PubMed Health⌕ Search

Biomedical subjects

R O Snyder

Publications and source records attributed to R O Snyder.

At least 19 recordsLinked to original sources
PubMed2004

Immunity to adeno-associated virus serotype 2 delivered transgenes imparted by genetic predisposition to autoimmunity.

Adeno-associated virus (AAV) is widely considered a promising vector for therapeutic gene delivery. This promise is based on previous studies assessing AAVs safety and toxicity, ability to infect nondividing cells, elicit a limited immune response and provide long-term gene expression. However, we now find that earlier studies underappreciated the degree of AAV immunogenicity as well as the extent to which genetic background, through regulation of immune responsiveness, influences the duration of gene expression and thereby the effectiveness of AAV-mediated gene therapy. We evaluated antibody responses in 12 mouse strains to AAV serotype 2 (AAV2) and AAV2-expressed transgene products including green fluorescent protein (GFP), human alpha1-antitrypsin and murine interleukin-10. As expected, all immunocompetent mice administered AAV2 developed serologic evidence of immune responsiveness to the virus. However, a previously unidentified serologic prozone effect was observed suggesting that the concentrations of anti-AAV2 antibodies may have historically been subject to marked underestimation. Furthermore, strains with genetic predisposition to autoimmunity (eg, NOD, NZW, MRL-lpr) specifically imparted a functionally deleterious immune response to AAV-delivered transgene products. These findings suggest that more thorough studies of anti-AAV immunity should be performed, and that genetic predisposition to autoimmunity should be considered when assessing AAV efficacy and safety in humans.

Animals↗
PubMed2000

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure.

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

Amino Acid Sequence↗
PubMed2000

Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction.

Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

Animals↗
PubMed2000

Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors.

PURPOSE: Gene transfer offers the potential to modify vein graft biology at the time of surgical implantation. Efficiency of gene delivery, stability of expression, and host responses are critical parameters for candidate vectors. We compared the effects of intraluminal exposure with adenovirus (AD) and adeno-associated virus (AAV) vectors on transgene expression and monocyte adhesion (MA) in treated vein segments. METHODS: Adult New Zealand white rabbits (N = 51) were anesthetized, and the jugular veins were cannulated bilaterally. Veins were gently distended with either vector (2.10(8) to 1.10(10) infective particles/mL) or vehicle (control) for 30 minutes, after which venous flow was restored. AD and AAV vectors encoding for the marker genes beta-galactosidase (LacZ) and green fluorescent protein (GFP) were used. Vessels were explanted 2 to 40 days postinfection for analysis of gene expression (X-gal staining, reverse transcriptase-polymerase chain reaction), MA, and immunohistochemistry. Ex vivo adhesion assays used (51)Cr-labeled THP-1 cells. Statistical significance was tested by using analysis of variance with a P value less than.05. RESULTS: All animals survived, and all treated veins were patent at sacrifice. Intraluminal exposure to AD at a titer of 1.10(9) resulted in near complete transduction of the endothelium at 2 days, with no detectable expression by day 14. At an equal titer of infectious particles, transgene expression was markedly less for AAV at 2 to 7 days, but improved at 2 weeks and persisted to 40 days. MA was significantly increased 2 days after AD exposure (2.7-fold vs control, *P <.002); AAV treatment had no discernible effect on MA. CONCLUSION: AD-mediated gene transfer to vein segments resulted in robust, transient gene expression that disappeared after 2 weeks. In comparison, AAV-mediated gene delivery was less efficient, but resulted in delayed onset, persistent expression beyond 30 days. AD exposure induced an early increase in MA to the vein surface that was not seen with AAV treatment. Current generations of both AD and AAV vectors have significant, albeit different, limitations for vascular gene therapy.

Adenoviridae↗
PubMed1999

Nerve growth factor expressed in the medial septum following in vivo gene delivery using a recombinant adeno-associated viral vector protects cholinergic neurons from fimbria-fornix lesion-induced degeneration.

Nerve growth factor (NGF) has been shown to support the survival of axotomized medial septal cholinergic neurons after aspirative lesions of the fimbria-fornix (FF). This survival effect has been achieved utilizing intraventricular and intraparenchymal delivery of the NGF protein. While the use of NGF for the treatment of the cholinergic deficits present in Alzheimer's disease shows promise based on its efficacy in animal models, concerns about side-effects of intraventricular NGF delivery in humans have been raised. In the present study, NGF was delivered directly to the medial septum via a recombinant adeno-associated viral vector (rAAV) encoding the cDNA for human NGF prior to a FF lesion in rats. This rAAV-mediated NGF delivery was shown to significantly attenuate the medial septal cholinergic cell loss observed in animals receiving an equivalent injection of a control rAAV vector.

Adenoviridae↗
PubMed1999

Progress in direct striatal delivery of L-dopa via gene therapy for treatment of Parkinson's disease using recombinant adeno-associated viral vectors.

Viral vectors have recently been used successfully to transfer genes and express different proteins in the brain. This review discusses the requirements to consider human clinical trials in which recombinant adeno-associated virus vectors are used to transfer the genes necessary to produce l-dihydroxyphenylalanine (l-dopa) directly into the striatum of Parkinson's patients. Preclinical data that apply to the criteria defined as prerequisite for clinical trials are discussed. Thus, in animal models using recombinant adeno-associated virus vectors it has been demonstrated that l-dopa can be synthesized in the striatum after in vivo transduction. In addition, these l-dopa levels are sufficient to affect behavior in a dopamine-deficient animal model, the expression is extremely long-lasting, and the ability to transcriptionally regulate tyrosine hydroxylase has been demonstrated but not fully characterized. However, while immune responses to recombinant adeno-associated virus infection in the periphery have been studied, direct assessment of the potential immune response in the brain has not been sufficiently defined. Therefore, the rationale for delivering l-dopa directly to the striatum to treat Parkinson's disease is sound and the preclinical data are promising but all the issues surrounding this strategy are not resolved.

Adenoviridae↗
PubMed1999

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease.

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinson's disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.

Animals↗
PubMed1999

Long-term restoration of striatal L-aromatic amino acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a rodent model of Parkinson's disease.

As a potential treatment for Parkinson's disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinson's disease patients may be an attractive gene therapy strategy.

Adenoviridae↗
PubMed1999

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Adenoviridae↗
PubMed1999

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Animals↗
PubMed1999

Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle.

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.

Animals↗
PubMed1998

Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease.

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.

Animals↗
PubMed1998

Regulation of gene expression in vivo following transduction by two separate rAAV vectors.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

3T3 Cells↗
PubMed1997

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats.

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson's disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson's disease.

Animals↗
PubMed1997

Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice.

Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.

Animals↗
PubMed1997

Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson's disease.

Current approaches to gene therapy of CNS disorders include grafting genetically modified autologous cells or introducing genetic material into cells in situ using a variety of viral or synthetic vectors to produce and deliver therapeutic substances to specific sites within the brain. Here we discuss issues related to the application of ex-vivo and in-vivo gene therapies as possible treatments for Parkinson's disease. Autologous monkey fibroblasts engineered ex-vivo to express tyrosine hydroxylase were grafted into MPTP-treated monkeys and found to express for up to 4 months. Adeno-associated (AAV) viral vectors expressing beta-galactosidase or tyrosine hydroxylase were introduced into monkey brains to determine the extent of infection and the types of cells infected by the vector at 21 days and 3 months. Gene expression was detected at both time points and was restricted to neurons in the striatum. These experiments demonstrate that two different approaches can be used to deliver proteins into the CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be considered for treating human disease.

Animals↗