Biology subjects

Kordasiewicz, H.

Publications and source records attributed to Kordasiewicz, H..

7 recordsLinked to original sources

bioRxiv2025

Analgesic actions of Intrathecal NaV 1.7 antisense in rats: loss of antagonist channel binding, message depletion, and neuraxial distribution of oligonucleotide

BackgroundGenome targeting strategies to address NaV 1.7 mediated signaling in nociceptive afferents produce highly selective and persistent analgesic outcomes. Here, we analyze the concentration-dependent effects of the reduction of primary afferent NaV 1.7 channel expression by intrathecal delivery of an antisense oligonucleotide on pain behaviors and the covariance of Scn9a knock-down on NaV 1.7 message expression and channel binding. MethodsMale Sprague-Dawley rats were implanted with lumbar intrathecal catheters and dosed with different NaV 1.7 ASO concentrations (100 to 3000 g;10 L). Pain behavior assays were conducted 0-28 days after ASO injections. Brain, spinal cords (SC) and dorsal root ganglia (DRGs) were collected. Quantification of ASO knock-down was assessed through RT-qPCR. NaV 1.7 expression was assessed by binding of NaV 1.7 fluorescent labeled antagonist (ATTO488PTx-II). Distribution studies were performed using anti-ASO antibody staining in brain, SCs and DRGs. ResultsNaV 1.7 message was detected in nerve, DRG and SC. Intrathecal ASO induced a concentration dependent gradient of knock down in DRGs (lumbar to cervical) of Scn9a mRNA and ATTO488PTx-II binding in small DRG neurons, and in spinal parenchyma, and a suppression of pain behaviors initiated by mechanical compression, inflammation and following intraplantar NaV1.7 agonist (OD1) or formalin. At 1000 {micro}g, there was a 47% reduction in phase 2 flinching, a 60% reduction in DRG mRNA and a 36% reduction in ATTO488PTx-II DRG binding in comparison with mismatch controls. Although marked changes were seen at the sensory ganglia level and spinal dorsal horn, no changes in NaV 1.7 binding or mRNA were detected in sciatic nerves. Reduction in DRG message displayed a rostrocaudal gradient that corresponded with ASO distribution. ConclusionsThe study presents how NaV 1.7 ASOs reduce primary afferent channel binding through an effective knock-down on Scn9a mRNA, and channel binding leading to a covariate reduction in pain behavior.

neuroscience↗

bioRxiv2024

Suppression of Huntington's Disease Somatic Instability by Transcriptional Repression and Direct CAG Repeat Binding

Huntingtons disease (HD) arises from a CAG expansion in the huntingtin (HTT) gene beyond a critical threshold. A major thrust of current HD therapeutic development is lowering levels of mutant HTT mRNA (mHTT) and protein (mHTT) with the aim of reducing the toxicity of these product(s). Human genetic data also support a key role for somatic instability (SI) in HTTs CAG repeat - whereby it lengthens with age in specific somatic cell types - as a key driver of age of motor dysfunction onset. Thus, an attractive HD therapy would address both mHTT toxicity and SI, but to date the relationship between SI and HTT lowering remains unexplored. Here, we investigated multiple therapeutically-relevant HTT-lowering modalities to establish the relationship between HTT lowering and SI in HD knock-in mice. We find that repressing transcription of mutant Htt (mHtt) provides robust protection from SI, using diverse genetic and pharmacological approaches (antisense oligonucleotides, CRISPR-Cas9 genome editing, the Lac repressor, and virally delivered zinc finger transcriptional repressor proteins, ZFPs). However, we find that small interfering RNA (siRNA), a potent HTT-lowering treatment, lowers HTT levels without influencing SI and that SI is also normal in mice lacking 50% of total HTT levels, suggesting HTT levels, per se, do not modulate SI in trans. Remarkably, modified ZFPs that bind the mHtt locus, but lack a repressive domain, robustly protect from SI, despite not reducing HTT mRNA or protein levels. These results have important therapeutic implications in HD, as they suggest that DNA-targeted HTT-lowering treatments may have significant advantages compared to other HTT-lowering approaches, and that interaction of a DNA-binding protein and HTTs CAG repeats may provide protection from SI while sparing HTT expression.

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bioRxiv2023

Totally tubular: ASO-mediated knock-down of G2019S-Lrrk2 modulates lysosomal tubule-associated antigen presentation in macrophages

Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinsons disease (PD). LRRK2 levels have become an appealing target for potential PD-therapeutics with LRRK2 antisense oligonucleotides (ASOs) now in clinical trials. However, LRRK2 has been suggested to play a fundamental role in peripheral immunity, and it is currently unknown if targeting increased LRRK2 levels in peripheral immune cells will be beneficial or deleterious. Furthermore, the precise role of LRRK2 in immune cells is currently unknown, although it has been suggested that LRRK2-mediated lysosomal function may be crucial to immune responses. Here, it was observed that G2019S macrophages exhibited increased stimulation-dependent lysosomal tubule formation (LTF) and MHC-II trafficking from the perinuclear lysosome to the plasma membrane in an mTOR dependent manner with concomitant increases in pro-inflammatory cytokine release. Both ASO-mediated knock down of mutant Lrrk2 and LRRK2 kinase inhibition ameliorated this phenotype and decreased these immune responses in control cells. Given the critical role of antigen presentation, lysosomal function, and cytokine release in macrophages, it is likely LRRK2-targetting therapies may have therapeutic value with regards to mutant LRRK2 but deleterious effects on the peripheral immune system, such as altered pathogen control and infection resolution.

neuroscience↗

bioRxiv2022

EMC10 reduction in human neurons and adult mouse brain rescues cellular and behavioral deficits linked to 22q11.2 deletion

Adults and children with the 22q11.2 Deletion Syndrome demonstrate cognitive, social and emotional impairments and high risk for schizophrenia. Work in mouse model of the 22q11.2 deletion provided compelling evidence for abnormal expression and processing of microRNAs. A major transcriptional effect of the microRNA dysregulation is up-regulation of Emc10, a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. We previously uncovered a key contribution of EMC10 in mediating the behavioral phenotypes observed in 22q11.2 deletion mouse models. Here we show that expression and processing of miRNAs is abnormal and EMC10 expression is elevated in neurons derived from 22q11.2 deletion carriers. Reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling in patient neurons. Furthermore, antisense oligonucleotide administration and normalization of Emc10 in the adult mouse brain not only alleviates cognitive deficits in social and spatial memory but remarkably sustains these improvements for over two months post injection, indicating its therapeutic potential. Broadly, our study integrates findings from both animal models and human neurons to elucidate the translational potential of modulating EMC10 levels and downstream targets as a specific venue to ameliorate disease progression in 22q11.2 Deletion Syndrome.

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bioRxiv2021

Gys1 antisense therapy rescues neuropathological bases of murine Lafora disease

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in the diseases mouse models. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analyzed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.

neuroscience↗

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

Convective forces increase rostral delivery of intrathecal antisense oligonucleotides in the cynomolgus monkey nervous system

BackgroundThe intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys. MethodsThere are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging. ResultsIt was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 {+/-} 1.6 cm3. ConclusionsThese results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

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