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1.
Aging Dis ; 2024 Feb 22.
Article in English | MEDLINE | ID: mdl-38421833

ABSTRACT

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), that starts with atrophic lesions in the outer retina that expand to cover the macula and fovea, leading to severe vision loss over time. Pigment Epithelium-Derived Factor (PEDF) has a diverse-range of properties, including its ability to promote cell survival, reduce inflammation, inhibit angiogenesis, combat oxidative stress, regulate autophagy, and stimulate anti-apoptotic pathways, making it a promising therapeutic candidate for GA. However, the relatively short half-life of PEDF protein has precluded its potential as a clinical therapy for GA since it would require frequent injections. Therefore, we describe administration of a PEDF gene, comparing and contrasting delivery routes, viral and non-viral vectors, and consider the critical challenges for PEDF as a neuroprotectant for GA.

2.
Mol Ther Methods Clin Dev ; 14: 206-216, 2019 Sep 13.
Article in English | MEDLINE | ID: mdl-31406701

ABSTRACT

A recent phase I-II, open-label trial of ProSavin, a lentiviral vector delivering the key enzymes in the dopamine biosynthetic pathway to non-dopaminergic striatal neurons, demonstrated safety and improved motor function in parkinsonian patients. However, the magnitude of the effect suggested that optimal levels of dopamine replacement may not have been achieved. OXB-102, a lentiviral vector with an optimized expression cassette for dopamine biosynthesis, has been shown to achieve a significantly higher dopamine yield than ProSavin. We assessed the efficacy of OXB-102 in the MPTP macaque model of Parkinson's disease (PD). At 6 months post-vector administration, all treated animals showed significant improvements in clinical scores and spontaneous locomotor activity compared to controls, with the highest recovery observed in the OXB-102 high-dose (HD) group. Positron emission tomography quantification of 6-[18F]-fluoro-L-m-tyrosine uptake showed a significant increase in amino acid decarboxylase activity for all treated animals, compared with controls, where the OXB-102 HD group showed the highest level of dopaminergic activity. A toxicology study in macaques demonstrated that the vector was safe and well tolerated, with no associated clinical or behavioral abnormalities and no immune response mounted against any transgene products. Overall, these data support the further clinical development of OXB-102 for the treatment of PD.

3.
Hum Gene Ther ; 29(6): 687-698, 2018 06.
Article in English | MEDLINE | ID: mdl-29361840

ABSTRACT

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye, corneal transplantation is one of the most successful clinical transplant procedures. However, in high-risk patients, which account for >20% of the 180,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient cornea. The main reason for graft failure is irreversible immunological rejection, and it is therefore unsurprising that neovascularization (NV; both pre and post grafting) is a significant risk factor for subsequent graft failure. NV is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the equine infectious anemia virus called pONYK1EiA, which subsequently prevents rejection by suppressing NV. Previously, it has been shown that rabbit donor corneas treated with pONYK1EiA substantially suppress corneal NV, opacity, and subsequent rejection in an aggressive rabbit model of cornea graft rejection. Here, efficacy data are presented in a second rabbit model, which more closely mirrors the clinical setting for high-risk corneal transplant patients, and safety data from a 3-month good laboratory practice toxicology and biodistribution study of pONYK1EiA-modified rabbit corneas in a rabbit corneal transplant model. It is shown that pONYK1EiA-modified rabbit corneas (OXB-202) significantly reduce corneal NV and the rate of corneal rejection in a dose-dependent fashion, and are tolerated with no adverse toxicological findings or significant biodistribution up to 13 weeks post surgery in these rabbit studies. In conclusion, angiogenesis is a valid target to prevent corneal graft rejection in a high-risk setting, and transplanted genetically modified corneas are safe and well-tolerated in an animal model. These data support the evaluation of OXB-202 in a first-in-human trial.


