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1.
EMBO Mol Med ; 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890537

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is a rare paediatric lysosomal storage disorder, caused by the progressive accumulation of heparan sulphate, resulting in neurocognitive decline and behavioural abnormalities. Anecdotal reports from paediatricians indicate a more severe neurodegeneration in MPS IIIA patients, following infection, suggesting inflammation as a potential driver of neuropathology. To test this hypothesis, we performed acute studies in which WT and MPS IIIA mice were challenged with the TLR3-dependent viral mimetic poly(I:C). The challenge with an acute high poly(I:C) dose exacerbated systemic and brain cytokine expression, especially IL-1ß in the hippocampus. This was accompanied by an increase in caspase-1 activity within the brain of MPS IIIA mice with concomitant loss of hippocampal GFAP and NeuN expression. Similar levels of cell damage, together with exacerbation of gliosis, were also observed in MPS IIIA mice following low chronic poly(I:C) dosing. While further investigation is warranted to fully understand the extent of IL-1ß involvement in MPS IIIA exacerbated neurodegeneration, our data robustly reinforces our previous findings, indicating IL-1ß as a pivotal catalyst for neuropathological processes in MPS IIIA.

2.
Hum Gene Ther ; 35(7-8): 232-242, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37212263

RESUMO

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.


Assuntos
Iduronato Sulfatase , Mucopolissacaridose II , Doenças do Sistema Nervoso , Vírus da Raiva , Camundongos , Animais , Mucopolissacaridose II/genética , Mucopolissacaridose II/terapia , Ácido Idurônico , Iduronato Sulfatase/genética , Glicoproteínas/genética , Peptídeos
3.
Mol Ther Methods Clin Dev ; 31: 101127, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-37920237

RESUMO

Mucopolysaccharidosis type II (MPSII) is a pediatric lysosomal storage disease caused by deficiencies in the IDS (iduronate-2-sulfatase) gene resulting in accumulation of glycosaminoglycans, multisystem disease, and profound neurodegeneration in severe forms. Although enzyme replacement therapy is available for somatic forms of disease, the inability of native IDS to pass the blood-brain barrier renders it ineffective for the brain. We previously demonstrated the short-term efficacy of a brain-targeted hematopoietic stem cell gene therapy approach to treat MPSII mice using lentiviral IDS fused to the blood-brain-barrier-crossing peptide ApoEII (IDS.ApoEII) in comparison with a lentivirus expressing native IDS and an unmanipulated bone marrow transplant. Here we evaluated the longevity of disease correction for 12-16 months following treatment. We observed sustained IDS enzyme activity in organs of long-term IDS.ApoEII-treated MPSII mice, similar to those analyzed 6 months post-treatment, with continued clearance of storage material in the brain and peripheral organs, maintained correction of astrogliosis, microgliosis, and correction of altered cytokines and chemokines. IDS.ApoEII also significantly reduced retinal atrophy, characteristic of MPSII. Overall, IDS.ApoEII resulted in systemic prevention of the MPSII phenotype, with no observed toxicity following treatment. This provides evidence of the sustained efficacy and safety of this treatment ahead of a recently opened clinical trial.

4.
Front Mol Biosci ; 9: 965089, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36172050

RESUMO

Mucopolysaccharide diseases are a group of paediatric inherited lysosomal storage diseases that are caused by enzyme deficiencies, leading to a build-up of glycosaminoglycans (GAGs) throughout the body. Patients have severely shortened lifespans with a wide range of symptoms including inflammation, bone and joint, cardiac, respiratory and neurological disease. Current treatment approaches for MPS disorders revolve around two main strategies. Enzyme replacement therapy (ERT) is efficacious in treating somatic symptoms but its effect is limited for neurological functions. Haematopoietic stem cell transplant (HSCT) has the potential to cross the BBB through monocyte trafficking, however delivered enzyme doses limit its use almost exclusively to MPSI Hurler. Gene therapy is an emerging therapeutic strategy for the treatment of MPS disease. In this review, we will discuss the various vectors that are being utilised for gene therapy in MPS as well as some of the most recent gene-editing approaches undergoing pre-clinical and clinical development.

5.
Genes (Basel) ; 12(5)2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922187

RESUMO

Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases.


