Biology subjects

Karda, R.

Publications and source records attributed to Karda, R..

9 recordsLinked to original sources

bioRxiv2025

Next Generation AAV-F Capsid gene therapy rescues disease pathology in a model of Pyruvate Dehydrogenase Complex Deficiency

Pyruvate dehydrogenase complex deficiency (PDHD) is a severe mitochondrial disorder most frequently caused by pathogenic variants in PDHA1, leading to neurodevelopmental delay and early mortality, necessitating brain-targeted interventions. Using a brain-specific Pdha1 knockout mouse model, we compared intracerebroventricular delivery of AAV9 capsid and a recently described synthetic neurotropic AAV-F capsid, both expressing human PDHA1 coding sequence driven by a constitutive CAG promoter. Newborn mice received, titre matched AAV9 or AAV-F or AAV9 at ten-fold higher dose. Low-dose AAV-F and high-dose AAV9 significantly improved survival, and restored PDH enzyme activity, metabolite profiles, and brain histopathology to near wild-type levels. However, treated mice showed reduced locomotion by P100 and impaired motor function. Importantly, AAV-F achieved broad CNS transduction with minimal liver expression, outperforming AAV9 at lower dose. There results support the therapeutic potential of AAV-based gene therapy for PDHD and highlighting AAV-F as a promising capsid for efficient, CNS specific delivery.

neuroscience↗

bioRxiv2025

AAV delivery of RNA editing machinery rescues SUDEP and seizure phenotype in a mouse model of Dravet Syndrome

Dravet syndrome (DS) is a severe childhood genetic epilepsy, caused by de novo heterozygous mutations in SCN1A, resulting in a loss-of-function of the voltage-gated sodium ion channel, Nav1.1. Nav1.1 is expressed in the brain, and at a lower level, in the heart. DS manifests in the first year of life. Patients exhibit tonic-clonic seizures, febrile seizures, cognitive decline, developmental delays, ataxia, and sudden unexpected death from epilepsy (SUDEP). We have developed a novel AAV-F mediated CRISPR-Cas-inspired RNA targeting system (CIRTS) preclinical treatment to increase endogenous Scn1a and ameliorate the disease phenotype in a clinically-relevant heterozygous loss-of-function mouse model of DS. We designed novel guide RNAs (gRNAs) to target the long non-coding RNA, (or natural antisense transcript) of Scn1a to increase the expression of Scn1a mRNA in DS mice. We show that intracerebroventricular and intravenous administration of AAV-F-CIRTS-gRNA9 to target the brain and the heart to neonatal Scn1a+/-mice resulted in a significant increase in survival, and a reduction in SUDEP, febrile seizures and seizure duration. These findings provide proof-of-concept evidence that an AAV-F-CIRTS mediated therapy hold promise as a potential treatment for DS.

neuroscience↗

bioRxiv2024

AAV9-mediated targeting of natural antisense transcript as a novel treatment for Dravet Syndrome

Dravet syndrome (DS) is a severe childhood onset developmental and epileptic encephalopathy which leads to life-long disability. Symptoms usually manifest in the first year of life and include prolonged severe seizures, developmental delay and severe intellectual disability. DS patients have an increased mortality rate, including sudden unexpected death in epilepsy (SUDEP). Approximately 90% of patients carry a heterozygous loss-of-function mutation in the SCN1A gene, which encodes a voltage-gated sodium ion channel, NaV1.1. The NaV1.1 channel is expressed in the brain and at a lower level, in the heart. Previous studies have identified a long non-coding RNA (lncRNA) which specifically downregulates SCN1A gene expression. This natural antisense transcript (NAT) can be modulated by AntagoNATs, small synthetic oligonucleotides developed to inhibit NAT function. In a DS mouse model, AntagoNATs were shown to modulate Scn1a expression by targeting the Scn1a NAT, improving seizure frequency after repeated administration. Here, we have developed novel AntagoNATs and incorporated these into a clinically relevant adeno-associated virus serotype 9 (AAV9) gene therapy vector, to test in a DS mouse model (Scn1a+/-) and provide a one-off treatment approach. Eighteen AntagoNATs were tested in vitro; from the best performing candidates, we selected two AntagoNAT sequences (K & H) for in vivo testing as they had the highest homology (90%) to human SCN1A NAT. We administered both vectors to newborn Scn1a+/- mice via intracerebroventricular (ICV) and intravenous (IV) injection to target the brain and heart. AAV9-AntagoNAT-H significantly increased survival, decreased febrile seizures and reduced spontaneous seizure frequency compared to the PBS control group. When administered at P14 by ICV and IV injection, AAV9-AntagoNAT-H increased survival. In this proof-of-concept study, we have demonstrated for the first time the delivery of AntagoNAT technology via an AAV9 vector and thus offering the possibility of a one-time treatment for DS patients.

molecular biology↗

bioRxiv2024

GENE THERAPY PREVENTS HEPATIC MITOCHONDRIAL DYSFUNCTION IN MURINE DEOXYGUANOSINE KINASE DEFICIENCY

Primary mitochondrial disorders are an uncommon cause of neonatal hepatic failure. Biallelic pathogenic variants of the gene encoding the mitochondrial localising enzyme deoxyguanosine kinase (DGUOK) cause hepatocerebral mitochondrial DNA depletion syndrome leading to acute neonatal liver failure and early mortality. There are currently no effective disease-modifying therapies. In this study, we developed an adeno-associated virus 9 (AAV9) gene therapy approach to treat a mouse model of DGUOK deficiency that recapitulates human disease. We delivered AAV9-hDGUOK gene therapy intravenously to newborn Dguok knock-out mice and showed that liver dysfunction was prevented in a dose dependent manner. Unexpectedly for neonatal delivery, durable and long-lasting liver transduction and RNA expression were demonstrated. Liver mitochondrial DNA depletion, deficiencies of oxidative phosphorylation complexes I, III and IV and liver transaminitis and survival were ameliorated in a dose-dependent manner.

