RESUMO
Individuals with neurofibromatosis type 1 develop rat sarcoma virus (RAS)-mitogen-activated protein kinase-mitogen-activated and extracellular signal-regulated kinase (RAS-MAPK-MEK)-driven nerve tumors called neurofibromas. Although MEK inhibitors transiently reduce volumes of most plexiform neurofibromas in mouse models and in neurofibromatosis type 1 (NF1) patients, therapies that increase the efficacy of MEK inhibitors are needed. BI-3406 is a small molecule that prevents Son of Sevenless (SOS)1 interaction with Kirsten rat sarcoma viral oncoprotein (KRAS)-GDP, interfering with the RAS-MAPK cascade upstream of MEK. Single agent SOS1 inhibition had no significant effect in the DhhCre;Nf1 fl/fl mouse model of plexiform neurofibroma, but pharmacokinetics (PK)-driven combination of selumetinib with BI-3406 significantly improved tumor parameters. Tumor volumes and neurofibroma cell proliferation, reduced by MEK inhibition, were further reduced by the combination. Neurofibromas are rich in ionized calcium binding adaptor molecule 1 (Iba1)+ macrophages; combination treatment resulted in small and round macrophages, with altered cytokine expression indicative of altered activation. The significant effects of MEK inhibitor plus SOS1 inhibition in this preclinical study suggest potential clinical benefit of dual targeting of the RAS-MAPK pathway in neurofibromas. SIGNIFICANCE STATEMENT: Interfering with the RAS-mitogen-activated protein kinase (RAS-MAPK) cascade upstream of mitogen activated protein kinase kinase (MEK), together with MEK inhibition, augment effects of MEK inhibition on neurofibroma volume and tumor macrophages in a preclinical model system. This study emphasizes the critical role of the RAS-MAPK pathway in controlling tumor cell proliferation and the tumor microenvironment in benign neurofibromas.
Assuntos
Neurofibroma Plexiforme , Neurofibroma , Neurofibromatose 1 , Animais , Camundongos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Neurofibroma/tratamento farmacológico , Neurofibroma Plexiforme/tratamento farmacológico , Neurofibromatose 1/tratamento farmacológico , Neurofibromatose 1/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/uso terapêutico , Microambiente Tumoral , Proteína SOS1/metabolismoRESUMO
Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Riboswitch , Camundongos , Humanos , Animais , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Terapia Genética , Interleucina-12/genética , Interleucina-12/metabolismo , Tetraciclina/farmacologiaRESUMO
Prion-like transmission of pathology in α-synucleinopathies like Parkinson's disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier.
RESUMO
Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Dispositivos Lab-On-A-Chip , Organoides/metabolismo , Retina/metabolismo , Transdução Genética , Biomarcadores , Técnicas de Cultura de Células , Técnicas de Cultura de Células em Três Dimensões , Diferenciação Celular , Imunofluorescência , Expressão Gênica , Genes Reporter , Terapia Genética , Humanos , Organoides/citologia , Retina/citologia , TransgenesRESUMO
Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.