Biology subjects

Rigo, F.

Publications and source records attributed to Rigo, F..

26 records · Page 2Linked to original sources

bioRxiv2020

Antisense oligonucleotide therapy for SCN2A gain-of-function epilepsy

The clinical spectrum associated with SCN2A de novo mutations (DNMs) continues to expand and includes autism spectrum disorder with or without seizures, in addition to early and late seizure onset developmental and epileptic encephalopathies (DEEs). Recent biophysical studies on SCN2A variants suggest that the majority of early seizure onset DEE DNMs cause gain of function. Gain of function in SCN2A, the principal sodium channel of excitatory pyramidal neurons, would result in heightened neuronal activity and is likely to underlie the pathology seen in early seizure onset DEE patients. Supratherapeutic dosing of the non-selective sodium channel blocker phenytoin, is effective in controlling seizures in these patients but does not impact neurodevelopment, raising the idea that more profound and specific reduction in SCN2A function could significantly improve clinical outcome. To test the potential therapeutic benefit of reducing SCN2A in early seizure onset DEE we centrally administered an antisense oligonucleotide (ASO) targeting mouse Scn2a (Scn2a ASO) to a mouse model of human SCN2A early seizure onset DEE. Mice were genetically engineered to harbour the human equivalent SCN2A p.R1882Q mutation (Q/+), one of the most recurrent mutations in early seizure onset DEE. Q/+ mice presented with spontaneous seizures at postnatal day (P) 1 and did not survive beyond P30. Intracerebroventricular Scn2a ASO administration into Q/+ mice between P1-2 (that reduced Scn2a mRNA levels by 50%) significantly extended lifespan and markedly reduced spontaneous seizures occurrence. Across a range of cognitive and motor behavioural tests, Scn2a ASO treated Q/+ mice were largely indistinguishable from wildtype (+/+) mice. Further improvements in survival and behaviour were seen by adjustment of dosing regimens during development. Scn2a ASO efficacy was also evident at the cellular level. Whole cell patch clamp recording showed that Scn2a ASO administration reversed changes in neuronal excitability in layer 2/3 pyramidal neurons of Q/+ mice to levels seen in +/+ mice. Safety was assessed in +/+ mice and showed a developmental stage dependent tolerability and a favourable therapeutic index. This study suggests that a human SCN2A gapmer ASO could profoundly and safely impact early seizure onset DEE patients and heralds a new era of precision therapy in neurodevelopmental disorders.

neuroscience↗

bioRxiv2020

ASO-based PKM Splice-switching Therapy Inhibits Hepatocellular Carcinoma Cell Growth

The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in the Warburg effect, which is characterized by the preference for aerobic glycolysis for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Previously, we developed antisense oligonucleotides (ASOs) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform and induce apoptosis in cultured glioblastoma cells. Here, we explore the potential of ASO-based PKM splice-switching as a targeted therapy for liver cancer. We utilize a more potent lead cEt/DNA ASO and demonstrate that it induces PKM splice-switching and inhibits the growth of cultured hepatocellular-carcinoma (HCC) cells. This PKM isoform switch increases pyruvate-kinase activity and alters glucose metabolism. The lead ASO and a second ASO targeting a non-overlapping site inhibit tumorigenesis in an orthotopic-xenograft HCC mouse model. Finally, a surrogate mouse-specific ASO induces Pkm splice-switching and inhibits HCC growth, without observable toxicity, in a genetic HCC mouse model. These results lay the groundwork for a potential ASO therapy for HCC. Statement of significanceAntisense oligonucleotides are used to force a change in PKM isoform usage in HCC, reversing the Warburg effect and inhibiting tumorigenesis.

cancer biology↗

bioRxiv2020

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing the field to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal, and non-neuronal cells. Therefore, it is critically important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and robust activity throughout the CNS in neurons, oligodendrocytes, astrocytes, and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

neuroscience↗

bioRxiv2020

Reduction of Nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding TDP-43, account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS (fALS)-causing mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimers disease, and Parkinsons disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a novel function of Nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and lifespan deficits in two distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared along the autophagy-lysosome axis, targeted reduction of Nlk represents a viable approach to therapy development for multiple neurodegenerative disorders.

