Adeno-associated virus-mediated gene transfer of arginine decarboxylase to the central nervous system prevents opioid analgesic tolerance.

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for site-specific overexpression of the presumptive synthetic enzyme for local therapeutic effects.

Comparative dose effectiveness of intravenous and intrathecal AAV9.CB7.hIDS, RGX-121, in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disease caused by iduronate-2-sulfatase (IDS) deficiency, leading to accumulation of glycosaminoglycans (GAGs) and the emergence of progressive disease. Enzyme replacement therapy is the only currently approved treatment, but it leaves neurological disease unaddressed. Cerebrospinal fluid (CSF)-directed administration of AAV9.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background.

Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine.

Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome.

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits.

Phenotypic Correction of Murine Mucopolysaccharidosis Type II by Engraftment of Lentiviral Vector-Transduced Hematopoietic Stem and Progenitor Cells.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase.

Comparative Effectiveness of Intracerebroventricular, Intrathecal, and Intranasal Routes of AAV9 Vector Administration for Genetic Therapy of Neurologic Disease in Murine Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration.

Intravenous delivery for treatment of mucopolysaccharidosis type I: A comparison of AAV serotypes 9 and rh10.

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of heparan and dermatan sulfate glycosaminoglycans (GAGs). Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, intellectual disability, and death by age 10. Current treatments for this disease include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT).

Intranasal Adeno-Associated Virus Mediated Gene Delivery and Expression of Human Iduronidase in the Central Nervous System: A Noninvasive and Effective Approach for Prevention of Neurologic Disease in Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector.

Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5.

We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles.

Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse.

Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.

Comparison of Endovascular and Intraventricular Gene Therapy With Adeno-Associated Virus-α-L-Iduronidase for Hurler Disease.

Background: Hurler disease (mucopolysaccharidosis type I [MPS-I]) is an inherited metabolic disorder characterized by deficiency of the lysosomal enzyme α-L-iduronidase (IDUA). Currently, the only therapies for MPS-I, enzyme replacement and hematopoietic stem cell transplantation, are generally ineffective for central nervous system manifestations.

Objective: To test whether brain-targeted gene therapy with recombinant adeno-associated virus (rAAV5)-IDUA vectors in an MPS-I transgenic mouse model would reverse the pathological hallmarks.

PR_b functionalized stealth liposomes for targeted delivery to metastatic colon cancer.

A gene delivery system was designed to carry a payload to integrin overexpressing cells. Branched-polyethyleneimine (bPEI) condensed plasmid DNA was encapsulated into targeted stealth liposomes, thereby combining the condensing and transfection properties of bPEI with the stealth and targeting properties of the liposomal carrier system. PR_b was used as a targeting ligand - a peptide we designed to bind specifically to the cancer cell surface marker αβ integrin - and such a robust receptor-ligand interaction achieved higher specificity than what has been previously reported for targeted delivery systems.

Direct gene transfer to the CNS prevents emergence of neurologic disease in a murine model of mucopolysaccharidosis type I.

The mucopolysaccharidoses (MPSs) are a group of 11 storage diseases caused by disruptions in glycosaminoglycan (GAG) catabolism, leading to their accumulation in lysosomes. Resultant multisystemic disease is manifested by growth delay, hepatosplenomegaly, skeletal dysplasias, cardiopulmonary obstruction, and, in severe MPS I, II, III, and VII, progressive neurocognitive decline. Some MPSs are treated by allogeneic hematopoietic stem cell transplantation (HSCT) and/or recombinant enzyme replacement therapy (ERT), but effectiveness is limited by central nervous system (CNS) access across the blood-brain barrier.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System.

Background: Metastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver.

Results: Liver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid.

Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Background: Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Gene expression in lung and liver after intravenous infusion of polyethylenimine complexes of Sleeping Beauty transposons.

Two methods of systemic gene delivery have been extensively explored, using the mouse as a model system: hydrodynamic delivery, wherein a DNA solution equivalent in volume to 10% of the mouse weight is injected intravenously in less than 10 sec, and condensation of DNA with polyethylenimine (PEI) for standard intravenous infusion. Our goal in this study was to evaluate quantitatively the kinetics of gene expression, using these two methods for delivery of Sleeping Beauty transposons. Transposons carrying a luciferase expression cassette were injected into mice either hydrodynamically or after condensation with PEI at a PEI nitrogen-to-DNA phosphate ratio of 7.

Systemic correction of storage disease in MPS I NOD/SCID mice using the sleeping beauty transposon system.

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice.

Adeno-associated virus type 2 vectors: transduction and long-term expression in cerebellar Purkinje cells in vivo is mediated by the fibroblast growth factor receptor 1 : bFGFR-1 mediates AAV2 transduction of Purkinje cells.

The receptor for adeno-associated virus serotype 2 (AAV2) in Purkinje cells has not been identified, but based on work carried out in non-neuronal cell lines, heparan sulfate proteoglycan is thought to act as a primary receptor, with basic fibroblast growth factor receptor-1 being reported as a co-receptor. In this study, using antibody interference and protein competition strategies, we show specific reduction in Purkinje cell transduction by AAV2 vector in the presence of these inhibitors. We also demonstrate AAV2-mediated transgene expression in Purkinje cells in vivo that extends out to one year post-injection.

Liver-directed gene therapy using the sleeping beauty transposon system.

Sleeping Beauty (SB) is a transposon system genetically reconstructed from teleost fish that mediates chromosomal integration of DNA sequences by a cut-and-paste mechanism. SB has been shown to mediate transposition in a variety of cells and tissues, has been used for the generation of transgenic animals and has been tested as a vector for gene therapy in several animal models of human disease. Here, we describe methods that we have developed for testing SB-mediated transposition, first in cultured mammalian cells, and then in vivo using a combination of rapid, high-volume tail vein injection for DNA delivery to the liver along with in vivo bioluminescence imaging to monitor sustained luciferase gene expression in individual animals.

Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection.

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse.

Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system.

The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems.

Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer.

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues.