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Find video protocols related to scientific articles indexed in Pubmed.
Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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Vector platforms for gene therapy of inherited retinopathies.
Prog Retin Eye Res
PUBLISHED: 08-12-2014
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Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina's compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs' limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations.
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Novel Adeno-associated Viruses Derived From Pig Tissues Transduce Most Major Organs in Mice.
Sci Rep
PUBLISHED: 06-17-2014
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Recently, development of Adeno-associated virus (AAV) vectors has been focusing on expanding the genetic diversity of vectors from existing sequences via directed evolution or epitope remapping. Apart from intelligent design, AAV isolation from natural sources remains an important source of new AAVs with unique biological features. In this study, several new AAV sequences were isolated from porcine tissues (AAVpo2.1, -po4, -po5, and -po6), which aligned in divergent new clades. Viral particles generated from these sequences displayed tissue tropism and transduction efficiency profile specific to each porcine-derived AAV. When delivered systemically, AAVpo2.1 targeted the heart, kidney, and muscle, AAVpo5 performed poorly but was able to transduce muscle fibers when injected intramuscularly, whereas AAVpo4 and -po6 efficiently transduced all the major organs sampled, contending with 'gold-standard' AAVs. When delivered systemically, AAVpo4 and -po6 were detected by polymerase chain reaction (PCR) and histochemical staining of the transgene product in adult mouse brain, suggesting that these vectors can pass through the blood-brain barrier with efficiencies that may be useful for the development of therapeutic approaches. Porcine tissues are antigenically similar to human tissues and by inference, porcine AAVs may provide fresh tools to contribute to the development of gene therapy-based solutions to human diseases.
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Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age.
Circulation
PUBLISHED: 06-02-2014
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Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2(R33Q/R33Q) (R33Q) mutation.
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Similar therapeutic efficacy between a single administration of gene therapy and multiple administrations of recombinant enzyme in a mouse model of lysosomal storage disease.
Hum. Gene Ther.
PUBLISHED: 04-11-2014
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Enzyme replacement therapy (ERT) has become the standard of care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSD manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI, we compared the efficacy of a single intravascular administration of an adeno-associated viral vector targeting liver to weekly infusions of human recombinant enzyme at the same doses used in mucopolysaccharidosis VI patients. While gene therapy results in increased and stable levels of circulating enzyme up to 1 year after vector administration, ERT has typical peak-and-drop serum kinetics. Both therapies similarly reduced glycosaminoglycan levels in urine and tissues including heart valves and myocardium, with gene therapy improving skeletal skull abnormalities slightly better, although not significantly, than ERT. Both therapies seem to similarly improve animal motor performance, with gene therapy possibly associated with less animal distress. Thus, a single vector administration that converts liver into a factory organ for systemic secretion of therapeutic proteins is at least as effective as ERT in a mouse model of LSD, potentially eliminating problems with compliance and costs. Only testing in humans will prove whether this holds true in a clinical setting.
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Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.
Mol. Ther.
PUBLISHED: 01-14-2014
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Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2?×?10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.
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Sensory-motor behavioral characterization of an animal model of Maroteaux-Lamy syndrome (or Mucopolysaccharidosis VI).
Sci Rep
PUBLISHED: 01-11-2014
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Maroteaux-Lamy disease, also known as mucopolysaccharidosis (MPS) VI, is an MPS disorder caused by mutations in the ARSB gene encoding for the lysosomal enzyme arysulfatase B (ARSB). Deficient ARSB activity leads to lysosomal accumulation of dermatan sulfate in a wide range of tissues and organs. There are various animal models of MPS VI that have been well characterized from a biochemical and morphological point of view. In this study, we report the sensory-motor characterization of MPS VI rats carrying homozygous null ARSB mutations. We show that adult MPS VI rats are specifically impaired in vertical activity and motor endurance. All together, these data are consistent with biochemical findings that show a major impairment in connective tissues, such as joints and bones. The behavioral abnormalities of MPS VI rats represent fundamental endpoints for studies aimed at testing the pre-clinical safety and efficacy of novel therapeutic approaches for MPS VI.
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Effective delivery of large genes to the retina by dual AAV vectors.
EMBO Mol Med
PUBLISHED: 12-15-2013
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Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAVs limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardts disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on "forced" packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAVs ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
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Combined rod and cone transduction by adeno-associated virus 2/8.
