ABSTRACT
Viral delivery of neurotrophins or other therapeutic genes is an attractive option for treating retinal degeneration. Regulated expression of these genes in the retina is needed to aid in dose delivery and to promote safety. To evaluate whether tetracycline (tet)-inducible transgenes encapsidated in recombinant adeno-associated viruses (rAAV) can provide controlled gene expression in vitro and in the rat retina, two viruses were constructed: a silencer/activator vector and an inducible doxycycline (dox)-responsive GFP vector. Combinations of these two viruses were subretinally injected into wild-type rats and dox was orally administered through the drinking water. Retinal GFP expression was monitored in vivo with a noninvasive fluorescence imaging method. Eyes were also examined by histology, Western analysis, and electroretinography. Subretinal injection of rAAV efficiently delivers inducible genes to both photoreceptors and retinal pigment epithelial cells. GFP expression was initially observed 1 week postinduction, and GFP protein was undetectable after removal of dox. In uninduced animals, GFP expression was negligible. The dox dosage was varied in vivo and showed a correlation to the level of GFP expression. Thus, transduction of retinal cells with tet-inducible vectors allows for tight regulation of gene expression.
Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Genes, Reporter , Retina/metabolism , Tetracycline/pharmacology , Animals , Blotting, Western , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Electroretinography , Gene Expression Regulation , Genetic Vectors , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Models, Genetic , Rats , Retinal Degeneration/therapy , Time Factors , Transduction, GeneticABSTRACT
The goal of these experiments was to evaluate the potential of the fibroblast growth factor family members FGF-5 and FGF-18 to rescue photoreceptors from cell death in retinal degenerative disease. Two strains of transgenic rats, expressing either a P23H or an S334ter rhodopsin mutation, were used as model systems. The neurotrophic growth factors were delivered by subretinal injection of adeno-associated virus vectors, driving expression of the genes with a constitutive CMV promoter. Morphological and functional analyses were performed to determine whether FGF-5 or FGF-18 overexpression could ameliorate cell death in the retina. Immunocytochemistry was used to determine the cellular sites of expression of the factors and to test for up-regulation of FGF receptors due to injection. Significant rescue from photoreceptor cell death was found after injections of vectors expressing either FGF-5 or FGF-18 in the animal models. Increased survival of photoreceptors did not produce a significant increase in electroretinographic responses, however, reflecting either trauma due to the surgery or a suppression of signaling due to expression of proteins. Three weeks after injections, both growth factors were localized to the inner and outer segments of photoreceptors, and the receptors FGFR1 and FGFR2 were also found to be up-regulated in these regions. No visible pathological changes were seen in the FGF-5- or FGF-18-treated eyes. These results indicate that the delivery of either FGF-5 or FGF-18 with adeno-associated virus protects photoreceptors from apoptosis in transgenic rat models of retinitis pigmentosa and that the rescue is probably mediated by conventional receptor tyrosine kinase pathways in photoreceptors.
Subject(s)
Dependovirus/genetics , Fibroblast Growth Factors/genetics , Retinal Degeneration/therapy , Retinitis Pigmentosa/therapy , Animals , Animals, Genetically Modified , Blotting, Western , Cell Death , Cell Line , Cell Survival , Cytomegalovirus/genetics , Disease Models, Animal , Electroretinography , Fibroblast Growth Factor 5 , Genetic Vectors/genetics , Humans , Immunohistochemistry , Models, Genetic , Plasmids/metabolism , Point Mutation , Promoter Regions, Genetic , Rats , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1 , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Fibroblast Growth Factor/biosynthesis , Receptors, Fibroblast Growth Factor/genetics , Retina/metabolism , Retina/pathology , Retinitis Pigmentosa/genetics , Rhodopsin/genetics , Signal Transduction , Transfection , Up-RegulationABSTRACT
PURPOSE: We evaluated adeno-associated virus (AAV)-mediated gene transfer of basic fibroblast growth factor (FGF-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. METHODS: Recombinant adeno-associated virus vector (rAAV) incorporating a constitutive cytomegalovirus (CMV) promoter was used to transfer the bovine FGF-2 gene to photoreceptors. AAV was administered by subretinal injection to transgenic rats (TgN S334ter-4) at postnatal day 15 (P15). Control eyes were uninjected, injected with PBS, or AAV-LacZ. Eyes were examined by histopathology, morphometric analysis, and electroretinography at P60. RESULTS: Expression of recombinant FGF-2 slowed the rate of photoreceptor degeneration. Morphologic studies demonstrated significantly more photoreceptors surviving in eyes injected with AAV-FGF-2 than in controls. Insignificant rescue effects were seen in retinas injected with buffer only. No significant inflammatory response or neovascularization was detected. Electroretinographic (ERG) responses of eyes injected with AAV-FGF-2 were increased compared with uninjected eyes; however, these amplitudes were not significantly larger than eyes receiving an AAV-LacZ control vector. CONCLUSIONS: Transduction of retinal cells with AAV-FGF-2 reduces the rate of photoreceptor degeneration in an S334ter-4 animal model. Despite the lack of significantly increased ERG amplitudes from eyes expressing FGF-2, a greater number of surviving photoreceptors was demonstrated. Delivery of FGF-2 using recombinant AAV has potential as a therapy for retinal degeneration.
