Search

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans. 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. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, i.e. indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, CNS-directed gene transfer in the murine models of MPS I and MPS II. Although validation is needed in human clinical trials, our results suggest that depending on the extent of neurologic decline prior to treatment, gene transfer could confer neurodevelopmental improvement in severe MPS I and MPS II patients when carried out after the onset of neurologic decline.

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 α5β1 integrin – and such a robust receptor–ligand interaction achieved higher specificity than what has been previously reported for targeted delivery systems. In the process of formulating the PR_b functionalized gene delivery vehicle, we developed a protocol to fully encapsulate condensed DNA in liposomes and accurately quantify the total DNA in the system. We demonstrate that compared to non-targeted stealth liposomes and non-encapsulated condensed DNA, the PR_b functionalized stealth liposomes mediated improved in vitro transfection specifically to colon cancer cells overexpressing the α5β1 integrin. Furthermore, when administered in vivo to metastatic tumor bearing mice, PR_b functionalized stealth liposomes outperformed non-targeted liposomes and delivered genes specifically to the tumor site.

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). However, these treatments do not address CNS manifestations of the disease. In this study we compared the ability of intravenously administered AAV serotypes 9 and rh10 (AAV9 and AAVrh10) for delivery and expression of the IDUA gene in the CNS. Adult C57BL/6 MPS I mice were infused intravenously with either AAV9 or AAVrh10 vector encoding the human IDUA gene. Treated animals demonstrated supraphysiological levels and widespread restoration of IDUA enzyme activity in the plasma and all organs including the CNS. High levels of IDUA enzyme activity were observed in the plasma, brain and spinal cord ranging from 10 to 100-fold higher than heterozygote controls, while levels in peripheral organs were also high, ranging from 1000 to 10,000-fold higher than control animals. In general, levels of IDUA expression were slightly higher in peripheral organs for AAVrh10 administered animals although these differences were not significant except for the lung. Levels of IDUA expression between AAV 9 and rh10 were roughly equivalent in the brain. Urinary and tissue GAGs were significantly reduced starting at 3 weeks after vector infusion, with restoration of normal GAG levels by the end of the study in animals treated with either AAV9 or rh10. These results demonstrate that non-invasive intravenous AAV9 or AAVrh10-mediated IDUA gene therapy is a potentially effective treatment for both systemic and CNS manifestations of MPS I, with implications for the treatment of other metabolic and neurological diseases as well.

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. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.

