Biology subjects

Rigo, F.

Publications and source records attributed to Rigo, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Delivery of small interfering RNA and antisense oligonucleotides across the blood-brain barrier with monovalent transferrin receptor 1 binding VHH-Fc fusion proteins

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

neuroscience↗

bioRxiv2025

Enhanced splicing modulation by NMA-modified antisense oligonucleotides

Aberrant RNA splicing contributes to many human diseases, and splice-switching antisense oligonucleotides (SSOs) are ideally suited as a therapeutic strategy to modulate splicing and restore normal gene expression. Nusinersen (Spinraza) has revolutionized the treatment of spinal muscular atrophy. It is a splice-switching oligonucleotide (SSO) that is modified with 2-O-methoxyethyl (MOE) modifications. Here, we introduce a next-generation ribose modification, 2'-O-[2-(methyl-amino)-2-oxoethyl] (NMA), which enhances the pharmacological properties of SSOs. We identified a long-lasting NMA-modified human candidate SSO, salanersen, that is 3-4-fold more potent than nusinersen in human SMN2 transgenic mice. To evaluate the generality of the NMA chemistry, we applied it to modulation of SCN1A exon 20N splicing, a therapeutic strategy for Dravet syndrome. An NMA-modified SSO is 3.5-fold more potent than STK-001, a MOE-modified SSO currently in clinical trials. Our data establish the NMA chemistry as a broadly applicable ribose modification that markedly improves the pharmacological profile of SSOs, supporting its development as a next-generation platform for splicing modulation therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/680653v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@122e426org.highwire.dtl.DTLVardef@1b0a56eorg.highwire.dtl.DTLVardef@3ce4e8org.highwire.dtl.DTLVardef@1d86378_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

TDP-43 pathology triggers SRRM4-dependent cryptic splicing of G3BP1 in ALS/FTD

Loss of nuclear TDP-43 is a defining feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet how this leads to selective neuronal vulnerability is poorly understood. Here, using human iPSC-derived neurons and a large multi-omics dataset of ALS/FTD patients, we demonstrate that TDP-43 pathology induces the inclusion of an in-frame cryptic exon in human G3BP1. The resulting CRYPTIC G3BP1 protein contains an additional 10-amino acids within the highly conserved NTF2L domain, which acts as a dominant negative and disrupts stress granule dynamics. We further show that cryptic exon inclusion in G3BP1 upon TDP-43 loss is enriched in neurons. Mechanistically, the loss of TDP-43 unmasks a binding site for the neuron-specific splicing regulator SRRM4 within intron 2 of G3BP1, enabling the inclusion of the cryptic exon. Collectively, our findings reveal that neuron-specific regulatory mechanisms intersect with TDP-43 -mediated splicing and suggest a mechanistic basis for the increased neuronal vulnerability observed in ALS/FTD.

neuroscience↗

bioRxiv2025

Therapeutic antisense oligonucleotide mitigates retinal dysfunction in a pig model of CLN3 Batten disease

CLN3 Batten disease is a lethal pediatric autosomal recessive neurodegenerative disease caused by mutations in the CLN3 gene. Typically, the disease manifests as vision loss early in life and progresses to neurological dysfunction and death in young adulthood. Therapeutic development has focused on treating the central nervous system. However, such therapies may not protect against vision loss, which has a significant impact on quality of life. We have shown that a splice-switching antisense oligonucleotide (ASO) delivered to the central nervous system can reduce neurological disease burden in mouse models of CLN3 disease. Here, we report on a similar ASO approach for treating CLN3 Batten disease retinal dysfunction in a pig model of the disease, which is more representative of human vision. A single intravitreal injection of ASO induces robust exon skipping in the retina for up to 12 months. The ASO treatment resulted in higher amplitudes on electroretinograms, suggesting mitigation of retinal dysfunction at early timepoints of disease. One ASO that efficiently induces exon skipping in vivo was well-tolerated and targets a region of CLN3 that is conserved in humans, making it a promising candidate for treating the disease in humans. Our findings demonstrate the potential utility of an ASO-based approach to treat retinal dysfunction in CLN3 Batten disease and generally supports the use of ASOs for treating eye diseases. One Sentence SummarySplice-switching antisense oligonucleotides delivered by intravitreal injection are safe and show efficacy in preventing early retinal dysfunction in a pig model of CLN3 Batten disease.

