Molecular Therapy-Methods & Clinical Development最新文献

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.

Galsome-NEO is a glycolipid-adjuvanted mRNA lipid nanoparticle (LNP) cancer vaccine encoding neo-epitopes for evaluation in a phase 1 study in patients with non-small cell lung cancer. To assess the safety of Galsome-NEO, a repeated-dose toxicity study was conducted in Wistar Han rats involving three intramuscular doses of 30 μg mRNA. A dose-escalation study in piglets tested three doses of 3, 15, and 100 μg mRNA. Rats showed a pronounced pro-inflammatory response, evidenced by cytokine secretion and an acute phase reaction. Clinical findings included temporary local reactions (maximum grade 3), elevated temperatures, and weight loss. In pigs, all doses were well tolerated. Blood analysis showed elevated alkaline phosphatase and decreased thrombocytes in rats, while pigs had reduced reticulocyte counts. Histology revealed hepatocyte vacuolation in rats and immune infiltration at injection sites in both species. In rats, blood and histology alterations resolved 3 weeks post dosing, except for immune infiltration in the connective tissue at injection sites in two females. Galsomes with mRNA encoding the Chlamydia trachomatis major outer membrane protein induced T cell responses in pigs. Natural killer T cell activation was observed in both species. These findings align with the safety data for the COVID-19 mRNA vaccine, Comirnaty, and demonstrate Galsomes' potential in large animals.

Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds promise to provide life-long remission following a single treatment. Here we report a multi-pronged anti-HIV-1 HSPC-based gene therapy designed to defend against and attack HIV-1 infection. We developed a lentiviral vector capable of co-expressing three anti-HIV-1 genes. Two are designed to prevent infection, including a short hairpin RNA (shRNA) (CCR5sh1005) to knock down HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor (C46). The third gene is a CD4-based chimeric antigen receptor (CAR) designed to attack HIV-1-infected cells. Our vector also includes a non-signaling truncated human epidermal growth factor receptor (huEGFRt) which acts as a negative selection-based safety kill switch against transduced cells. Anti-HIV-1 vector-transduced human CD34⁺ HSPC efficiently reconstituted multi-lineage human hematopoietic cells in humanized bone marrow/liver/thymus (huBLT) mice. HIV-1 viral load was significantly reduced (1-log fold reduction, p < 0.001) in transplanted huBLT mice. Anti-huEGFR monoclonal antibody cetuximab (CTX) administration significantly reduced huEGFRt⁺ vector-modified cells (>4-fold reduction, p < 0.01) in huBLT mice. These results demonstrate that our strategy is highly effective for HIV-1 inhibition, and that CTX-mediated negative selection can deplete anti-HIV-1 vector-modified cells in the event of unwanted adverse effects in huBLT mice.

Plant-derived lipid nanoparticles (PDNPs) are nano-sized particles isolated from various edible plants that contain bioactive components involved in regulating biological responses. Here, we isolated maca-derived lipid nanoparticles (MDNPs) from Lepidium meyenii Walp (maca), evaluated their therapeutic effects using two representative lethal models of sepsis, and determined their multimodal anti-inflammatory mechanism that relied on broad sequestration and neutralization of multiple pro-inflammatory cytokines and acute phase proteins (APPs) through formation of a protein corona. Lipidomics of MDNPs revealed triacylglycerols and phytoceramides as major constituents. In vitro studies showed that MDNPs were non-toxic, reduced macrophage activation, and sequestered lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, while mitigating nuclear factor kappa B (NF-κB) activity. In a pre-established LPS-induced endotoxemia model, MDNP treatment significantly reduced systemic pro-inflammatory cytokines, reduced organ damage, and increased survival. Untargeted proteomics and bioinformatics analysis identified an enrichment in APPs present in MDNP protein coronas and corresponding inflammatory pathways modulated. The efficacy of MDNPs were further tested using a lethal polymicrobial sepsis model, where treatment significantly improved survival even in the absence of antibiotics. This study identifies MDNPs as an effective strategy capable of inducing potent anti-inflammatory responses, offering significant therapeutic potential for diseases such as sepsis, while informing the future design of synthetic lipid nanoparticles.

Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors' infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeLa-based cell lines that stably encode the rep and cap genes from AAV serotype 2. Co-infection with wild-type (WT) adenoviruses provides the helper functions for expression of these genes, and the use of quantitative PCR (qPCR)/droplet digital PCR (ddPCR) serves as the endpoint method for the detection of infectious events. However, TCID50 assays using these HeLa-based rep cap trans-complementing cell lines have traditionally been regarded as challenging due to high variability, stability of the integrated genes, and safety concerns associated with the use of WT helper viruses. Here we developed a novel method for infectious titration of rAAV using our vector "tetracycline-enabled self-silencing adenovirus" (TESSA); we engineered it to deliver and express the AAV2 rep genes and adenoviral helper functions for rAAV genome replication, independent of the cell type. This approach allows the infectious titration of rAAV serotypes in cell lines permissive to adenovirus but without the production of adenoviral particles for improved safety, therefore benefiting GMP analytical requirements for rAAV gene therapies.