Hum. Gene Ther.
PUBLISHED: 10-30-2013
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Abstract Gene transfer to both cone and rod photoreceptors (PRs) is essential for gene therapy of inherited retinal degenerations that are caused by mutations in genes expressed in both PR types. Vectors based on the adeno-associated virus (AAV) efficiently transduce PRs of different species. However, these are predominantly rods and little is known about the ability of the AAV to transduce cones in combination with rods. Here we show that AAV2/8 transduces pig cones to levels that are similar to AAV2/9, and the outer nuclear layer (mainly rods) to levels that are on average higher, although not statistically significant, than both AAV2/5 and AAV2/9. We additionally found that the ubiquitous cytomegalovirus (CMV), but not the PR-specific GRK1 promoter, transduced pig cones efficiently, presumably because GRK1 is not expressed in pig cones as observed in mice and humans. Indeed, the GRK1 and CMV promoters transduce a similar percentage of murine cones with the CMV reaching the highest expression levels. Consistent with this, the AAV2/8 vectors with either the CMV or the GRK1 promoter restore cone function in a mouse model of Leber congenital amaurosis type 1 (LCA1), supporting the use of AAV2/8 for gene therapy of LCA1 as well as of other retinal diseases requiring gene transfer to both PR types.
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AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently.
Hum Gene Ther Methods
PUBLISHED: 10-11-2013
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Abstract During the production of some adeno-associated virus (AAV) serotypes, a large amount of vectors is found in the medium of producing cells. For their purification, previous protocols used tangential flow filtration (TFF) of the medium followed by iodixanol gradient centrifugation. Taking advantage of the higher purity of the medium than the cell-derived material as the source of AAV, we tested a simple method that combines production of large culture medium volumes containing AAV from cell stacks with medium clarification+TFF without further time-consuming and nonscalable centrifugation. To test this, we selected AAV2/8, which is emerging as a favored serotype for transduction of liver, muscle, and retina and abundantly found in the extracellular medium. We show that yields and in vitro infectivity of AAV2/8 vectors produced from the culture medium using this method are higher than those of vectors purified from the same cell lysate using a conventional CsCl2 gradient ultracentrifugation-based method, although purity appears inferior. In addition, we found that the transduction efficiency of AAV2/8 purified from medium was similar to that of AAV2/8 purified from the same cell lysate in the murine liver, muscle, and retina. Considering that the purification protocol from the medium we describe requires 3?hr as opposed to the 63?hr of a conventional two-round CsCl2-gradient ultracentrifugation+desalting, we conclude that TFF of the medium containing AAV2/8 represents a quick and scalable method to purify research-grade vectors for use in animal models.
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AAV-mediated Liver-specific MPV17 Expression Restores mtDNA Levels and Prevents Diet-induced Liver Failure.
Mol. Ther.
PUBLISHED: 06-11-2013
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Mutations in human MPV17 cause a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS) hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients eventually develop slowly progressive peripheral and central neuropathy. The physiological role of Mpv17, including its functional link to mitochondrial DNA (mtDNA) maintenance, is still unclear. We show here that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in critical tissues, i.e. liver, of a Mpv17 knockout mouse model. On a standard diet, Mpv17(-/-) mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet (KD) leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus (AAV)-mediated gene replacement, the Mpv17 knockout mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation (OXPHOS) proficiency, and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.
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Use of a lower dosage liver-Detargeted AAV vector to prevent hamster muscular dystrophy.
Hum. Gene Ther.
PUBLISHED: 04-04-2013
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The BIO14.6 hamster carries a mutation in the delta sarcoglycan gene causing muscular dystrophy and cardiomyopathy. The disease can be prevented by systemic delivery of delta sarcoglycan cDNA using adeno-associated viruses (AAVs). However, all AAVs also target the liver, raising concerns about their therapeutic efficacy in human applications. We compared the AAV2/8 with the chimeric AAV2/2i8, in which the 585-QQNTAP-590 motif of the AAV8 serotype was added to the heparan sulfate receptor footprint of the AAV2 strain. Both vectors carrying the human delta sarcoglycan cDNA were delivered into 24 14-day-old BIO14.6 hamsters. We followed transgene expression in muscle and liver for 7 months. We detected a sustained ectopic expression of delta sarcoglycan in the liver when using AAV2/8 but not AAV2/2i8. Genomic copies of AAV2/2i8 were not detectable in the liver, while at least 100-fold more copies of AAV2/8 were counted. In contrast, the hamster skeletal muscle expressed more delta sarcoglycan using AAV2/2i8 and were still healthy after 7 months at the lower dosage. We conclude that this chimeric vector is a robust option for safer and longer-term diseased muscle targeting.