Hurler disease or mucopolysaccharidosis type I (MPS-I) is an autosomal-recessive lysosomal storage disease caused by a defect in α-L-iduronidase (IDUA).1,2 It is the most common of the mucopolysaccharidoses, with an incidence of 1 per 75 000 live births.3 Deficiency of the IDUA enzyme leads to a pathological accumulation of glycosaminoglycans (GAG) in the brain and other organs. Typical central nervous system (CNS) features are severe mental retardation, leptomeningeal fibrosis with obstructive hydrocephalus, arachnoid cysts, and sensorineural deafness. Although systemic features of MPS-I can be treated with enzyme replacement therapy or hematopoietic stem cell transplantation, there is currently no accepted treatment for the CNS features. A caveat is that intervention in the neonatal period with bone marrow or cord blood transplantation has shown limited improvement in cognitive performance, likely through cross-correction by stem cells that migrate to the perivascular space.4,5 However, even in the best-case scenario, the existing treatment options do not reverse abnormal cognitive development, are associated with numerous long-term complications, and are prohibitively expensive for many patients. The pathological hallmark of MPS-I in the brain is the accumulation of GAG inside neurons, where they are increased up to 5-fold, as well as in mesodermally derived tissues of the perivascular space, leptomeninges, and choroid plexus, where they are often increased 10-fold and form so-called clear-cell periadventitial inclusions.6 As a result of pathological accumulation of GAG, there is a secondary perturbation of β-galactosidase (originally thought to be the main defect in Hurler disease),7 and consequently, the neuronal inclusions in Hurler disease are made up primarily of gangliosides (GM2, GM3) which are not usually present in substantial amounts in normal brain.8,9 Another downstream enzyme, β-hexosaminidase (defective in Tay-Sachs disease), which breaks down GM2, is upregulated in MPS-I, and normalization of this enzyme was used as a secondary outcome measure in canine gene therapy studies for Hurler disease.10 Our inability to achieve high-level neuronal gene expression throughout the large volume of the human brain is currently an impediment to more effective gene therapy for many neurodegenerative diseases. The purpose of this study was to test the feasibility of minimally invasive viral vector-based CNS gene therapy in Hurler disease and, more specifically, to compare endovascular with intracerebral ventricular (ICV) delivery. Recombinant adeno-associated viral vector (rAAV) was chosen because it is nonpathogenic and is able to express genes in the CNS at very high levels.11 It exists in more than 10 serotypes, most of which specifically transduce neurons when injected intraparenchymally, but some are capable of transducing astrocytes, microglia, ependyma, and other cells to various degrees. To maximize the unit dose per animal, we used a transgenic mouse model12 rather than one of the larger animal models,13,14 with an aggressive endovascular dosing regimen of 1 trillion genomic particles (g.p.) per mouse. We chose rAAV5 vector, which has an advantage of high-level neuronal expression and cellular internalization by the platelet-derived growth factor receptor,15 which also targets ependymal, choroid plexus, and perivascular cells affected in Hurler disease. Another theoretical benefit of rAAV5 is that neutralizing antibody titers are the lowest among AAV serotypes.16 Endovascular gene therapy offers widespread access to the brain but generally requires blood-brain barrier disruption, and until recently, results in animal models were disappointing. Rapoport and colleagues17,18 were the first to show reversible osmotic opening of the blood-brain barrier, and their technique of hyperosmolar disruption remains in widespread use. This method was adapted for clinical use in intra-arterial (IA) chemotherapy for brain tumors19 but has never been used clinically for gene therapy. Endovascular techniques have been used in rats20–22 or mice23 to deliver viral vectors to the brain, but gene expression was significantly less than with intraparenchymal delivery, and the microsurgical techniques are demanding. As a result, this approach was abandoned by the majority of gene therapists, even as microcatheter techniques and vector technology advanced. ICV gene therapy is appealing because the ependymal and meningeal surface area is large and may be particularly suited to Hurler disease, which involves these structures. The original clinical application of ICV gene therapy in 1996 used a nonviral vector with Ommaya reservoirs in patients with Canavan disease together with intravenous mannitol.24 The benefit of mannitol for this route of delivery is the greater bulk flow through the interstitial space and increased permeability of the ependyma, rather than effects on the blood-brain barrier. Systemic mannitol was shown in animal models to increase spread of vectors with ICV or intraparenchymal delivery.24–26 However, as a result of perceived limitations of the ICV route with viral vectors, an intraparenchymal approach with multiple rAAV2 injections and systemic mannitol was subsequently adopted for human use.27,28 At this time, the ICV approach deserves a closer look with new vector technology and techniques to optimize transependymal flow. Our study used rAAV5 vectors in adult animals to deliver foreign genes to the brain via IA or ICV delivery, with mannitol treatment used in both cases. In the first case, mannitol is administered directly to arterial vessels to transiently disrupt the endothelial tight junctions and to allow penetration of vector, whereas in the latter case, mannitol is used to improve bulk flow of rAAV5 through ependyma and interstitial space. We initially tested dose-response and biodistribution using rAAV5–green fluorescent protein (GFP) and then assessed the tolerability and efficacy of a very high unit dose (5 × 1013 viral particles per kg) of the therapeutic vector rAAV5-IDUA. With IA delivery, blood-brain barrier disruption was performed with 25% or 29.1% mannitol, and with ICV delivery, we used 25% mannitol injected systemically or directly into the cerebral ventricle. Our goal with ICV delivery was not merely to assess the use of mannitol, because this is already known to increase vector spread,24,29 but rather to compare the endovascular and ICV delivery routes using the best available techniques. Using either delivery paradigm, we significantly reversed brain pathology in animals with a Hurler phenotype.

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. To provide a high level of gene product to the CNS, we tested neonatal intracerebroventricular (ICV) infusion of an adeno-associated virus (AAV) serotype 8 vector transducing the human α-L-iduronidase gene in MPS I mice. Supranormal levels of iduronidase activity in the brain (including 40x normal levels in the hippocampus) were associated with transduction of neurons in motor and limbic areas identifiable by immunofluorescence staining. The treatment prevented accumulation of GAG and GM3 ganglioside storage materials and emergence of neurocognitive dysfunction in a modified Morris water maze test. The results suggest the potential of improved outcome for MPSs and other neurological diseases when a high level of gene expression can be achieved by direct, early administration of vector to the CNS.

