RESUMEN
In the era of genomic medicine, diagnoses of rare paediatric neurological diseases are increasing. Many are untreatable and life-limiting, leading to an exceptional increase in gene therapy development. It is estimated that 20 gene therapy products will have received approval from the US Food and Drug Administration by 2025. With viral gene therapy considered a potential single-dose cure for patients with spinal muscular atrophy type 1 as one example, and contemporaneously tragically resulting in the deaths of three male children with X-linked myotubular myopathy receiving high-dose gene therapy in 2020, what is the current state of gene therapy? What is behind the decades of hype around viral gene therapy and is it high impact, but high risk? In this review, we outline principles of viral gene therapy development and summarize the most recent clinical evidence for the therapeutic effect of gene therapy in paediatric neurological diseases. We discuss adeno-associated virus and lentiviral vectors, antisense oligonucleotides, emerging genetic editing approaches, and current limitations that the field still faces. What this paper adds Viral gene therapy development and clinically used transgenes, regulatory elements, capsids, dosage, and delivery routes are summarized. Viral gene therapy for 18 childhood neurological disorders involving over 600 children in 40 clinical trials are reviewed.
Asunto(s)
Enfermedades Genéticas Congénitas/terapia , Terapia Genética/métodos , Vectores Genéticos , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/terapia , Ensayos Clínicos como Asunto , Dependovirus/genética , Humanos , Lentivirus/genética , Transducción Genética/métodos , Transfección/métodosRESUMEN
Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example, Cocal virus, have been proposed as alternative LV envelopes with possible advantages over VSVind.G. Well-characterized antibodies that recognize VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here, we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralizes G proteins from three strains of VSV, as well as Cocal and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralizes only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , Reacciones Cruzadas , Epítopos/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Pruebas de Neutralización , Filogenia , Homología de Secuencia , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.
Asunto(s)
Adhesión Celular/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Neurofibromina 2/genética , Proteínas Supresoras de Tumor/genética , Proliferación Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Inhibición de Contacto/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Neoplasias/patología , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Ratones Transgénicos/genética , Animales , Técnicas Biosensibles/métodos , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Inflamación/genética , Luciferasas de Luciérnaga/genética , Ratones , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Virus Formadores de Foco en el Bazo/genética , Transcripción Genética/genéticaRESUMEN
TMEM16F is a Ca2+ -gated ion channel that is required for Ca2+ -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca2+, but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca2+ activated membrane trafficking.
Asunto(s)
Anoctaminas/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Anoctaminas/genética , Sistemas CRISPR-Cas/genética , Línea Celular , Citometría de Flujo , Humanos , Células Jurkat , Lentivirus/genética , Microscopía Confocal , Proteínas de Transferencia de Fosfolípidos/genética , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136-370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.