Assuntos
Cromatina/genética , Epigênese Genética/genética , RNA/genética , Transcrição Gênica/genética , Animais , DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Processamento Pós-Transcricional do RNA/genética
6.
Curr Cancer Drug Targets ; 21(4): 326-352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33504307

RESUMO

The advent of new genome-wide sequencing technologies has uncovered abnormal RNA modifications and RNA editing in a variety of human cancers. The discovery of reversible RNA N6-methyladenosine (RNA: m6A) by fat mass and obesity-associated protein (FTO) demethylase has led to exponential publications on the pathophysiological functions of m6A and its corresponding RNA modifying proteins (RMPs) in the past decade. Some excellent reviews have summarized the recent progress in this field. Compared to the extent of research into RNA: m6A and DNA 5-methylcytosine (DNA: m5C), much less is known about other RNA modifications and their associated RMPs, such as the role of RNA: m5C and its RNA cytosine methyltransferases (RCMTs) in cancer therapy and drug resistance. In this review, we will summarize the recent progress surrounding the function, intramolecular distribution and subcellular localization of several major RNA modifications, including 5' cap N7-methylguanosine (m7G) and 2'-O-methylation (Nm), m6A, m5C, A-to-I editing, and the associated RMPs. We will then discuss dysregulation of those RNA modifications and RMPs in cancer and their role in cancer therapy and drug resistance.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Metiltransferases , Neoplasias , Processamento Pós-Transcricional do RNA/fisiologia , RNA/metabolismo , Epigênese Genética/genética , Humanos , Metiltransferases/classificação , Metiltransferases/genética , Metiltransferases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Edição de RNA/fisiologia
7.
Neuro Oncol ; 22(9): 1289-1301, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32227096

RESUMO

BACKGROUND: Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. METHODS: Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. RESULTS: Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. CONCLUSION: These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. KEY POINTS: 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Criança , Dactinomicina/farmacologia , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas , Fatores de Transcrição SOXB1/genética
8.
Mol Ther Methods Clin Dev ; 13: 399-413, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31044143

RESUMO

Hematopoietic stem cell gene therapy is a promising therapeutic strategy for the treatment of neurological disorders, since transplanted gene-corrected cells can traffic to the brain, bypassing the blood-brain barrier, to deliver therapeutic protein to the CNS. We have developed this approach for the treatment of Mucopolysaccharidosis type IIIA (MPSIIIA), a devastating lysosomal storage disease that causes progressive cognitive decline, leading to death in early adulthood. In a previous pre-clinical proof-of-concept study, we demonstrated neurological correction of MPSIIIA utilizing hematopoietic stem cell gene therapy via a lentiviral vector encoding the SGSH gene. Prior to moving to clinical trial, we have undertaken further studies to evaluate the efficiency of gene transfer into human cells and also safety studies of biodistribution and genotoxicity. Here, we have optimized hCD34+ cell transduction with clinical grade SGSH vector to provide improved pharmacodynamics and cell viability and validated effective scale-up and cryopreservation to generate an investigational medicinal product. Utilizing a humanized NSG mouse model, we demonstrate effective engraftment and biodistribution, with no vector shedding or transmission to germline cells. SGSH vector genotoxicity assessment demonstrated low transformation potential, comparable to other lentiviral vectors in the clinic. This data establishes pre-clinical safety and efficacy of HSCGT for MPSIIIA.

9.
Hum Gene Ther Methods ; 30(2): 44-52, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30963787

RESUMO

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.


Assuntos
Bestrofinas/fisiologia , Parvovirinae/genética , Bestrofinas/genética , Dependovirus , Vetores Genéticos , Células HEK293 , Humanos , Transdução Genética
10.
ACS Chem Neurosci ; 10(1): 18-20, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30136572

RESUMO

Neurological lysosomal storage diseases are rare, inherited conditions resulting mainly from lysosomal enzyme deficiencies. Current treatments, such as enzyme replacement therapy and hematopoietic stem cell transplantation, fail to effectively treat neurological disease due to insufficient brain delivery of the missing enzyme. Ex vivo gene therapy approaches to overexpress the missing enzyme in hematopoietic stem cells prior to transplant are an emerging technology that has the potential to offer a viable therapy for patients with these debilitating diseases.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Doenças por Armazenamento dos Lisossomos/terapia , Doenças do Sistema Nervoso/terapia , Animais , Encéfalo/fisiologia , Técnicas de Transferência de Genes/tendências , Terapia Genética/tendências , Transplante de Células-Tronco Hematopoéticas/tendências , Células-Tronco Hematopoéticas/fisiologia , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças do Sistema Nervoso/genética
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