Subject(s)
Dependovirus/physiology , Fibroblast Growth Factor 2/therapeutic use , Genetic Therapy/methods , Retinal Degeneration/therapy , Animals , Animals, Genetically Modified , Defective Viruses , Electroretinography , Enzyme-Linked Immunosorbent Assay , Fibroblast Growth Factor 2/metabolism , Fluorescent Antibody Technique, Indirect , Gene Transfer Techniques , Genetic Vectors , Microscopy, Fluorescence , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/pathology , Photoreceptor Cells, Vertebrate/physiology , Photoreceptor Cells, Vertebrate/virology , Rats , Rats, Sprague-Dawley , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Degeneration/physiopathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Adenoviridae , Antiparkinson Agents/metabolism , Corpus Striatum , Genetic Therapy , Genetic Vectors , Levodopa/genetics , Animals , Humans , Recombinant Proteins/geneticsABSTRACT
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.
Subject(s)
Dependovirus/genetics , Gene Expression Regulation , Genetic Therapy/methods , Genetic Vectors/genetics , 3T3 Cells , Animals , Antibody Formation , Cells, Cultured , Erythropoietin/biosynthesis , Erythropoietin/genetics , Female , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Hematocrit , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Muscle, Skeletal/metabolism , Promoter Regions, Genetic , T-Lymphocytes, Cytotoxic/immunology , Tetracycline/pharmacology , Trans-Activators/genetics , TransgenesABSTRACT
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.
Subject(s)
Dependovirus , GTP Cyclohydrolase/genetics , Gene Transfer Techniques , Parkinson Disease, Secondary/therapy , Tyrosine 3-Monooxygenase/genetics , Animals , Apomorphine , Behavior, Animal/drug effects , Corpus Striatum/cytology , Corpus Striatum/enzymology , Disease Models, Animal , Dopamine Agents/metabolism , Dopamine Agonists , Gene Expression Regulation, Enzymologic , Humans , Levodopa/genetics , Levodopa/metabolism , Male , Microdialysis , Neurons/enzymology , Parkinson Disease, Secondary/enzymology , Rats , Rats, Inbred F344 , Recombinant Proteins/genetics , RotationABSTRACT
To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.
Subject(s)
GTP Cyclohydrolase/metabolism , Genetic Therapy , Levodopa/biosynthesis , Parkinson Disease, Secondary/therapy , Tyrosine 3-Monooxygenase/metabolism , 3T3 Cells , Animals , Antioxidants/metabolism , Antiparkinson Agents/pharmacology , Apomorphine/pharmacology , Behavior, Animal/drug effects , Biopterins/analogs & derivatives , Biopterins/metabolism , Corpus Striatum/chemistry , Corpus Striatum/enzymology , Corpus Striatum/pathology , Dihydroxyphenylalanine/metabolism , Disease Models, Animal , Fibroblasts/cytology , Fibroblasts/enzymology , Fibroblasts/transplantation , Gene Expression Regulation, Enzymologic/physiology , Gliosarcoma , Humans , Male , Mice , Microdialysis , Parkinson Disease, Secondary/metabolism , Parkinson Disease, Secondary/surgery , Rats , Rats, Inbred F344 , Recombinant Fusion Proteins/physiology , Retroviridae/genetics , Transformation, Genetic , Transgenes/physiology , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/transplantationABSTRACT
The no-on-transient-A gene was identified by independently isolated mutations causing visual-response abnormalities or courtship song defects. The nonA(diss) mutant is abnormal for all of these phenotypes. The pleiotropic effects of this dissonance allele dovetail with the widespread tissue expression of the nonA products. This gene, which encodes a putative RNA-binding protein, is expressed at essentially all stages of the life cycle. To determine whether the behavioral and physiological abnormalities exhibited by nonA mutant adults have a developmental etiology or are the result of an impaired mature nervous (or perhaps neuro-muscular) system, a conditional form of this gene was constructed. Animals from the resulting hsp-nonA (cDNA) transgenic strain were subjected to heat-shock regimes such that the gene's coding sequences were activated during development only, solely in the imaginal stage, or both. Surprisingly, expression during any of these time periods effected rescue of the visual-response and the courtship-song defects.