molecular biology↗

bioRxiv2024

Reliability of canopy photography for forest ecology and biodiversity studies

Understory is a key component of forest biodiversity. The structure of the forest stand and the horizontal composition of the canopy play a major role on the light regime of the understory, which in turn affects the abundance and the diversity of the understory plant community. Reliable assessments of canopy structural attributes are essential for forest research and biodiversity monitoring programs, as well as to study the relationship between canopy and understory plant communities. Canopy photography is a widely used method but it is still not clear which photographic techniques is better suited to capture canopy attributes at stand-level that can be relevant in forest biodiversity studies. For this purpose, we collected canopy structure and understory plant diversity data on 51 forest sites in the north-eastern Italian Alps, encompassing a diversity of forest types. Canopy images were acquired using both digital cover (DCP) and hemispherical (DHP) photography. Canopy structural attributes were then compared to tree species composition data to evaluate whether they were appropriate to differentiate between forest types. Additionally, we tested what canopy attributes derived from DCP and DHP best explained the species composition of vascular plants growing in the understory. We found that hemispherical canopy photography was most suitable to capture differences in forest types, which was best expressed by variables such as leaf inclination angle and canopy openness. On our sites, DHP-based canopy attributes were also able to better distinguish between different conifer forests. Leaf clumping was the most important attribute for determining plant species distribution of the understory, indicating that diverse gap structures create different microclimate conditions enhancing diverse plant species with different ecological strategies. This study supports the reliability of canopy photography in forest ecology and biodiversity monitoring, but also provide insights for increasing understory diversity in managed forests of high conservation value. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/610276v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@15b706forg.highwire.dtl.DTLVardef@329571org.highwire.dtl.DTLVardef@f13605org.highwire.dtl.DTLVardef@11b816_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2023

G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in C9orf72 ALS/FTD human neurons

The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.

neuroscience↗

bioRxiv2023

Targeting Transferrin Receptor to Transport Antisense Oligonucleotides Across the Blood-Brain Barrier

Antisense oligonucleotides (ASO) are promising therapies for neurological disorders, though they are unable to cross the blood-brain barrier (BBB) and must be delivered directly to the central nervous system (CNS). Here, we use a human transferrin receptor (TfR)-binding molecule to transport ASO across the BBB in mice and non-human primates, termed oligonucleotide transport vehicle (OTV). Systemically delivered OTV drives significant, cumulative, and sustained knockdown of the ASO target across multiple CNS regions and all major cell types. Further, systemic OTV delivery enables more uniform ASO biodistribution and knockdown compared to two other clinically relevant ASO delivery routes: a standard, high affinity TfR antibody, or direct ASO delivery to the CSF. Together, our data support systemically delivered OTV as a potential therapeutic platform for neurological disorders. One-Sentence SummarySystemically dosed OTV delivered via TfR1 targeting shows widespread and cumulative target knockdown in the mouse and NHP CNS.

neuroscience↗

bioRxiv2022

Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation

The human mRNA most affected by TDP-43 loss-of-function is transcribed from the STMN2 gene and encodes stathmin-2 (also known as SCG10), whose loss is a neurodegenerative disease hallmark. Here using multiple in vivo approaches, including transient antisense oligonucleotide (ASO)-mediated suppression, chronic shRNA-mediated depletion in aging mice, and germline deletion, we establish stathmin-2 to be essential for acquisition and maintenance of neurofilament-dependent structuring of axoplasm critical for maintaining diameter and conduction velocity of large-myelinated axons. Sustained stathmin-2 loss from an otherwise mature adult nervous system is demonstrated over a time course of eight months to initiate and drive motor neuron disease that includes 1) shrinkage in inter-neurofilament spacing that is required to produce a three-dimensional space filling array that defines axonal caliber, 2) collapse of mature axonal caliber with tearing of outer myelin layers, 3) reduced conduction velocity, 4) progressive motor and sensory deficits (including reduction of the pain transducing neuropeptide CGRP), and 5) muscle denervation. Demonstration that chronic stathmin-2 reduction is itself sufficient to trigger motor neuron disease reinforces restoration of stathmin-2 as an attractive therapeutic approach for TDP-43-dependent neurodegeneration, including the fatal adult motor neuron disease ALS.

