Miguel Sena-Esteves

Miguel Sena-Esteves
University of Massachusetts Medical School | UMMS · Department of Neurology

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213
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Introduction
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Publications

Publications (213)
Article
Adeno-associated virus (AAV)-mediated gene therapies have provided promising treatments for numerous neurological disorders. Redosing of AAV to the central nervous system (CNS) is an attractive research area due to both the somewhat immunologically privileged status of the CNS as well as the possibility of reduced glial transgene expression over ti...
Article
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Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP...
Article
Full-text available
Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months wi...
Article
Full-text available
Transformative results of adeno-associated virus (AAV) gene therapy in patients with spinal muscular atrophy and Leber’s congenital amaurosis led to approval of the first two AAV products in the United States to treat these diseases. These extraordinary results led to a dramatic increase in the number and type of AAV gene-therapy programs. However,...
Article
GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal β-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral vector provides pronounced therapeut...
Article
Huntington's disease (HD) is a devasting, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene. Inactivation of the mutant allele by CRISPR-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neurona...
Article
Tay-Sachs disease (TSD) is a fatal neurodegenerative disease caused by a deficiency of the enzyme β-N-acetylhexosaminidase A (HexA). TSD naturally occurs in Jacob sheep is the only experimental model of TSD. TSD in sheep recapitulates neurologic features similar to juvenile onset and late onset TSD patients. Due to the paucity of human literature o...
Article
Full-text available
Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effecti...
Preprint
Full-text available
Tay-Sachs Disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Preclinical work demonstrated safety and efficacy of CNS gene therapy using AAVrh8-HEXA/HEXB. Here we describe an expanded access trial in two patients with infantile TSD (IND 18225). Case TSD-001 demonstrated neurodevelopmental regression...
Article
Epidemiological studies and work in transgenic animals suggest that transthyretin (TTR) levels in plasma and/or CSF may modulate Alzheimer’s disease (AD) presentation and/or progression. AD patients exhibit lower levels of TTR in CSF compared to age‐matched healthy individuals. Similarly, genetic reduction of TTR in mouse models of AD is associated...
Article
Full-text available
Abstract Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of β-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson’s disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and...
Article
AAV virions are built from three major capsid proteins, VP1, VP2, and VP3, at a ratio of 1:1:18. On a silver-stained SDS-polyacrylamide gel, VP1, VP2, and VP3 should be the only visible bands in a highly purified recombinant adeno-associated virus (rAAV) preparation, migrating at approximately 87, 73, and 62 kDa, respectively. This protocol describ...
Article
Negative staining is a simple and rapid method for studying the morphology and ultrastructure of small particulate specimens (e.g., viruses, bacteria, cell fragments, and isolated macromolecules such as proteins and nucleic acids). The technique described in this protocol involves allowing particles or fragments of cells to settle onto a support fi...
Article
Centrifugation to equilibrium in cesium chloride gradients has been used for more than 40 yr to purify viruses. The application of high G-forces for a long period of time to a solution of CsCl generates a density gradient that allows separation of empty, partially packaged, and fully packaged viral particles from cellular debris, proteins, and nucl...
Article
Sandhoff disease (SD) is caused by decreased function of the enzyme β‐N‐acetylhexosaminidase, resulting in accumulation of GM2 ganglioside in tissues. Neural tissue is primarily affected and individuals with the infantile form of the disease generally do not survive beyond 4 years of age. Current treatments address neurometabolic deficits to improv...
Article
The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-hexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5...
Article
Over the years, many different viral vector systems have been developed to take advantage of the specific biological properties and tropisms of a large number of mammalian viruses. As a result, researchers wanting to introduce and/or express genes in mammalian cells have many options, as discussed here.
Article
Thalamic infusion of adeno-associated viral (AAV) vectors has been shown to have therapeutic effects in neuronopathic lysosomal storage diseases. Preclinical studies in sheep model of Tay-Sachs disease demonstrated that bilateral thalamic injections of AAV gene therapy are required for maximal benefit. Translation of thalamic injection to patients...
Article
Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determin...
Article
This protocol is used to determine the concentration of DNase-resistant vector genomes (i.e., packaged in the capsid) in purified recombinant adeno-associated virus (rAAV) preparations. The protocol begins with treatment of the vector stock with DNase I to eliminate unencapsidated AAV DNA or contaminating plasmid DNA. This is followed by a heat tre...
Article
This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.
Article
This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.
Article
This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.
Article
The most commonly used method for production of recombinant adeno-associated virus (rAAVs) in research laboratories is by transient triple transfection of 293 cells with AAV cis and trans plasmids and an adenovirus helper plasmid. This protocol describes the processes required to prepare the transfected cell suspension for virus purification.
Article
The sensitivity of an assay for replication-competent adenoviruses (RCAs) can often be enhanced by biological amplification of the RCAs with serial passage. Here, we describe an extension of this technique, termed "concentration passage," in which RCA replicated during the first plating of the vector is collected and concentrated onto one-tenth of...
Article
The easiest way to confirm the structure and identity of genomic DNA isolated from purified adenoviral recombinants is restriction enzyme digestion and gel electrophoresis. This analysis entails comparing the restriction patterns of the adenoviral vector DNA with that plasmid that was used to initiate the entire rescue and expansion process. The in...
Article
Full-text available
GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8...
Article
AAV gene therapy for neurological diseases was revolutionized by the discovery that AAV9 crosses the blood-brain barrier (BBB) after systemic administration. Transformative results have been documented in various inherited diseases, but overall neuronal transduction efficiency is relatively low. The recent development of AAV-PHP.B with ~60-fold hig...
Article
Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of gene...
Article
Full-text available
Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in tr...
Article
Full-text available
GM1 gangliosidosis is an autosomal recessive neurodegenerative disorder caused by the deficiency of lysosomal β-galactosidase (β-gal) and resulting in accumulation of GM1 ganglioside. The disease spectrum ranges from infantile to late onset and is uniformly fatal, with no effective therapy currently available. Although animal models have been usefu...
Article
Here we describe the preparation of quantitative polymerase chain reaction (qPCR) standards.
Article
Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected f...
Article
Full-text available
Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospin...
Article
Full-text available
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mammalian target of rapamycin (mTOR)-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal...
Article
This protocol describes how to generate an infectious adenovirus vector by direct ligation and cloning. This is the first step in the production of a recombinant adenovirus vector. The protocol begins with a convenient and efficient double-selection procedure based on antibiotic resistance and identification of green fluorescent protein (GFP)-negat...
Article
After generating the recombinant adenovirus plasmid by direct cloning, the viral genome must be released from the plasmid for virus rescue and expansion in corresponding packaging cell lines. This protocol describes how to process and rescue a recombinant adenovirus and grow the rescued virus to large scale.
Article
Cesium chloride gradient centrifugation is the most widely used method for purification of recombinant adenovirus. This protocol describes the entire process, from the preparation and clarification of crude viral lysate to the formulation and storage of purified virus.
Article
Full-text available
Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential...
Article
Full-text available
Background: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artific...
Article
Full-text available
The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV...
Article
Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease since...
Article
Glioblastoma (GBM) is the most common and lethal form of primary brain tumor with dismal median and two-year survivals of 14.5 months and 18%, respectively. The paucity of new therapeutic agents stems from the complex biology of a highly adaptable tumor that uses multiple survival and proliferation mechanisms to circumvent current treatment approac...
Article
The titer of a lentivirus vector is often expressed in transducing units per milliliter. This is a functional titer that reflects the lentivirus' ability to transduce a particular cell line under specific conditions. Transduction of other cell lines is likely to be different and will require optimization. 293T cells are used for production of lenti...
Article
The most commonly used method for production of retrovirus and lentivirus vectors in research laboratories is of transient transfection of 293T cells with a lentivirus vector plasmid, packaging genome plasmid(s), and an envelope expression plasmid. High-titer stocks are obtained by concentration of vector supernatants by ultracentrifugation.
Article
The potential emergence of replication-competent lentiviruses (RCLs) during vector production and the significant biosafety risk that this represents has led to the development of lentivirus vector production systems to minimize the risk of generating RCLs. Second- and third-generation lentivirus vector production systems appear to be safe, because...
Article
Full-text available
Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of beta-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside mig...
Article
Full-text available
Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-leng...
Article
Full-text available
The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dl...
Article
Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). Tay-Sachs disease also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 tesla MRI, echocardiogr...
Article
GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder caused by an enzyme deficiency of β-galactosidase (β-gal) leading to toxic accumulation of GM1 ganglioside in the central nervous system (CNS) and progressive neurodegeneration. Adeno-associated virus (AAV) mediated gene delivery of lysosomal enzymes to the CNS has shown great...
Article
This study is the first to show progressive restrictive pulmonary disease in the most commonly used ALS mouse model—the SOD1G93A mouse.
Article
GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers us...
Article
GM2 gangliosidoses, including Tay-Sachs disease (TSD) and Sandhoff disease (SD), are lysosomal storage disorders caused by deficiencies in β-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system dysfunction (CNS). Studies in mice, cats, and sheep have indicated safety and widespread distribution of H...
Article
Full-text available
The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously we have shown that intracranial interferon-beta (IFNβ) gene therapy by locally administered adeno-associated viral vectors (AAV) suc...
Poster
Bilateral thalamic delivery of AAVrh10-CBA-mβgal vector is effective to supply supra-physiological levels of βgal to the cerebrum at doses as low as 1E09 vg (2E12vg/kg brain weight). ICV delivery is most effective route for enzyme delivery to the spinal cord, albeit relatively high doses seem to be necessary to restore >50% of normal βgal activity...
Poster
GM1 gangliosidosis is a hereditary neurodegenerative disease caused by a mutation of the lysosomal enzyme β-galactosidase (βgal), with the most common form being fatal by the age of 4 years old. In a well characterized feline model of GM1 gangliosidosis (GM1), AAV mediated gene therapy results in approximately 6 fold increase in lifespan after thal...
Article
Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, a...
Article
Full-text available
BACKGROUND: Mutant huntingtin (mHTT) is encoded by the Huntington's disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy. OBJECTIVE: To...
Article
Background: The genetic mutation in Huntington's disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in i...
Article
Full-text available
Background: Mutant huntingtin (mHTT) is encoded by the Huntington’s disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy. Objective: To...
Article
Full-text available
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies h...
Article
Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of...
Article
Background: Reducing mutant huntingtin (mHTT) in neurons may be a therapy for Huntington's disease (HD). Elevating NUB1 protein reduced mHTT levels in cell and fly models of HD through a proteasome dependent mechanism. Objective: To examine the effects of augmenting NUB1 in HD mouse striatum on mHTT levels. Methods: Striata of HDQ175/Q7 mice w...
Article
Background Complete surgical removal of all glioblastoma (GBM) cells is impossible due to extensive infiltration into brain parenchyma that ultimately leads to tumor recurrence. The current standard of care affords modest improvements in survival. New therapeutic interventions are needed to prevent recurrence. Local AAV-hIFNβ gene delivery to the b...