Subject(s)
Drosophila/genetics , Gene Expression Regulation/physiology , Genes, Insect , Sexual Behavior, Animal/physiology , Vision Disorders/genetics , Vocalization, Animal/physiology , Animals , Animals, Genetically Modified , Female , Male , Mutation , Phenotype , Time FactorsABSTRACT
The Drosophila no-on-transient A (nonA) gene is involved in the visual behaviors and courtship song of the fly. The NONA polypeptide contains two copies of the RNA-recognition motif (RRM), a hallmark of proteins involved in RNA binding, and an adjacent conserved charged region. This 311-amino-acid region is found in four other proteins and largely overlaps the Drosophila-Behavior/Human Splicing (or DBHS) domain. The newest family member, Drosophila nAhomo, was discovered in a database search, and encodes a protein with 80% identity to NONA. In this study, three nonA mutations generated by chemical mutagenesis were sequenced and found to fall within the conserved region. Site-directed mutagenesis of the two RRMs, and within a (conserved) charged region located C-terminal to them, was performed to determine the significance of these domains with respect to whole-organismal phenotypes. Behavior and viability were assessed in transformed flies, the genetic background of which lacks the nonA locus. Point mutations of amino acid 548 in the charged region confirmed the etiology of the nonAdiss courtship-song mutation and showed that a milder substitution at this site produced intermediate singing behavior, although it failed to rescue visual defects. Mutagenesis of the RRM1 domain resulted in effects on viability, vision, and courtship song. However, amino acid substitutions in RNP-II of RRM2 led to near-normal phenotypes, and the in vivo nonA mutations located in or near RRM2 caused visual defects only. Thus, we suggest that the first RRM could be important for all functions influenced by nonA, whereas the second RRM may be required primarily for normal vision.
Subject(s)
Amino Acids/pharmacology , Genes/genetics , RNA-Binding Proteins/metabolism , Visual Cortex/physiology , Amino Acid Sequence , Animals , Cloning, Molecular , Courtship , Drosophila , Immunohistochemistry , Molecular Sequence Data , MutationABSTRACT
We have performed a genetic analysis of the 14C region of the X chromosome of Drosophila melanogaster to isolate loss of function alleles of no-on-transient A (nonA; 14C1-2; 1-52.3). NONA is a nuclear protein common to many cell types, which is present in many puffs on polytene chromosomes. Sequence data suggest that the protein contains a pair of RNA binding motifs (RRM) found in many single-strand nucleic acid binding proteins. Hypomorphic alleles of this gene, which lead to aberrant visual and courtship song behavior, still contain normally distributed nonA RNA and NONA protein in embryos, and in all available alleles NONA protein is present in puffs of third instar larval polytene chromosomes. We find that complete loss of this general nuclear protein is semilethal in hemizygous males and homozygous cell lethal in the female germline. Surviving males show more extreme defects in nervous system function than have been described for the hypomorphic alleles. Five other essential genes that reside within this region have been partially characterized.
Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Nuclear Proteins/genetics , X Chromosome , Alleles , Animals , Animals, Genetically Modified , Base Sequence , Chromosome Mapping , Chromosomes/ultrastructure , Crosses, Genetic , DNA/chemistry , DNA Primers , Drosophila melanogaster/embryology , Drosophila melanogaster/radiation effects , Embryo, Nonmammalian/physiology , Female , Genes, Lethal , Genetic Complementation Test , Male , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/physiology , Organ Specificity , Phenotype , Polymerase Chain Reaction , X Chromosome/radiation effects , X-Rays , ZygoteABSTRACT
Genetic and molecular results are here presented revealing that the dissonance (diss) courtship song mutation is an allele of the no-on-transient-A (nonA) locus of Drosophila melanogaster. diss (now called nonAdiss) was originally isolated as a mutant with aberrant pulse song, although it was then noted to exhibit defects in responses to visual stimuli as well. The lack of transient spikes in the electroretinogram (ERG) and optomotor blindness associated with nonAdiss are shown to be similar to the visual abnormalities caused by the original nonA mutations. nonAdiss failed to complement either the ERG or optomotor defects associated with four other nonA mutations. However, all four of these nonA mutants--which were isolated on visual criteria alone--sang a normal courtship song. nonAdiss complemented at least three of the nonA mutations with regard to the singing phenotype, as assessed by a new method for temporal analysis of the male's pulse song. Both visual and song abnormalities caused by nonAdiss were rescued by P-element-mediated transformation with overlapping 11 and 16 kilobase (kb) fragments of genomic DNA (originally cloned from the nonA locus by Jones and Rubin, 1990). Analysis of behavioral phenotypes in transformed flies carrying mutagenized versions of the 11 kb genomic fragment (in a nonAdiss genomic background) localized the rescuing DNA to a region containing an open reading frame that encodes a polypeptide (NONA) with similarity to a family of RNA-binding proteins. Immunohistochemical determination of NONA's spatial and temporal expression revealed that it is localized to the nuclei of cells in many neural and non-neural tissues, at all stages of the life cycle after very early in development. Genetic connections between the control of two quite different behaviors--reproductive and visual--are discussed, along with precedences for generally expressed gene products playing roles in specific behaviors.