cell biology↗

bioRxiv2022

The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells

The Myc proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by Myc transcriptionally upregulates the core pre-mRNA splicing machinery and causes mis-regulation of alternative splicing. However, our understanding of how splicing changes are directed by Myc is limited. We performed a signaling pathway-guided splicing analysis to identify Myc dependent splicing events. These included an HRAS cassette exon repressed by Myc across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of hnRNP H gene expression with Myc hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, hnRNP F expression showed a positive correlation with Myc hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal new mechanisms for Myc-dependent regulation of splicing, and point to new possible therapeutic targets in prostate cancers. SIGNIFICANCE STATMENTMyc Transformation by the proto-oncogene c-Myc causes dysregulation of the pre-mRNA splicing reaction in cancer, but it is not known how mRNA isoform changes are directed by Myc. Here, we use bioinformatics to identify a splicing event in another proto-oncogene, HRAS, that is regulated by Myc across multiple tumor types. We identify new splicing regulators, hnRNPs H and F, that control this HRAS exon by binding to enhancer elements within its downstream intron. Additional pan-cancer bioinformatic analyses show hnRNP H expression to be anti- correlated with Myc hallmarks, consistent with the reduced splicing of the HRAS exon in Myc driven cancer. These findings uncover new mechanisms by which Myc can alter splicing in cancer cells and provide new molecular targets for potential therapeutics.

molecular biology↗

bioRxiv2022

Early activation of cellular stress and death pathways caused by cytoplasmic TDP-43 in the rNLS8 mouse model of ALS/FTD

TAR DNA binding protein 43 (TDP-43) pathology is a key feature of over 95% of amyotrophic lateral sclerosis (ALS) and nearly half of frontotemporal dementia (FTD) cases. The pathogenic mechanisms of TDP-43 dysfunction are poorly understood, however activation of cell stress pathways may contribute to pathogenesis. We therefore sought to identify which cell stress components are critical for driving disease onset and neurodegeneration in ALS/FTD. We studied the rNLS8 transgenic mouse model, which expresses human TDP-43 with a genetically-ablated nuclear localisation sequence within neurons of the brain and spinal cord resulting in cytoplasmic TDP-43 pathology and progressive motor dysfunction. Amongst numerous cell stress-related biological pathways profiled using qPCR arrays, several critical ISR effectors, including CCAAT/enhancer-binding homologous protein (Chop/Ddit3) and activating transcription factor 4 (Atf4), were upregulated in the cortex of rNLS8 mice prior to disease onset. This was accompanied by early up-regulation of anti-apoptotic gene Bcl2 and diverse pro-apoptotic genes including BH3-interacting domain death agonist (Bid). However, pro-apoptotic signalling predominated after onset of motor phenotypes. Notably, pro-apoptotic caspase-3 protein was elevated in the cortex of rNLS8 mice at later disease stages, suggesting that downstream activation of apoptosis drives neurodegeneration following failure of early protective responses. Unexpectedly, suppression of Chop in the brain and spinal cord using antisense oligonucleotide-mediated silencing had no effect on overall TDP-43 pathology or disease phenotypes in rNLS8 mice. Cytoplasmic TDP-43 accumulation therefore causes very early activation of ISR and both anti-and pro-apoptotic signalling that switches to predominant pro-apoptotic activation later in disease. These findings suggest that precise temporal modulation of cell stress and death pathways may be beneficial to protect against neurodegeneration in ALS and FTD. Key pointsO_LIISR genes Atf4 and Chop, anti-apoptotic Bcl2 and pro-apoptotic gene Bid, Bim, Noxa were upregulated in the cortex of rNLS8 mice prior to disease onset C_LIO_LIKnockdown of Chop had limited effects on TDP-43 pathology and did not alter motor deficits in rNLS8 mice C_LIO_LIBoth anti-and pro-apoptotic genes are upregulated prior to disease onset, and switches to activation of pro-apoptotic signalling at later disease stages C_LIO_LICaspase-3 activation likely drives neurodegeneration in the cortex of rNLS8 mice C_LI

neuroscience↗

bioRxiv2022

Antisense oligonucleotide therapy rescues disturbed brain rhythms and sleep in juvenile and adult mouse models of Angelman syndrome

