Biology subjects

Cook, A. L.

Publications and source records attributed to Cook, A. L..

10 recordsLinked to original sources

bioRxiv2026

Computational drug repurposing identifies N-acetylglucosamine as a potential therapeutic compound for CLN3 Batten disease

Batten disease, also known as neuronal ceroid lipofuscinoses, is one of the most common causes of childhood dementia. It is characterized by the accumulation of lipofuscin in lysosomes, leading to loss of brain cell function, onset of dementia-like symptoms, vision loss and seizures and has extremely limited treatment options. Here, we performed computational drug repurposing analysis to identify existing compounds that may target Batten disease risk genes. A total of 81 candidate compounds were identified, 6 of which were selected based on clinical tractability for downstream testing in Batten disease (CLN3) iPSC-derived models. After confirming disease phenotype and drug candidate safety, CLN3 brain cell cultures treated with and without drug candidates underwent bulk RNA-seq to identify drug responses. One of the candidate drugs N-acetylglucosamine (GlcNAc) significantly upregulated Batten disease risk gene CLN5 expression and several other lysosomal markers within CLN3 brain cells, and modulated several pathways implicated in lysosomal storage disorders. Importantly, GlcNAc significantly reduced lipofuscin burden in both CLN3 iPSC-derived neurons and astrocytes, supporting its investigation as an additional therapy for Batten disease.

neuroscience↗

bioRxiv2025

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorders like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation (CLN3{Delta}ex7-8) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence SummaryAcid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

neuroscience↗

bioRxiv2024

Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease.

PurposeCLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. MethodsIsogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9{-}mediated biallelic deletion of exons 7 and 8. A transgenic CLN3{Delta}7-8/{Delta}7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3{Delta}7-8/{Delta}7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3{Delta}7/8 and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age. ResultsCLN3{Delta}7-8/{Delta}7-8 RPE (CLN3 RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3{Delta}7-8/{Delta}7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age. ConclusionsCLN3{Delta}7-8/{Delta}7-8 mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

cell biology↗

bioRxiv2023

Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD. This screen implicated disruption of kinase SMG1s phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from truncating mutations in vivo and in vitro. Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable HLA class I-associated peptides from NMD-downregulated proteins on the surface of cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases. One Sentence SummaryDisruption of the nonsense-mediated decay pathway with a newly developed SMG1 inhibitor with in-vivo activity increases the expression of T-cell targetable cancer neoantigens resulting from truncating mutations.

cancer biology↗

bioRxiv2023

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

BackgroundPericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and PDGF receptor-beta inhibitors impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive meditators. iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

cell biology↗

bioRxiv2023

HDAC6 inhibition as a mechanism to prevent axon degeneration in the mSOD1G93A mouse model of ALS.

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One dichotomy emerging in the field is that protection of the neuronal cell soma itself does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of individual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a potential mechanism to stabilise axons and prevent axonal loss and neurodegeneration in ALS. This study has utilized an orally dosed HDAC6 specific inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1G93A mouse model of ALS. Furthermore, co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1G93A mice, reduced lower motor neuron degeneration in the lumbar spinal cord of female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS. HighlightsO_LIACY-738 inhibits HDAC6 and leads to increased microtubule acetylation in spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment reduces lower motor neuron degeneration in the lumbar spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment restores peripheral nerve axon puncta size of mSOD1G93A mice. C_LIO_LIACY-738 treatment does not prevent overt motor function decline mSOD1G93A mice. C_LIO_LIRiluzole treatment partially restores peripheral nerve axon puncta size in mSOD1G93A mice. C_LI

neuroscience↗

bioRxiv2022

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 {Delta} 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

neuroscience↗

bioRxiv2021

An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

cancer biology↗

bioRxiv2020

Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing.

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vitro and in vivo.

bioengineering↗

bioRxiv2018

Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing \"kamikaze\" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas9), after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9, the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive \"kamikaze\" CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.

genetics↗