotypic model to evaluate AAV transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

Saponins are a class of phytocompounds known for their amphiphilic properties. Here, we have evaluated incorporation of 40 saponins into a model lipid nanoparticle (LNP) formulation and evaluated their performance in vitro and in vivo. We reasoned that the surfactant activity of saponins could be beneficial in the context of cell and gene therapy due to the disruption of the intracellular membranes. We established formulation methodology to incorporate saponins into LNPs and measured their endosomal disruption and transfection efficiency with DNA barcode and mRNA cargoes. We identified two saponins-quillaic acid and macranthoidin B-that increase the LNP transfection efficiency and endosomal disruption. Saponin formulations demonstrated cargo-dependent activation of the innate immune system, as measured by the cell-based assays of interferon regulatory factor (IRF) and NF-κB pathway activation. Quillaic acid LNPs resulted in higher titers of anti-OVA IgG2a in the vaccination studies compared to a "naive" LNP control, which suggests a more Th1-biased immunopathology of these vaccines. As Th2-biased vaccines can trigger an allergic response, an mRNA vaccine with a balanced Th1/Th2 response is more favorable for translation into the clinic. Overall, quillaic acid may serve as an adjuvant for mRNA vaccines and potentially decrease the risk of vaccine-associated adverse events.

Adeno-associated viral (AAV) vectors have been established as a safe and effective delivery vehicle for gene therapy. However, current methods for AAV production using adherent approaches are suboptimal due to their reliance on a substantial number of plastic-based flasks, manual labor, and a significant manufacturing footprint. Consequently, a protocol for generating AAV2 was developed on the Quantum, a semi-automated closed hollow-fiber bioreactor platform. In this system, Human Embryonic Kidney 293T cells were successfully expanded and transfected to produce an average crude AAV2 titer of 4.92 × 10¹⁴ viral particles and 6.81 × 10¹³ viral genomes from 1.2 L of harvested cell lysate. The application of a standard AAV downstream process confirmed normal processability of the material. A cost of goods model comparing the Quantum bioreactor with the current standard HYPERStack36 and Corning CellSTACK 10-layer systems demonstrated that the Quantum bioreactor reduced the number of open steps by more than 40-fold, production time by up to 3.6-fold (HYPERStack36) and 7.5-fold (CellSTACK 10-layer), and costs by up to 2-fold (HYPERStack36) and 20.7-fold (CellSTACK 10-layer). Therefore, the Quantum bioreactor is an effective alternative to plastic flasks for the manufacturing of AAVs at both R&D and early translational scale, as it reduces production time, operating costs, and process risk.

Muscles, traditionally recognized for their role in locomotion and breathing, also participate in tissue communication. Extracellular microRNAs (miRNA) have been identified as key players in intercellular and inter-organ communication in muscle and other tissues. We have previously shown that intramuscular administration of an antagomiR led to the repression of target miRNA in neighboring skeletal muscles. This study investigated whether antagomiRs could be delivered to distant muscle and other tissues following intramuscular administration. We designed antagomiRs targeting a muscle-specific miRNA, miR-133b; a ubiquitously expressed miRNA, miR-16; and a scrambled oligonucleotide. Although all sequences were detected in neighboring skeletal muscles and distant tissues following intramuscular administration, antagomiR-133b showed the highest accumulation and efficacy in various tissues. This is the first study to provide evidence that intramuscular administration of antagomiRs could be utilized to achieve efficient and widespread distribution in tissues. This in turn could form the basis for alternative future therapeutic approaches.