UBE3A encodes ubiquitin protein ligase E3A, and in neurons its expression from the paternal allele is repressed by the UBE3A antisense transcript (UBE3A-ATS). This leaves neurons susceptible to loss-of-function of maternal UBE3A. Indeed, Angelman syndrome, a severe neurodevelopmental disorder, is caused by maternal UBE3A deficiency. A promising therapeutic approach to treating Angelman syndrome is to reactivate the intact paternal UBE3A by suppressing UBE3A-ATS. Prior studies show that many neurological phenotypes of maternal Ube3a knockout mice can only be rescued by reinstating Ube3a expression in early development, indicating a restricted therapeutic window for Angelman syndrome. Here we report that reducing Ube3a-ATS by antisense oligonucleotides in juvenile or adult maternal Ube3a knockout mice rescues the abnormal electroencephalogram rhythms and sleep disturbance, two prominent clinical features of Angelman syndrome. Importantly, the degree of phenotypic improvement correlates with the increase of Ube3a protein levels. These results indicate that the therapeutic window of genetic therapies for Angelman syndrome is broader than previously thought, and electroencephalogram power spectrum and sleep architecture should be used to evaluate the clinical efficacy of therapies.

neuroscience↗

bioRxiv2021

Antisense oligonucleotides targeting UBE3A-ATS restore expression of UBE3A by relieving transcriptional interference

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. In neurons, the paternal allele of UBE3A is silenced in cis by the long noncoding RNA, UBE3A-ATS. Unsilencing paternal UBE3A by reducing UBE3A-ATS is a promising therapeutic approach for the treatment of AS. Here we show that targeted cleavage of UBE3A-ATS using antisense oligonucleotides (ASOs) restores UBE3A and rescues electrophysiological phenotypes in human AS neurons. We demonstrate that cleavage of UBE3A-ATS results in termination of its transcription by displacement of RNA Polymerase II. Reduced transcription of UBE3A-ATS allows transcription of UBE3A to proceed to completion, providing definitive evidence for the transcriptional interference model of paternal UBE3A silencing. These insights into the mechanism by which ASOs restore UBE3A inform the future development of nucleotide-based approaches for the treatment of AS, including alternative strategies for cleaving UBE3A-ATS that can be developed for long-term restoration of UBE3A function.

genetics↗

bioRxiv2021

The long non-coding RNA MaTAR20 promotes mammary tumor growth by regulating angiogenesis pathways

Long non-coding RNAs (lncRNAs) are an emerging class of regulatory molecules that have been shown to play important roles in tumorigenesis and cancer progression. Here, we studied the recently identified lncRNA Mammary Tumor Associated RNA 20 (MaTAR20) in mammary cancer progression. A CRISPR/Cas9 knockout of MaTAR20 in the metastatic 4T1 cell line led to reduced cancer cell proliferation and increased cell surface adhesion compared to control cells. Consistent with these knockout results antisense oligonucleotide (ASO) mediated knockdown of MaTAR20 resulted in reduced growth and invasion in 4T1 cells, and in primary mammary tumor organoids derived from the MMTV-PyMT mouse model of breast cancer. Injection of MaTAR20-specific ASOs subcutaneously into tumor bearing MMTV-PyMT mice resulted in smaller and highly necrotic tumors in comparison to mice injected with a scrambled control ASO. To investigate the molecular mechanism by which MaTAR20 acts to advance mammary tumor progression, we applied a combination of RNA-sequencing and RNA-pulldown coupled to DNA-sequencing. These analyses demonstrated that the nuclear retained lncRNA is associated with several essential cancer signaling pathways such as VEGF signaling. In particular, MaTAR20 directly binds to and regulates the expression of Tnfsf15. Our results indicate that MaTAR20 is an important driver of mammary tumor progression and represents a promising new therapeutic target.

cancer biology↗

bioRxiv2021

PHAROH lncRNA regulates c-Myc translation in hepatocellular carcinoma via sequestering TIAR

Hepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is over-expressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a c-Myc motif in their promoter. C-MYC is decreased at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases c-MYC translation. In summary, our data suggest that PHAROH regulates c-MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma.

cancer biology↗

bioRxiv2021

Antisense therapy in a new rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment.