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Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2.
Ophthalmology
PUBLISHED: 03-06-2013
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The aim of this study was to show the clinical data of long-term (3-year) follow-up of 5 patients affected by Leber congenital amaurosis type 2 (LCA2) treated with a single unilateral injection of adeno-associated virus AAV2-hRPE65v2.
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Photoreceptor degeneration in mice: adeno-associated viral vector-mediated delivery of erythropoietin.
Methods Mol. Biol.
PUBLISHED: 03-05-2013
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The exogenous delivery of erythropoietin (EPO) and EPO derivatives (EPO-Ds) represents a valuable strategy to protect the retina from degeneration. In this chapter we describe a method to deliver EPO and the EPO derivative S100E in the light-damage model of induced retinal degeneration using adeno--associated viral (AAV) vectors and to evaluate the functional and morphological protection of the retina from light damage.
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Recombinant vectors based on porcine adeno-associated viral serotypes transduce the murine and pig retina.
PLoS ONE
PUBLISHED: 02-08-2013
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Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.
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Gene therapy for mucopolysaccharidosis type VI is effective in cats without pre-existing immunity to AAV8.
Hum. Gene Ther.
PUBLISHED: 01-30-2013
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Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.
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Myosin7a deficiency results in reduced retinal activity which is improved by gene therapy.
PLoS ONE
PUBLISHED: 01-01-2013
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Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1(-/-)) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1(-/-) photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1(-/-) retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1(-/-) photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1(-/-) retina is effective.
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Worsening of cardiomyopathy using deflazacort in an animal model rescued by gene therapy.
PLoS ONE
PUBLISHED: 05-19-2011
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We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients.
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MicroRNA-restricted transgene expression in the retina.
PLoS ONE
PUBLISHED: 04-05-2011
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Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions.
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Non-erythropoietic erythropoietin derivatives protect from light-induced and genetic photoreceptor degeneration.
Hum. Mol. Genet.
PUBLISHED: 03-19-2011
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Given the high genetic heterogeneity of inherited retinal degenerations (IRDs), a wide applicable treatment would be desirable to halt/slow progressive photoreceptor (PR) cell loss in a mutation-independent manner. In addition to its erythropoietic activity, erythropoietin (EPO) presents neurotrophic characteristics. We have previously shown that adeno-associated viral (AAV) vector-mediated systemic EPO delivery protects from PR degeneration. However, this is associated with an undesired hematocrit increase that could contribute to PR protection. Non-erythropoietic EPO derivatives (EPO-D) are available which allow us to dissect erythropoiesiss role in PR preservation and may be more versatile and safe than EPO as anti-apoptotic agents. We delivered in animal models of light-induced or genetic retinal degeneration either intramuscularly or subretinally AAV vectors encoding EPO or one of the three selected EPO-D: the mutant S100E, the helix A- and B-derived EPO-mimetic peptides. We observed that (i) systemic expression of S100E induces a significantly lower hematocrit increase than EPO and provides similar protection from PR degeneration, and (ii) intraocular expression of EPO-D protects PR from degeneration in the absence of significant hematocrit increase. On the basis of this, we conclude that erythropoiesis is not required for EPO-mediated PR protection. However, the lower efficacy observed when EPO or S100E is expressed intraocularly rather than systemically suggests that hormone systemic effects contribute to PR protection. Unlike S100E, EPO-mimetic peptides preserve PR only when given locally, suggesting that different EPO-D have a different potency or mode of action. In conclusion, our data show that subretinal delivery of AAV vectors encoding EPO-D protects from light-induced and genetic PR degeneration.
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Efficacy of a combined intracerebral and systemic gene delivery approach for the treatment of a severe lysosomal storage disorder.
Mol. Ther.
PUBLISHED: 02-15-2011
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Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.
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The human visual cortex responds to gene therapy-mediated recovery of retinal function.
J. Clin. Invest.
PUBLISHED: 02-01-2011
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Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.
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Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa.
EMBO Mol Med
PUBLISHED: 01-26-2011
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Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a repression-replacement strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.