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. Gene expression in the lungs and liver after hydrodynamic delivery resulted in exponential decay with a half-life of about 35-40 hr between days 1 and 14 postinjection. The decay kinetics of gene expression after PEI-mediated gene delivery were more complex; an initial decay rate of 6 hr was followed by a more gradual loss of activity. Consequently, the liver became the primary site of gene expression about 4 days after injection of PEI-DNA, and by 14 days expression in the liver was 10-fold higher than in the lung. Overall levels of gene expression 2 weeks postinjection were 100- to 1000-fold lower after PEI-mediated delivery compared with hydrodynamic injection. These results provide insight into the relative effectiveness and organ specificity of these two methods of nonviral gene delivery when coupled with the 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. The stages of this protocol are preparation of DNA-PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA-PEI complexes to analysis of transient transgene expression.

The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.

Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, α1-antitrypsin deficiency, emphysema, and cancer. DNA-mediated gene transfer to the lung has been previously demonstrated, but anticipated effectiveness has been limited by low gene transfer efficiencies and by transient expression of the transgene. Here, we combine plasmid-based gene transfer with the integrating capacity of the nonviral Sleeping Beauty (SB) transposon vector system to mediate gene insertion and long-term gene expression in mouse lung. We observed transgene expression after 24 h in lungs of all animals injected with the luciferase transposon (pT/L), but expression for up to 3 months required codelivery of a plasmid encoding the Sleeping Beauty transposase. We also observed long-term expression in pT/L-injected animals transgenic for SB transposase. Transgene expression was localized to the alveolar region of the lung, with transfection including mainly type II pneumocytes. We used a linker-mediated PCR technique to recover transposon flanking sequences, demonstrating transposition of pT/L into mouse chromosomal DNA of the lung.

Mucopolysaccharidosis type I (MPS I) is a progressive, multisystemic, inherited metabolic disease caused by deficiency of alpha-L-iduronidase (IDUA). The most severe form of this disease (Hurler syndrome) results in death by age 10. Current treatments for this disease are ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. Our goal is to supplement current therapy, and treat CNS manifestations of the disease, by AAV-mediated gene delivery and expression of IDUA. We have taken a non-invasive and effective approach to the treatment of CNS disease by intranasal administration of an IDUA-encoding AAV9 vector. Adult IDUA-deficient mice were immunotolerized at birth with human iduronidase, to prevent anti-IDUA immune response, and at 3 months of age were infused intranasally with AAV9-IDUA vector. Animals sacrificed 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Neurocognitive testing using the Barnes maze demonstrated that treated IDUA-deficient mice were not different from normal control animals, while untreated IDUA-deficient mice exhibited a significant learning deficit. There was strong IDUA immunofluorescence staining observed in tissue sections of the nasal epithelium and olfactory bulb, but no staining was observed in other portions of the brain. This indicates that the widespread distribution of IDUA enzyme most likely was the result of enzyme diffusion from sites of transduction and IDUA expression in the olfactory bulb and the nasal epithelium into deeper areas of the brain. In order to increase access, delivery and vector distribution throughout the brain, IDUA-deficient animals were pretreated with intranasal infusions of an absorption enhancer. At different time points following pretreatment, animals were infused intranasally with AAV9 or AAVrh10 vector encoding IDUA. Animals were sacrificed at 2 months post-infusion, brains microdissected, and assayed for IDUA enzyme activity, clearance of glycosaminoglycans, and immunofluorescence staining for IDUA and GFP. This novel, non-invasive strategy for intranasal AAV9-IDUA administration could potentially be used to treat CNS manifestations of MPS I.

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. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

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.

The receptor for adeno-associated virus sero-type 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. These results provide new information on fibro-blast growth factor receptor-1 involvement in AAV2-mediated transduction of Purkinje cells and have important implications for AAV-mediated treatment of various cerebellar disorders.

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.

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. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells. Disclosure of potential conflicts of interest is found at the end of this article.