Alexander disease (AxD) is a devastating leukodystrophy caused by gain of function mutations in GFAP, and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that Gfap-targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report we introduce a new rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with approximately 15 % dying of unknown cause between 6 to 12 weeks of age. In this model, a single treatment with Gfap-targeted ASO provides long lasting suppression, reverses GFAP pathology, and depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. This is the first report of an animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease. We use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

neuroscience↗

bioRxiv2020

Relevance of Antibodies against the Chicken Anaemia Virus

BackgroundChicken Infectious Anaemia (CIA) Virus (CAV) curtails the function of multiple immune compartments. Mortality due to blatant infection is controlled in broilers by passive immunization derived from vaccinated breeders. Therefore, chicks are often assessed by serology to determine maternally-derived antibodies (MDA). MethodsA vaccine overdose-induced model of CIA. The model replicated the most common features of the disease. This model was used to determine the role of MDA in the protection of chicks. Hatchlings were tested for anti-CAV by ELISA and were sorted into groups based on antibody levels. SPF chicks were used as a no-antibody control. ResultsLower specific antibody levels seemed to facilitate viral entry into the thymus, but viral levels, CD4+ and CD8+ counts, thymus architecture, and haematocrit were preserved by MDA, regardless of its levels. ConclusionLevels of MDA are not correlated with CIA, but are important for CAV infection. IMPORTANCEVaccination is paramount in broiler production. Many of the vaccines are given to broiler breeders, instead of to the broilers themselves. This is cost-effective and practical, since in vaccinating one breeder hundreds of broilers are born with maternally-derived protection. To assess the quality of maternal immunity, antibodies are measured in their chicks. For Chicken Anaemia, this does not seem to suffice to verify protection. This viral disease is very common, and measuring maternal immunity against it determines whether to purchase chicks from a breeder farm. In this study, we verified that antibodies are not correlated with protection from the disease, and therefore should not be used as the sole parameter in assessing immunity against Chicken Anaemia in broilers.

immunology↗

bioRxiv2020

Antisense oligonucleotide therapy for KCNT1 encephalopathy

Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo mutations in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. The homozygous p.P924L (L/L) mouse model recapitulates the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provides a proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

neuroscience↗

bioRxiv2020

Splice-switching of the insulin receptor in rhabdomyosarcoma: Rescuing the IR-B isoform for better treatment options.

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with poor prognosis for metastasis and recurrent disease. Large scale sequencing endeavors demonstrate that RMS tumors have limited mutations and a dearth of driver mutations that are precisely targetable. However, IGF2 signaling is known to be grossly altered in RMS. The IGF2 signalling molecule binds both its innate IGF1 receptor as well as the insulin-receptor-variant-A (IR-A) with high affinity. Mitogenic and proliferative signalling via the canonical IGF2 pathway is therefore augmented by IR-A. The insulin receptor (IR) which is a transmembrane tyrosine-kinase receptor exists in two alternatively spliced isoforms, IR-A and IR-B. In this study, we show that RMS patients express increased IR-A compared to control tissues that express predominantly the IR-B isoform. We also found that Hif1a is significantly increased in RMS tumors, portraying their hypoxic phenotype. Furthermore, the alternative-splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a hypoxia-responsive-element, which is also the binding site for the RNA-binding protein CUG-BP1. We designed Splice-Switching-Oligonucleotides (SSO) against this binding site to decrease the levels of IR-A in RMS cell-lines and consequently rescue the IR-B expression levels. SSO treatment resulted in significant reductions in proliferation, migration and angiogenesis. Our data show promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. Our data reveal that RMS tumors use IR alternative-splicing as yet another survival strategy which can be exploited as therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

cancer biology↗