Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord.

Current and emerging targeted therapies for spinal muscular atrophy.

Introduction: Spinal muscular atrophy (SMA) is a progressive neurodegenerative disorder caused by insufficiency or total absence of the survival motor neuron protein due to a mutation in the gene. The copy number of its paralog, , influences disease onset and phenotype severity. Current therapeutic approaches include viral and non-viral modalities affecting gene expression.

Lentiviral-Induced Spinal Cord Gliomas in Rat Model.

Intramedullary spinal cord tumors are a rare and understudied cancer with poor treatment options and prognosis. Our prior study used a combination of PDGF-B, HRAS, and p53 knockdown to induce the development of high-grade glioma in the spinal cords of minipigs. In this study, we evaluate the ability of each vector alone and combinations of vectors to produce high-grade spinal cord gliomas.

Controlling the Release of Neurotrophin-3 and Chondroitinase ABC Enhances the Efficacy of Nerve Guidance Conduits.

Nerve guidance conduits (NGCs) have the potential to replace autografts in repairing peripheral nerve injuries, but their efficacy still needs to be improved. The efficacy of NGCs is augmented by neurotrophic factors that promote axon growth and by enzymes capable of degrading molecules that inhibit axon growth. In the current study, two types of NGCs loaded with factors (both neurotrophin-3 and chondroitinase ABC) are constructed and their abilities to repair an 8 mm gap in the rat sciatic nerve are examined.

Lot-to-Lot Variation in Adeno-Associated Virus Serotype 9 (AAV9) Preparations.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot.

Minimally Invasive Injection to the Phrenic Nerve in a Porcine Hemidiaphragmatic Paralysis Model: A Pilot Study.

Background: Neurodegenerative diseases and spinal cord injury can affect respiratory function often through motor neuron loss innervating the diaphragm. To reinnervate this muscle, new motor neurons could be transplanted into the phrenic nerve (PN), allowing them to extend axons to the diaphragm. These neurons could then be driven by an optogenetics approach to regulate breathing.

Progress in gene and cell therapies for the neuronal ceroid lipofuscinoses.

Introduction: The neuronal ceroid lipofuscinoses (NCLs) are a subset of lysosomal storage diseases (LSDs) that cause myoclonic epilepsy, loss of cognitive and motor function, degeneration of the retina leading to blindness, and early death. Most are caused by loss-of-function mutations in either lysosomal proteins or transmembrane proteins. Current therapies are supportive in nature.

Gene, Stem Cell, and Alternative Therapies for SCA 1.

Spinocerebellar ataxia 1 is an autosomal dominant disease characterized by neurodegeneration and motor dysfunction. In disease pathogenesis, polyglutamine expansion within Ataxin-1, a gene involved in transcriptional repression, causes protein nuclear inclusions to form. Most notably, neuronal dysfunction presents in Purkinje cells.

Gene Therapy for the Treatment of Neurological Disorders: Amyotrophic Lateral Sclerosis.

Gene therapy is a powerful tool for treating diseases, including neurological disorder such at amyotrophic lateral sclerosis. When delivered to the CNS, gene therapy vectors can provide prosurvival signals to neurons, knock down the expression of toxic proteins, or restore lost function. How to best deliver this type of therapeutic depends on the nature of the disease and the expected function of the transgene.

Fetal Brain-directed AAV Gene Therapy Results in Rapid, Robust, and Persistent Transduction of Mouse Choroid Plexus Epithelia.

Fetal brain-directed gene addition represents an under-appreciated tool for investigating novel therapeutic approaches in animal models of central nervous system diseases with early prenatal onset. Choroid plexuses (CPs) are specialized neuroectoderm-derived structures that project into the brain's ventricles, produce cerebrospinal fluid (CSF), and regulate CSF biochemical composition. Targeting the CP may be advantageous for adeno-associated viral (AAV) gene therapy for central nervous system disorders due to its immunoprivileged location and slow rate of epithelial turnover.

L-threo-dihydroxyphenylserine corrects neurochemical abnormalities in a Menkes disease mouse model.

Objective: Menkes disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting adenosine triphosphatase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine beta hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes disease, mottled-brindled (mo-br), we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology.

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy.

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking.

Increased frequency of congenital heart defects in Menkes disease.

ATP7A is a copper-transporting ATPase critical for central and peripheral nervous system function. Mutations in ATP7A cause Menkes disease and occipital horn syndrome (OHS), allelic X-linked recessive conditions that feature vascular abnormalities ascribed to low activity of lysyl oxidase, a copper-dependent enzyme. From a recently created Menkes disease/OHS patient registry, we identified four of 95 patients with major congenital heart defects (4.