genetics↗

bioRxiv2023

Creation of de novo cryptic splicing for ALS/FTD precision medicine

A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe "TDP-REG", a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF in vitro and in vivo, including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the UNC13A cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals. One-Sentence SummaryWe engineer TDP-43-regulated cryptic exons, enabling exceptionally precise activation of gene therapies in diseased neurons.

molecular biology↗

bioRxiv2023

Spinal cord pathology in a Dravet Syndrome mouse model

SummaryO_ST_ABSObjectivesC_ST_ABSDravet syndrome is a severe epileptic encephalopathy that begins in early childhood. More than 80% of patients with Dravet syndrome exhibit a haploinsufficiency in SCN1A, which encodes the voltage-gated sodium ion channel NaV1.1. The epilepsy is believed be caused by specific deficit of SCN1A in inhibitory interneurons of the hippocampus. However, the aetiology of other symptoms including gait disturbances, ataxia, cardiac issues and dysautonomia is less clear. MethodsIn an Scn1a knock-out (Scn1a-/-) mouse model which recapitulates clinical phenotypes, we assessed NaV1.1 and neuroinflammation throughout the central nervous system. ResultsConsistent with current understanding, wild-type expression of NaV1.1 transcript and protein were absent in knock-out mice in the prefrontal cortex, striatum, hippocampus, thalamus, and cerebellum. Increased GFAP was detected in the brain only in the hippocampus. Transcript and protein were detected in wild-type cervical, thoracic and lumbar spinal cord but not in knock-out mice. Unexpectedly, GFAP was increased in all three spinal regions. Therefore, we proceeded to perform transcriptomic analysis of cortex, hippocampus and spinal cord. Pathways associated with monooxygenase activity, fatty acid ligases and lactate transporters were highly dysregulated in the spinal cord. ConclusionThe existence and relevance of pathology of the spinal cord in Dravet syndrome has received scant attention. Our findings are consistent with some systemic symptoms of Dravet syndrome, with the benefits of treatments which may modulate the astrocyte-neuron lactate shuttle such as Stiripentol and ketogenic dietary regimes, and with the efficacy of intrathecal delivery of therapeutics. Key PointsO_LIDecrease of endogenous Scn1a and NaV1.1 expression in Scn1a-/- mice has a widespread impact on the gene expression profile in the spinal cord. C_LIO_LIIncreased GFAP expression observed in the spinal cord of Scn1a-/- mice. C_LIO_LIDifferentially expressed genes related to monooxygenase activity, fatty acid ligases and lactate transporters in cervical spinal cord of Scn1a-/- mice. C_LI

molecular biology↗

bioRxiv2019

Cervical gene delivery of the antimicrobial peptide, Human β-defensin (HBD)-3, in a mouse model of ascending infection-related preterm birth

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space: ascent from the vagina is the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) help to constitute a barrier which prevents ascending infection. We investigated whether expression of the AMP, human {beta}-defensin-3 (HBD3), in the cervical mucosa prevented bacterial ascent from the vagina into the uterine cavity of pregnant mice. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control (AAV8 GFP), was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent E.coli (E.coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, cellular events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. In addition, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 hours post-E.coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. There was also an increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be considered a possible candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth.

immunology↗

bioRxiv2018

Gene therapy in argininosuccinic aciduria

Argininosuccinate lyase (ASL) belongs to the liver-based urea cycle detoxifying ammonia, and the citrulline-nitric oxide cycle synthesising nitric oxide (NO). ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia and a multi-organ disease with neurocognitive impairment. Current therapeutic guidelines aim to control ammonaemia without considering the systemic NO imbalance. Here, we observed a neuronal disease with oxidative/nitrosative stress in ASL-deficient mouse brains. A single systemic injection of gene therapy mediated by an adeno-associated viral vector serotype 8 (AAV8) in adult or neonatal mice demonstrated the long-term correction of the urea cycle and the citrulline-NO cycle in the brain, respectively. The neuronal disease persisted if ammonaemia only was normalised but was dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This was correlated with behavioural improvement and a decrease of the cortical cell death rate. Thus, the cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress not mediated by hyperammonaemia, which is reversed by AAV gene transfer targeting the brain and the liver, acting on two different metabolic pathways via a single vector delivered systemically. This approach provides new hope for hepatocerebral metabolic diseases.

genetics↗

bioRxiv2018

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

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 NF{kappa}B binding sequence for bioluminescence and biosensor evaluation.\n\nAn 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-NF{kappa}B mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days.\n\nTo further restrict biosensor activity to the CNS, we performed intracerebroventricular injection of each vector. We observed greater restriction of bioluminescence to the head and spine with both vectors. Immunohistochemistry revealed strongest expression in cells of neuronal morphology. LPS administration stimulated a 4-fold increase over baseline bioluminescence.\n\nWe have validated a novel biosensor technology in an AAV system by using an NF{kappa}B response element and revealed its potential to monitor signalling pathway in a non-invasive manner using a model of LPS-induced inflammation. This technology employs the 3Rs of biomedical animal research, complements existing germline-transgenic models and may be applicable to other rodent disease models with the use of different response elements.

genomics↗