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AAV-mediated gene replacement, either alone or in combination with physical and pharmacological agents, results in partial and transient protection from photoreceptor degeneration associated with betaPDE deficiency.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-01-2011
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Mutations in the PDE6B gene cause recessive, severe retinitis pigmentosa (RP). PDE6B encodes the ? subunit of the rod-specific phosphodiesterase (?PDE), which, when absent, results in toxic levels of intracellular Ca(2+) and photoreceptor cell death. Ca(2+) blockers, such as nilvadipine, as well as light restriction, slow photoreceptor degeneration in animal models of ?PDE deficiencies. The goal of the study was to evaluate the efficacy of AAV2/5- or AAV2/8-mediated gene replacement in combination with nilvadipine and/or with light restriction in the rd10 mouse bearing homozygous pde6b mutations.
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Long-term amelioration of feline Mucopolysaccharidosis VI after AAV-mediated liver gene transfer.
Mol. Ther.
PUBLISHED: 11-30-2010
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Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.
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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Hum. Gene Ther.
PUBLISHED: 09-16-2010
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A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18?x?10¹¹ particles/ml; 95% confidence interval [CI], 7.89?x?10¹¹ to 1.05?x?10¹² particles/ml), vector genomes ({X}, 3.28?x?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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MicroRNAs involved in molecular circuitries relevant for the Duchenne muscular dystrophy pathogenesis are controlled by the dystrophin/nNOS pathway.
Cell Metab.
PUBLISHED: 03-23-2010
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In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program.
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AAV-mediated gene supply for treatment of degenerative and neovascular retinal diseases.
Curr Gene Ther
PUBLISHED: 02-15-2010
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Common blinding diseases that are currently untreatable include conditions characterized by progressive neuronal degeneration, such as retinitis pigmentosa, leber congenital amaurosis or glaucoma, or characterized by ocular neovascularization, like wet age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. The pathogenic mechanisms underlying either neuronal degeneration or new vessel formation may be similar and independent of the mutation underlying the disease, thus allowing to test therapeutic strategies acting downstream of the primary causative event. Gene supply is the delivery of a gene that can prevent or arrest disease progression without being directly implicated in the disease pathogenesis. To this end, one of the most efficient and safe retinal gene delivery vehicles derives from the small adeno-associated virus (AAV). We review studies on AAV-mediated gene supply of: neurotrophic/antiapoptotic factors to prevent retinal neurons degeneration, and anti-angiogenic molecules to inhibit retinal neovascularization. Successful gene supply may represent a one-fit-all treatment for inherited and acquired blinding diseases.
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Gene therapy for Lebers congenital amaurosis is safe and effective through 1.5 years after vector administration.
Mol. Ther.
PUBLISHED: 12-01-2009
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The safety and efficacy of gene therapy for inherited retinal diseases is being tested in humans affected with Lebers congenital amaurosis (LCA), an autosomal recessive blinding disease. Three independent studies have provided evidence that the subretinal administration of adeno-associated viral (AAV) vectors encoding RPE65 in patients affected with LCA2 due to mutations in the RPE65 gene, is safe and, in some cases, results in efficacy. We evaluated the long-term safety and efficacy (global effects on retinal/visual function) resulting from subretinal administration of AAV2-hRPE65v2. Both the safety and the efficacy noted at early timepoints persist through at least 1.5 years after injection in the three LCA2 patients enrolled in the low dose cohort of our trial. A transient rise in neutralizing antibodies to AAV capsid was observed but there was no humoral response to RPE65 protein. The persistence of functional amelioration suggests that AAV-mediated gene transfer to the human retina does not elicit immunological responses which cause significant loss of transduced cells. The persistence of physiologic effect supports the possibility that gene therapy may influence LCA2 disease progression. The safety of the intervention and the stability of the improvement in visual and retinal function in these subjects support the use of AAV-mediated gene augmentation therapy for treatment of inherited retinal diseases.
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Age-dependent effects of RPE65 gene therapy for Lebers congenital amaurosis: a phase 1 dose-escalation trial.
Lancet
PUBLISHED: 10-23-2009
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Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Lebers congenital amaurosis.
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AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).
Mol. Ther.
PUBLISHED: 05-12-2009
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Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.
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Ocular gene therapy: current progress and future prospects.