Background The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII. Methods We constructed SB transposon plasmids for high-level expression of human β-glucuronidase (hGUSB) or α-L-iduronidase (hIDUA). Plasmids were delivered with and without SB transposase to mouse liver by rapid, high-volume tail-vein injection. We studied the duration of expressed therapeutic enzyme activity, transgene presence by PCR, lysosomal pathology by toluidine blue staining and cell-mediated immune response histologically and by immunohistochemical staining. Results Transgene frequency, distribution of transgene and enzyme expression in liver and the level of transgenic enzyme required for amelioration of lysosomal pathology were estimated in MPS I and VII mice. Without immunomodulation, initial GUSB and IDUA activities in plasma reached > 100-fold of wild-type (WT) levels but fell to background within 4 weeks post-injection. In immunomodulated transposon-treated MPS I mice plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of MPS I mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, some of which were CD4- or CD8-positive. Conclusions Our results demonstrate the feasibility of achieving prolonged expression of lysosomal enzymes in the liver and reversing MPS disease in adult mice with a single dose of therapeutic SB transposons. Copyright © 2007 John Wiley & Sons, Ltd.

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. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAGs). In the severe form of the disease (Hurler syndrome), death results by age 10. Current treatments for this disease include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality.We have taken a novel approach to treat neurologic disease associated with Hurler syndrome, using intranasal administration of an IDUA-encoding AAV9 vector. A CAGS regulated AAV9-IDUA vector was infused intranasally into adult mice (2-3 months of age) that had been immunotolerized at birth with Aldurazyme to prevent anti-IDUA immune response. Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain. QPCR analysis of vector genomes indicated only background levels in all portions of the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain. This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze. Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape. Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals. There was no significant difference between heterozygote animals and treated animals, while latency to escape was significantly different between these two groups and MPS I deficient animals (P

The Sleeping Beauty (SB) transposon system combines the advantages of non-viral plasmid based gene transfer with an integrative ability, thus leading to long term expression of the transgene. Our goal is to use the SB transposon system to deliver antiangiogenic and immunostimulatory genes, and to test their antitumor effectiveness in an animal model of colorectal cancer metastases to the liver.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

Methotrexate (MTX) is an effective antitumor agent that has been demonstrated to be particularly useful in the treatment of hematopoietic neoplasms but causes substantial hematologic and gastrointestinal toxicity. We previously demonstrated that transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase (DHFR) into animals preconditioned by irradiation substantially protected recipient mice from the toxic side effects of methotrexate administration. Here we test the use of methotrexate itself as a preconditioning agent for engraftment of drug-resistant transgenic marrow, subsequently conferring drug resistance upon recipient animals. Administration of methotrexate beginning 1 or 2 weeks prior to or on the same day as transplantation with drug-resistant DHFR transgenic marrow did not allow sufficient engraftment to confer drug resistance to most unirradiated recipients. A small number of animals were curiously protected from lethal MTX toxicity but exhibited extremely low hematocrits and were not engrafted with stem cells, as indicated by low engraftment levels assessed in secondary transplant recipients. However, we subsequently found that MTX preconditioning allowed sufficient engraftment of DHFR transgenic marrow to confer drug resistance if MTX administration was withdrawn at the time of bone marrow transplantation (BMT) and withheld until 2 weeks post-transplant. Quantitative molecular analysis of primary and secondary recipients indicated a stem cell engraftment level of approximately 1%, consistent with previous studies demonstrating that a low level of DHFR transgenic cell engraftment was sufficient to confer drug resistance in recipient animals. We conclude that MTX can be used as a preconditioning agent for subsequent engraftment of hematopoietic stem cells, in this case conferring resistance to MTX.