neuroscience↗

bioRxiv2020

G4C2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

Nucleocytoplasmic transport, controlled by the nuclear pore complex, has recently emerged as a pathomechanism underlying neurodegenerative diseases including C9orf72 ALS/FTD. However, little is known about the underlying molecular events and the underlying biology in human neurons. Using super resolution structured illumination microscopy of twenty three nucleoporins in nuclei from C9orf72 iPSC derived neurons and postmortem human tissue we identify a unique subset of eight nucleoporins lost from human neuronal nuclei. POM121, an integral transmembrane nucleoporin, appears to coordinate the composition of the nucleoporins within human neuronal nuclei ultimately impacting nucleocytoplasmic transport, and subsequent cellular toxicity in C9orf72 iPSNs. These data suggest that POM121 is a critical nucleoporin in the maintenance of the nuclear localization of specific nucleoporins in human neurons. Moreover, loss of nuclear POM121, as a result of expanded C9orf72 ALS/FTD repeat RNA, initiates a pathological cascade affecting nucleoporin composition within neuronal nuclei, nuclear pore complex function, and overall downstream neuronal survival.

neuroscience↗

bioRxiv2020

MaTAR25 LncRNA Regulates the Tensin1 Gene to Impact Breast Cancer Progression

Misregulation of long non-coding RNA genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin 1 (Tns1) to regulate its expression in trans. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. The human ortholog of MaTAR25, LINC01271, is upregulated with human breast cancer stage and metastasis. SIGNIFICANCELncRNAs have great potential to reveal new regulatory mechanisms of function as well as having exciting therapeutic capacity given their ease of being targeted by nucleic acid drugs. Our study of MaTAR25, and its human ortholog LINC01271, reveal an unexpected function of this lncRNA in breast cancer progression by regulating Tns1 gene expression, whose protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. We identified LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and cancer metastasis. Our findings demonstrate that LINC01271 represents an exciting therapeutic target to alter breast cancer progression.

cancer biology↗

bioRxiv2018

Therapeutic suppression of proteolipid protein rescues Pelizaeus-Merzbacher Disease in mice

Mutations in proteolipid protein 1 (PLP1) result in failure of myelination and severe neurological dysfunction in the X-linked pediatric leukodystrophy Pelizaeus-Merzbacher disease (PMD). The majority of PLP1 variants, including supernumerary copies and various point mutations, lead to early mortality. However, PLP1-null patients and mice display comparatively mild phenotypes, suggesting that reduction of aberrant PLP1 expression might provide a therapeutic strategy across PMD genotypes. Here we show, CRISPR-Cas9 mediated germline knockdown of Plp1 in the severe jimpy (Plp1jp) point mutation mouse model of PMD rescued myelinating oligodendrocytes, nerve conduction velocity, motor function, and lifespan to wild-type levels, thereby validating PLP1 suppression as a therapeutic approach. To evaluate the therapeutic potential of Plp1 suppression in postnatal PMD mice, we tested antisense oligonucleotides (ASOs) that stably decrease mouse Plp1 mRNA and protein in vivo. Administration of a single intraventricular dose of Plp1-targeted ASOs to postnatal jimpy mice increased myelination, improved motor behavior, and extended lifespan through an 8-month endpoint. Collectively, these results support the development of PLP1 suppression as a disease-modifying therapy for most PMD patients. More broadly, we demonstrate that RNA therapeutics can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, opening a new treatment modality for myelin disorders.

neuroscience↗

bioRxiv2018

Delivery of GalNAc-conjugated splice-switching ASOs to non-hepatic cells through ectopic expression of asialoglycoprotein receptor

Splice-switching antisense oligonucleotides (ASOs) are promising therapeutic tools to target various genetic diseases, including cancer. However, in vivo delivery of ASOs to orthotopic tumors in cancer mouse models or to certain target tissues remains challenging. A viable solution already in use is receptor-mediated uptake of ASOs via tissue-specific receptors. For example, the asialoglycoprotein receptor (ASGP-R) is exclusively expressed in hepatocytes. Triantennary GalNAc (GN3)-conjugated ASOs bind to the receptor and are efficiently internalized by endocytosis, enhancing ASO potency in the liver. Here we explore the use of GalNAc-mediated targeting to deliver therapeutic splice-switching ASOs to cancer cells that ectopically express ASGP-R, both in vitro and in tumor mouse models. We found that ectopic expression of the major isoform ASGP-R1 H1a is sufficient to promote uptake and increase GN3-ASO potency to various degrees in all tested cancer cells. We show that cell-type specific glycosylation of the receptor does not affect its activity. In vivo, GN3-conjugated ASOs specifically target subcutaneous xenograft tumors that ectopically express ASGP-R1, and modulate splicing significantly more strongly than unconjugated ASOs. Our work shows that GN3-targeting is a useful tool for proof-of-principle studies in orthotopic cancer models, until endogenous receptors are identified and exploited for efficiently targeting cancer cells.

molecular biology↗