Trends Mol Med
PUBLISHED: 04-22-2009
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As gene therapy begins to produce its first clinical successes, interest in ocular gene transfer has grown owing to the favorable safety and efficacy characteristics of the eye as a target organ for drug delivery. Important advances also include the availability of viral and non-viral vectors that are able to efficiently transduce various ocular cell types, the use of intraocular delivery routes and the development of transcriptional regulatory elements that allow sustained levels of gene transfer in small and large animal models after a single administration. Here, we review recent progress in the field of ocular gene therapy. The first experiments in humans with severe inherited forms of blindness seem to confirm the good safety and efficacy profiles observed in animal models and suggest that gene transfer has the potential to become a valuable therapeutic strategy for otherwise untreatable blinding diseases.
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Abnormal autophagy, ubiquitination, inflammation and apoptosis are dependent upon lysosomal storage and are useful biomarkers of mucopolysaccharidosis VI.
Pathogenetics
PUBLISHED: 02-20-2009
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Lysosomal storage diseases are characterized by intracellular accumulation of metabolites within lysosomes. Recent evidence suggests that lysosomal storage impairs autophagy resulting in accumulation of polyubiquitinated proteins and dysfunctional mitochondria, ultimately leading to apoptosis. We studied the relationship between lysosome storage and impairment of different intracellular pathways and organelle function in mucopolysaccharidosis VI, which is characterized by accumulation of dermatan sulfate and signs of visceral and skeletal but not cerebral involvement.
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Disease rescue and increased lifespan in a model of cardiomyopathy and muscular dystrophy by combined AAV treatments.
PLoS ONE
PUBLISHED: 02-19-2009
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The BIO14.6 hamster is an excellent animal model for inherited cardiomyopathy, because of its lethal and well-documented course, due to a spontaneous deletion of delta-sarcoglycan gene promoter and first exon. The muscle disease is progressive and average lifespan is 11 months, because heart slowly dilates towards heart failure.
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Pharmacological read-through of nonsense ARSB mutations as a potential therapeutic approach for mucopolysaccharidosis VI.
J. Inherit. Metab. Dis.
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Mucopolysaccharidosis type VI (MPS VI) is a severe lysosomal storage disorder without central nervous system involvement caused by arylsulfatase B (ARSB) deficiency. MPS VI is characterized by dysostosis multiplex, corneal clouding, heart valve defects and urinary excretion of glycosaminoglycans (GAGs). The current treatment for MPS VI is enzyme replacement therapy (ERT) which has limited efficacy on bone, joints and heart valve disease, as well as high costs. A potential therapeutic approach for the subgroup of MPS VI patients that carry nonsense mutations is to enhance stop-codon read-through, using small molecules, to restore production of the full-length ARSB protein. In this study we investigated whether two compounds known to induce stop codon read-through, the aminoglycoside gentamicin and PTC124, can promote read-through of four different ARSB nonsense mutations (p.R315X, p.R327X, p.Q456X and p.Q503X) associated with MPS VI and enable the synthesis of full-length functional ARSB protein in patients fibroblast cell lines. Our study demonstrates that PTC124 but not gentamicin, increases the level of ARSB activity in three MPS VI patient fibroblast cell lines. In two of them the levels of ARSB activity obtained were significantly higher than in untreated cells, reaching ?2.5 % of those detected in wild-type fibroblasts and resulting in significant reduction of lysosomal size. Since even small increases in enzyme activity can dramatically influence the clinical phenotype of MPS VI, our study suggests that pharmacological read-through may be combined with ERT potentially increasing therapeutic efficacy in those patients bearing nonsense ARSB mutations.
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Effective AAV-mediated gene therapy in a mouse model of ethylmalonic encephalopathy.
EMBO Mol Med
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Ethylmalonic encephalopathy (EE) is an invariably fatal disease, characterized by the accumulation of hydrogen sulfide (H(2)S), a highly toxic compound. ETHE1, encoding sulfur dioxygenase (SDO), which takes part in the mitochondrial pathway that converts sulfide into harmless sulfate, is mutated in EE. The main source of H(2)S is the anaerobic bacterial flora of the colon, although in trace amount it is also produced by tissues, where it acts as a gasotransmitter. Here, we show that AAV2/8-mediated, ETHE1-gene transfer to the liver of a genetically, metabolically and clinically faithful EE mouse model resulted in full restoration of SDO activity, correction of plasma thiosulfate, a biomarker reflecting the accumulation of H(2)S, and spectacular clinical improvement. Most of treated animals were alive and well >6-8 months after birth, whereas untreated individuals live 26 ± 7 days. Our results provide proof of concept on the efficacy and safety of AAV2/8-mediated livergene therapy for EE, and alike conditions caused by the accumulation of harmful compounds in body fluids and tissues, which can directly be transferred to the clinic.
