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R J Samulski

Publications and source records attributed to R J Samulski.

At least 19 recordsLinked to original sources
PubMed2016

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Animals↗
PubMed2006

Immune responses to AAV in a phase I study for Canavan disease.

BACKGROUND: Canavan disease is a rare leukodystrophy with no current treatment. rAAV-ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder. METHODS: Subjects received intracranial infusions via six cranial burr holes. Adeno-associated virus, serotype 2 (AAV2), mediated intraparenchymal delivery of the human aspartoacylase cDNA at a maximum dose of 1 x 10(12) vector genomes per subject. The immune response and safety profiles were monitored in the follow-up of ten subjects. RESULTS: Following rAAV2 administration, we found no evidence of AAV2 neutralizing antibody titers in serum for the majority of subjects tested (7/10). In a subset (3/10) of subjects, low to moderately high levels of AAV2 neutralizing antibody with respect to baseline were detected. In all subjects, there were minimal systemic signs of inflammation or immune stimulation. In subjects with catheter access to the brain lateral ventricle, cerebrospinal fluid was examined and there was a complete absence of neutralizing antibody titers with no overt signs of brain inflammation. CONCLUSIONS: rAAV2 vector administration to the human CNS appears well tolerated. The low levels of immune response to AAV2 detected in 3/10 subjects in this study suggest at this dose and with intraparenchymal administration this approach is relatively safe. Long-term monitoring of subjects and expansion to phase II/III will be necessary in order to make definitive statements on safety and efficacy.

Amidohydrolases↗
PubMed2005

Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector.

Small interfering RNAs (siRNAs) are potentially powerful tools for therapeutic gene regulation. DNA cassettes encoding RNA polymerase III promoter-driven hairpin siRNAs allow long-term expression of siRNA in targeted cells. A variety of viral vectors have been used to deliver such cassettes to cells. Here we report on the development and use of a self-complementary recombinant adeno-associated virus (scAAV) vector for siRNA delivery into mammalian cells. We demonstrate that this modified vector efficiently delivers siRNA into multidrug-resistant human breast and oral cancer cells and suppresses MDR1 gene expression. This results in rapid, profound, and durable reduction in the expression of the P-glycoprotein multidrug transporter and a substantial reversion of the drug-resistant phenotype. This research suggests that scAAV-based vectors can be very effective agents for efficient delivery of therapeutic siRNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse.

Mitochondrial myopathy, associated with muscle weakness and progressive external ophthalmoplegia, is caused by mutations in mitochondria oxidative phosphorylation genes including the heart-muscle isoform of the mitochondrial adenine nucleotide translocator (ANT1). To develop therapies for mitochondrial disease, we have prepared a recombinant adeno-associated viral vector (rAAV) carrying the mouse Ant1 cDNA. This vector has been used to transduce muscle cells and muscle from Ant1 mutant mice, which manifest mitochondrial myopathy. AAV-ANT1 transduction resulted in long-term, stable expression of the Ant1 transgene in muscle precursor cells as well as differentiated muscle fibers. The transgene ANT1 protein was targeted to the mitochondrion, was inserted into the mitochondrial inner membrane, formed a functional ADP/ATP carrier, increased the mitochondrial export of ATP and reversed the histopathological changes associated with the mitochondrial myopathy. Thus, AAV transduction has the potential of providing symptomatic relief for the ophthalmoplegia and ptosis resulting from paralysis of the extraocular eye muscles cause by mutations in the Ant1 gene.

Adenine Nucleotide Translocator 1↗
PubMed2003

Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.

An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.

Animals↗
PubMed2001

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Adenoviridae↗
PubMed2001

Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury.

The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress.

Adenoviruses, Human↗
PubMed2001

Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat.

Adeno-associated virus (AAV) is the only known eukaryotic virus capable of targeted integration in human cells. An AAV Rep binding element (RBE) and terminal resolution site (trs) identical to the viral terminal repeats required for AAV DNA replication are located on chromosome (ch) 19. Both ch-19 RBE and trs elements have been shown to be essential for viral targeting to this locus. To characterize the role of the AAV inverted terminal repeat (ITR) cis-acting sequences in targeted integration an AAV trs mutant incapable of supporting viral replication was tested. Wild-type and mutant substrates were assayed for targeted integration after cotransfection in the presence or absence of Rep. Our results demonstrated that, in the presence of Rep78, both ITR substrates targeted to ch-19 with similar frequency. Molecular characterization of the mutant ITR integrants confirmed the presence of the trs mutation in the majority of samples tested. Complementation analysis confirmed that the mutant targeted viral genomes were unable to rescue and replicate. In addition, Rep78 induced extensive rearrangement and amplification of ch-19 sequences independent of wild-type or mutant targeting substrate. These studies demonstrate that Rep-dependent nicking of the viral cis-acting trs sequence is not a prerequisite for site-specific recombination and suggests AAV targeting is mediated by Rep78/68-dependent replication from the ch-19 origin of replication (ori). These studies have significant impact toward the understanding of AAV site-specific recombination and the development of targeting vectors.

Chromosomes, Human, Pair 19↗
PubMed2001

Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors.