ATP7A gene addition to the choroid plexus results in long-term rescue of the lethal copper transport defect in a Menkes disease mouse model.

Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age.

Somatic mosaicism in Menkes disease suggests choroid plexus-mediated copper transport to the developing brain.

The primary mechanism of copper transport to the brain is unknown, although this process is drastically impaired in Menkes disease, an X-linked neurodevelopmental disorder caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Potential central nervous system entry routes for copper include brain capillary endothelial cells that originate from mesodermal angioblasts and form the blood-brain barrier, and the choroid plexuses, which derive from embryonic ectoderm, and form the blood-cerebrospinal fluid barrier. We exploited a rare (and first reported) example of somatic mosaicism for an ATP7A mutation to shed light on questions about copper transport into the developing brain.

Molecular correlates of epilepsy in early diagnosed and treated Menkes disease.

Epilepsy is a major feature of Menkes disease, an X-linked recessive infantile neurodegenerative disorder caused by mutations in ATP7A, which produces a copper-transporting ATPase. Three prior surveys indicated clinical seizures and electroencephalographic (EEG) abnormalities in a combined 27 of 29 (93%) symptomatic Menkes disease patients diagnosed at 2 months of age or older. To assess the influence of earlier, presymptomatic diagnosis and treatment on seizure semiology and brain electrical activity, we evaluated 71 EEGs in 24 Menkes disease patients who were diagnosed and treated with copper injections in early infancy (≤6 weeks of age), and whose ATP7A mutations we determined.

Translational read-through of a nonsense mutation in ATP7A impacts treatment outcome in Menkes disease.

Protein translation ends when a stop codon in a gene's messenger RNA transcript enters the ribosomal A site. Mutations that create premature stop codons (nonsense mutations) typically cause premature translation termination. An alternative outcome, read-through translation (or nonsense suppression), is well known in prokaryotic, viral, and yeast genes but has not been clearly documented in humans except in the context of pharmacological manipulations.

Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R.

Menkes disease is a fatal neurodegenerative disorder of infancy caused by defects in an X-linked copper transport gene, ATP7A. Evidence from a recent clinical trial indicates that favorable response to early treatment of this disorder with copper injections involves mutations that retain some copper transport capacity. In three unrelated infants, we identified the same mutation, G727R, in the second transmembrane segment of ATP7A that complemented a Saccharomyces cerevisiae copper transport mutant, consistent with partial copper transport activity.

Neonatal diagnosis and treatment of Menkes disease.

Background: Menkes disease is a fatal neurodegenerative disorder of infancy caused by diverse mutations in a copper-transport gene, ATP7A. Early treatment with copper injections may prevent death and illness, but presymptomatic detection is hindered by the inadequate sensitivity and specificity of diagnostic tests. Exploiting the deficiency of a copper enzyme, dopamine-beta-hydroxylase, we prospectively evaluated the diagnostic usefulness of plasma neurochemical levels, assessed the clinical effect of early detection, and investigated the molecular bases for treatment outcomes.

AAV vector integration sites in mouse hepatocellular carcinoma.

Adeno-associated viruses (AAV) are promising gene therapy vectors that have little or no acute toxicity. We show that normal mice and mice with mucopolysaccharidosis VII (MPS VII) develop hepatocellular carcinoma (HCC) after neonatal injection of an AAV vector expressing b-glucuronidase. AAV proviruses were isolated from four tumors and were all located within a 6-kilobase region of chromosome 12.

Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome.

Background: Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the underlying molecular mechanisms.

Methods: We measured biochemical markers of impaired copper transport in five patients from two unrelated families and used RNase protection, quantitative reverse transcription (RT)-PCR, Western blot analysis and yeast complementation studies to characterise two ATP7A missense mutations, A1362D and S637L.

Central nervous system-directed AAV2/5-mediated gene therapy synergizes with bone marrow transplantation in the murine model of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is a rapidly progressing inherited neurodegenerative disorder caused by a deficiency in galactosylceramidase activity. Previous studies in the murine model of GLD (Twitcher mouse) have shown that both bone marrow transplantation (BMT) and central nervous system (CNS)-directed gene therapy can be moderately effective at ameliorating certain aspects of GLD. As BMT and CNS-directed gene therapy target fundamentally different tissues, we tested the hypothesis that combining these disparate therapies would be more efficacious than either therapy alone.