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Sustained reduction of hyperbilirubinemia in Gunn rats after adeno-associated virus-mediated gene transfer of bilirubin UDP-glucuronosyltransferase isozyme 1A1 to skeletal muscle.
Hum. Gene Ther.
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Crigler-Najjar syndrome is an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels, but liver transplantation is the only permanent treatment. Muscle-directed gene therapy has several advantages, including easy and safe access through simple intramuscular injections, and has been investigated in human clinical trials. In this study, we have investigated the efficacy of adeno-associated viral (AAV) vector-mediated muscle-directed gene therapy in the preclinical animal model of Crigler-Najjar syndrome, that is the Gunn rat. Serotype 1 AAV vector expressing rat UGT1A1 under the control of muscle-specific creatine kinase promoter was injected at a dose of 3×10(12) genome copies/kg into the muscles of Gunn rats and resulted in expression of UGT1A1 protein and functionally active enzyme in injected muscles. AAV-injected Gunn rats showed an approximately 50% reduction in serum bilirubin levels as compared with saline-treated controls, and this reduction was sustained for at least 1 year postinjection. Increased excretion of alkali-labile metabolites of bilirubin in bile and urine was detected in AAV-injected animals. High-performance liquid chromatography analysis of bile from AAV-injected Gunn rats showed a metabolite with retention time close to that of bilirubin diglucuronide. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type 1.
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Gene therapy of inherited retinopathies: a long and successful road from viral vectors to patients.
Hum. Gene Ther.
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Inherited retinopathies (IRs) are common and untreatable blinding conditions inherited mostly as monogenic due to mutations in genes expressed in retinal photoreceptors (PRs) and in retinal pigment epithelium (RPE). Over the last two decades, the retina has emerged as one of the most favorable target tissues for gene therapy given its small size and its enclosed and immune-privileged environment. Different types of viral vectors have been developed, especially those based on the adeno-associated virus (AAV), which efficiently deliver therapeutic genes to PRs or RPE upon subretinal injections. Dozens of successful proofs of concept of the efficacy of gene therapy for recessive and dominant IRs have been generated in small and large models that have paved the way to the first clinical trials using AAV in patients with Leber congenital amaurosis, a severe form of childhood blindness. The results from these initial trials suggest that retinal gene therapy with AAV is safe in humans, that vision can be improved in patients that have suffered from severe impairment of visual function, in some cases for decades, and that readministration of AAV to the subretinal space is feasible, effective, and safe. However, none of the trials could match the levels of efficacy of gene therapy observed in a dog model of the disease, suggesting that there is room for improvement. In conclusion, these results bode well for further testing of AAV-mediated retinal gene therapy in patients with other monogenic and complex forms of blindness.
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Correlation between photoreceptor layer integrity and visual function in patients with Stargardt disease: implications for gene therapy.
Invest. Ophthalmol. Vis. Sci.
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To perform a clinical characterization of Stargardt patients with ABCA4 gene mutation, and to investigate the correlation between the inner and outer segment (IS/OS) junction morphology and visual acuity, fundus lesions, electroretinogram abnormalities, and macular sensitivity.
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Impact of age at administration, lysosomal storage, and transgene regulatory elements on AAV2/8-mediated rat liver transduction.
PLoS ONE
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Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients.We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels.As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies.
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AAV2 gene therapy readministration in three adults with congenital blindness.
Sci Transl Med
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Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant adeno-associated virus (AAV) carrying the RPE65 gene (AAV2-hRPE65v2) prompted us to determine whether it was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis due to mutations in the RPE65 gene 1.7 to 3.3 years after they had received their initial subretinal injection of AAV2-hRPE65v2. Results (through 6 months) including evaluations of immune response, retinal and visual function testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious after previous exposure to AAV2-hRPE65v2.
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Viral gene transfer rescues arrhythmogenic phenotype and ultrastructural abnormalities in adult calsequestrin-null mice with inherited arrhythmias.
Circ. Res.
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Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.