Top of pageAbstract The purpose of this project was to evaluate the Sleeping Beauty (SB) transposon for delivery of the human |[alpha]|-L-iduronidase ( IDUA) gene to chromosomes in the livers of IDUA-deficient mice for long- term expression and correction of MPS type I. We chose the hydrodynamics-based DNA injection, which targets mainly the liver. We constructed SB transposon, plasmid-based vectors and injected them into idua-/-mice. Because our goal was in part to determine the efficacy of transposition as a way of providing long-term expression of IDUA protein, control groups of MPS I mice did not get the transposase. Blood samples for plasma isolation were collected 1 day after treatment and once every 2 weeks thereafter. Plasma IDUA activities reached >100-fold of wild type levels on day 1 following treatment, but were essentially gone in all mice 4 weeks following gene delivery. IDUA activity was not detected in the livers of these mice 3 months after plasmid administration. We examined the duration of the transposon-delivered human IDUA DNA maintenance and enzyme activity over 6 months in the liver of wild type (WT) C57Bl/6 mice that express IDUA. As in the MPS I mice, plasma and liver IDUA activity reached supra-normal levels on day 1 and remained at this level for the first week, but still dropped dramatically within 2 weeks and by 4 weeks were indistinguishable from background. Using DNA PCR, we found that the maintenance of the IDUA transgene mirrored the IDUA activity time-line. Even though transposition was confirmed by an excision assay, the PCR product was detectable only for the first two weeks, but not thereafter. It appears that cells that express the IDUA gene are cleared from the liver of treated mice by 4 weeks following injection, which suggests induction of an immune response either to the therapeutic protein or/and the cells that express the therapeutic gene. This hypothesis is supported by our observations that in all cyclophosphamide immune-suppressed MPS I mice, the initial 1-day plasma activity levels dropped more than 100-fold by 2 weeks, but then persisted at 20-500% WT activity in some mice. IDUA levels were stable (up to 5-fold higher than in the WT mice) in SB-transposase-treated MPS I mice whereas without SB, IDUA levels declined to near-background over 3 months. These persistent IDUA activity levels were sufficient to reduce the number and size of pathologic inclusions in the liver seen by toluidine blue staining. Secondary elevation of murine |[beta]|-glucuronidase, another lysosomal enzyme, was reduced in the liver, spleen and kidney and correlated with degree of reduction of lysosomal pathology in these organs. Thus, with immune suppression, a single dose of the SB transposon system can result in partial to complete biochemical correction of IDUA activity in the livers of treated adult MPS I mice.

The Sleeping Beauty (SB) transposon system has been demonstrated to mediate gene insertion and long-term expression, suggesting the potential application of this plasmid based system for non-viral gene therapy. All reported studies of SB-mediated gene transfer to somatic cells have supplied the transposase by expression of co-injected plasmid DNA. Although successful for both in vitro and in vivo transposition, there is a lack of control with respect to the transient and long-term levels of transposase expressed in a target cell. There is also the potential for genomic integration of the transposase encoding DNA, resulting in sustained transposase expression and potential destabilizing effects on the integrated transgene or host genome integrity. We address these problems by supplying the transposase-encoding molecule in the form of mRNA. Rapid, high pressure injection of m7G-capped luciferase (Luc) mRNA through the tail vein of mice resulted in efficient luciferase expression with respect to a DNA element where the Caggs promoter controlled luciferase transcription. Using this system as a model for efficient gene transfer in the liver, we have taken steps to optimize delivery/expression of the mRNA by generating constructs to promote stability and translation. Real-time whole body in vivo imaging of Luc mRNA translation was conducted to measure the effect of an IRES and UTR sequences on luciferase expression. These studies determined that incorporation of 5' and 3' UTR elements from the Xenopus -globin gene were essential to achieve peak luciferase expression levels from an otherwise unprotected transcript. Co-delivery of these mRNAs with an inhibitor of or a substrate for endogenous RNases provided luciferase expression sufficient to mimic levels achieved using promoters known to provide optimum transposase expression for in vivo transposition. Using this genetic arrangement, we have co-delivered SB transposase mRNA with a transposon encoding the Luc gene (driven by the Caggs promoter) to the liver of transgenic mice in which transcription of Cre recombinase is under the control of an interferon response element. To distinguish between plasmid-mediated expression and expression mediated by transposition, we have engineered this transposon encoding plasmid with lox-P sites positioned such that Cre-mediated recombination excises a sequence which includes the Caggs promoter, unless it has been segregated away from the plasmid by the SB transposase (Score et al., Mol. Ther. 7: S9, 2003). We found that pIpC induction of Cre recombinase caused a 1600-fold reduction in luciferase expression in animals infused with the transposon alone. While co-delivery of transposase mRNA resulted in a moderate degree of inadvertent Cre induction, we observed only a 45-fold reduction in luciferase expression following analogous pIpC administration. This remaining luciferase expression is most likely attributable to SB mediated transposition. These results provide the first evidence for use of SB transposase mRNA to mediate transposition and long-term expression in an in vivo setting.

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. Animals that were injected with a low dose (10 μg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 μg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 μg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid. Conclusion These results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

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. Results: Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. Conclusions: The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.