We previously reported that direct intramuscular injection of non-serotype-2 AAV vectors, especially AAV serotype 1 (AAV1), resulted in expression of supranormal levels of canine F9 in immunodeficient mice. Here we test the ability of the AAV1-F9 vector to deliver sustained expression and correction of factor IX (FIX) deficiency in genetically engineered hemophilic mice. Intramuscular injection of AAV1-F9 resulted in 100-1000 times more canine F9 in plasma of recombinant AAV1-F9 mice compared with injection of AAV2-F9. Assessment of clotting activity by activated partial thromboplastin time confirmed that circulating canine FIX was indeed functional. Moreover, phenotypic correction assayed by tail clip challenge resulted in survival of all AAV1-F9 treated animals, in contrast to naive mice and 50% of AAV2-treated hemophilia B mice, which failed to survive. Administration of cyclophosphamide (CTX) was required to suppress formation of anti-canine FIX antibodies for AAV2-treated animals, whereas it was dispensable for those treated with AAV1-F9. This difference in immunogenicity further emphasizes the usefulness of serotype-specific vectors. Finally, we report that correction of the hemophilia phenotype using AAV1-F9 was complete and persistent (over 8 months), a result that underscores the value of continued exploration of alternative AAV serotype vectors.

Animals↗
PubMed2001

Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.

Adeno-associated virus (AAV) vectors package single-stranded genomes and require host-cell synthesis of the complementary strand for transduction. However, when the genome is half wild-type size, AAV can package either two copies, or dimeric inverted repeat DNA molecules. Dimeric, or self-complementary molecules (scAAV) should spontaneously reanneal, alleviating the requirement for host-cell DNA synthesis. We generated and characterized scAAV vectors in order to bypass the rate-limiting step of second-strand synthesis. In vitro, scAAV vectors were five- to 140-fold more efficient transducing agents than conventional rAAV, with a 5.9:1 particle to transducing unit ratio. This efficiency is neither greatly increased by co-infection with Ad, nor inhibited by hydroxyurea, demonstrating that transduction is independent of DNA synthesis. In vivo, scAAV expressing erythropoietin resulted in rapid and higher levels of hematocrit than a conventional single-stranded vector. These novel scAAV vectors represent a biochemical intermediate in rAAV transduction and should provide new insights into the biology of vector transduction.

Animals↗
PubMed2001

Delivery of the Cu/Zn-superoxide dismutase gene with adenovirus reduces early alcohol-induced liver injury in rats.

BACKGROUND AND AIMS: Alcohol-induced liver injury is associated with an increase in oxidants from a variety of possible sources. Therefore, it was hypothesized that increased and stable expression of the antioxidant enzyme Cu/Zn-superoxide dismutase (SOD1) would diminish oxygen free radicals and reduce alcohol-induced liver injury. METHODS: To test this hypothesis, rats were given recombinant adenovirus containing Cu/Zn-superoxide dismutase (Ad.SOD1) or beta-galactosidase (Ad.lacZ) and fed ethanol enterally for 3 weeks. RESULTS: SOD was increased significantly 3-5-fold over endogenous levels in both hepatocytes as well as Kupffer cells 3 weeks after infection. Serum transaminase levels and pathology were elevated significantly in Ad.lacZ-treated animals by using an intragastric feeding model. This effect was blunted significantly in Ad.SOD1-infected animals. Importantly, electron spin resonance-detectable free-radical adducts caused by ethanol were also decreased by SOD1 overexpression. Moreover, the increase in nuclear factor kappaB (NFkappaB), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 messenger RNA (mRNA) caused by ethanol was blunted in animals treated with Ad.SOD1. CONCLUSIONS: These data support the hypothesis that oxidant production is critical in early alcohol-induced liver injury and that gene delivery of antioxidant enzymes may be useful in prevention and treatment.

Adenoviridae↗
PubMed2000

Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat.

BACKGROUND: Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad-SOD1) would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation was tested. METHODS: Some donors were infected with Ad-SOD1, whereas untreated grafts and livers infected with the indicator gene lacZ encoding bacterial beta-galactosidase (Ad-lacZ) served as controls. After orthotopic liver transplantation, survival, serum transaminases, and histopathology were evaluated. RESULTS: Approximately 80% of hepatocytes expressed beta-galactosidase 72 hr after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. After transplantation, 20-25% of rats treated with Ad-lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8 hr after transplantation in Ad-SOD1 rats were only 40% of those in controls, which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad-lacZ-infected organs were necrotic 8 hr after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad-SOD1. CONCLUSIONS: This study provides clear evidence for the first time that gene therapy with Ad-SOD1 increases survival and decreases hepatic injury after liver transplantation. Genetic modification of the liver represents a future approach to protect organs against injury where oxygen-derived free radicals are involved.

Adenoviridae↗
PubMed2000

The use of adeno-associated virus to circumvent the maturation-dependent viral transduction of muscle fibers.

Muscle-based gene therapy using adenovirus, retrovirus, and herpes simplex virus has been hindered by viral cytotoxicity, host immune response, and the maturation-dependent viral transduction of muscle fibers. The development of new mutant vectors has greatly reduced the toxicity and the immune rejection problems, but the inability of viral vectors to penetrate and transduce mature myofibers remains an important issue. Research has been focused on the characterization of barriers to viral transduction in mature myofibers to develop strategies to circumvent the maturation-dependent viral transduction of myofibers. Here, we report that adeno-associated virus (AAV) can be used to overcome the maturation-dependent viral transduction of myofibers. We have investigated by which mechanism AAV can penetrate and efficiently transduce mature muscle fibers, and have shown that this viral vector is not blocked by the basal lamina and that AAV transduction of myofibers is independent of myoblast mediation. Although AAV can efficiently transduce mature myofibers, a differential transduction is still observed among the different types of myofibers that correlates with the expression of the heparan sulfate proteoglycan receptors, the muscle maturity, the number of viral particles used, and the time postinjection. The identification of the mechanisms by which AAV transduces mature myofibers will help in the development of strategies to achieve an efficient muscle-based gene therapy for inherited and acquired diseases.

Animals↗
PubMed2000

Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.

We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (1) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo. In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 microg/ml followed by type 5 (6.52 microg/ml), type 3 (3.27 microg/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.

Animals↗