Humanized mice are valuable preclinical models for immuno-oncology research because they allow modeling of human immune cells and human tumors in vivo. Myeloid cells are highly abundant in many tumors and have been associated with tumor progression, metastasis, and therapy resistance. Next-generation humanized mice have been generated to improve the development, diversity, and function of human myeloid cells. In this study, we analyzed human immune cell development and myeloid cell composition in NSG-Quad and MISTRG-6 mice. NSG-Quad mice supported the development of tissue-resident and tumor-infiltrating human macrophages at levels almost comparable to those of MISTRG-6 mice. However, the development of human CD4⁺ and CD8⁺ T cells was impaired in the blood and spleen but not in the tumor of NSG-Quad mice. In a subset of NSG-Quad mice, human monocytes exhibited increased cellular granularity and elevated expression of activation and checkpoint molecules, consistent with a monocyte hyperactivation syndrome. Our study provides a comprehensive comparative analysis of the frequency and characteristics of circulating, tissue-resident, and tumor-infiltrating myeloid cell populations in NSG-Quad and MISTRG-6 mice, which is key to accurately design and interpret human tumor xenograft studies, particularly with regard to faithful reconstruction of the human tumor-immune microenvironment and preclinical testing.

One of the many challenges for islet transplantation as a treatment for type 1 diabetes is inflammation that contributes to islet de-differentiation and death. Innate immune cells such as monocytes and macrophages secrete tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS), and IL-6, which directly contribute to islet dysfunction. Attenuation of the early inflammatory response post-transplantation may protect cell survival and subsequent function. Herein, we investigate the development of anti-TNF-α antisense-oligonucleotide-conjugated polylactide-co-glycolide nanoparticles (PLG-aTNF-α NPs) as an anti-inflammatory therapy after stem-cell-derived islet transplantation. PLG-aTNF-α NPs are shelf stable and successfully reduce TNF-α secretion and expression in inflammatory macrophages. Synergy between the aTNF-α antisense oligonucleotide and the polylactide-co-glycolide NPs results in further knockdown of IL-1β, IL-6, iNOS, and IL-12 in vitro indicating PLG-aTNF-α NPs may protect against the inflammatory cascade in vivo. In a diabetic mouse model, stem-cell-derived islets transplanted to the peritoneal fat were protected after treatment with PLG-aTNF-α NPs compared with PLG NPs alone. Tnfα and I l 1β expression was reduced in mice treated with PLG-aTNF-α NPs, indicating inflammation was reduced after transplant. PLG-aTNF-α NPs reduce TNF-α and protect islets, supporting their potential use as a therapeutic in islet transplantation.

Recent advancements in gene insertion have shifted from DNA-repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. Although I-PGI does not require DNA double-strand breaks (DSBs) for activity, linear cargo, like AAV, stimulates DNA end-joining activity after integration. To mitigate potential risks from DSBs, we developed two circular AAV cargos capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated seamless cargo integration in primary human hepatocytes (PHHs) and robust DSB-free insertion structures in vivo. We then investigated the delivery of a packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD exhibited functional seamless gene insertion in PHHs and showed cargo efficacy in vivo. Together, these findings provide evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.

Advanced therapy medicinal products (ATMPs) require rigorous quality management to mitigate risks associated with their development and use in hospitals. This study aimed to identify guidelines, standards of practice, and practical experiences in ATMPs quality management in hospital settings. An integrative scoping review under PRISMA guidelines retrieved 14 studies from four databases, 144 quality management guidelines and standards, and risk management reports for approved ATMPs from three government agencies and six organizations. Thirteen models or programs of quality management practices for ATMPs were identified across 25 hospital-based settings in six countries. Major aspects of ATMPs included clinical quality and translation, logistics management, hospital preparation, and patient care. Primary goals of ATMPs management within hospitals involved regulatory compliance and accreditation with national and regional requirements and implementing and maintaining the standardized operational practices. Four priority actions to enhance the quality of ATMPs management were as follows: (1) risk-based procedures and strategies; (2) strengthening of the skills and knowledge of healthcare professionals and technical staff; (3) validation and maintenance of qualified storage, manufacturing, and delivery facilities; and (4) support for a documentation system. In summary, understanding key components of ATMPs management offers valuable insights for developing an adaptive quality management ecosystem supporting clinical translation.