Subject(s)
Cell- and Tissue-Based Therapy , Corneal Transplantation/adverse effects , Genetic Engineering , Graft Rejection/prevention & control , Angiostatins/metabolism , Animals , Cell Count , Corneal Neovascularization/pathology , Corneal Neovascularization/therapy , Corneal Opacity , Culture Media , Endostatins/metabolism , Endothelial Cells/pathology , Female , Genetic Vectors/metabolism , Graft Rejection/pathology , Graft Rejection/physiopathology , HEK293 Cells , Humans , Intraocular Pressure , Keratoplasty, Penetrating , Rabbits , Risk Factors , Tissue Distribution
4.
Lancet ; 385 Suppl 1: S75, 2015 Feb 26.
Article in English | MEDLINE | ID: mdl-26312897

ABSTRACT

BACKGROUND: Inherited, iatrogenic, and metabolic corneal disease could be potentially treated by supplying a functional gene or changing the expression levels of specific genes. Viral-based gene therapy is efficient, but restricted by evoking immune responses and inflammation. This study aimed to transfect mouse cornea with a non-viral based (oscillating magnetofection) method. METHODS: Cultured mouse corneas were treated with magnetic nanoparticles (MNP) tethered to CAG promoter and green fluorescent protein (GFP) reporter plasmids exposed to a 1 Hz, 2 Hz, and 4 Hz oscillating magnetic field for 30 min and 60 min and in three DNA:MNP ratios (1:2, 1:1, 2:3). Corneas were cultured for up to 3 days and their green fluorescent channel intensity and number of GFP-positive cells were recorded. Transfection efficiency was estimated as the percentage of GFP-positive cells per total cells in a microscopic field. FINDINGS: Control experiments with absent magnetic exposure showed no GFP-positive cells. The optimum condition was recorded at 3:2 DNA:MNP ratio, 1 Hz magnetic oscillation, and 30 min duration of magnetic exposure (mean GFP-positive endothelial cell count 191·7 [SD 54·5], p=0·009; mean green fluorescent intensity 85·3 [SD 48·5]; and average transfection efficiency 23·3% [range 10·6-30·9]). INTERPRETATION: A novel non-viral method of transfecting cornea, magnetofection, is demonstrated and gives proof of principle for its translation into corneal gene therapy. FUNDING: Welsh Clinical Academic Training Scheme.

5.
PLoS One ; 9(4): e94272, 2014.
Article in English | MEDLINE | ID: mdl-24705452

ABSTRACT

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.


Subject(s)
Genetic Therapy , Genetic Vectors/genetics , Infectious Anemia Virus, Equine/genetics , Usher Syndromes/genetics , Usher Syndromes/therapy , Animals , Cell Line , Disease Models, Animal , Female , Gene Order , Genetic Vectors/administration & dosage , Genetic Vectors/metabolism , Humans , Macaca , Male , Mice , Mice, Knockout , Myosin VIIa , Myosins/genetics , Phenotype , Photoreceptor Cells/metabolism , Photoreceptor Cells/pathology , Protein Transport , Retina/metabolism , Retina/pathology , Transducin/metabolism
6.
Invest Ophthalmol Vis Sci ; 54(6): 4061-71, 2013 Jun 12.
Article in English | MEDLINE | ID: mdl-23620430

ABSTRACT

PURPOSE: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS: Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS: Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS: In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Genetic Vectors , Infectious Anemia Virus, Equine/genetics , Macular Degeneration/congenital , Photoreceptor Cells, Vertebrate/metabolism , Transduction, Genetic , Animals , Blotting, Western , Body Fluids/metabolism , Cytomegalovirus/genetics , Electroretinography , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression , Genetic Therapy , Green Fluorescent Proteins/genetics , Intraocular Pressure , Macaca mulatta , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/physiopathology , Male , Polymerase Chain Reaction , Rabbits , Stargardt Disease , Tissue Distribution , Transfection
7.
Mol Ther Nucleic Acids ; 1: e60, 2012 Dec 11.
Article in English | MEDLINE | ID: mdl-23232328

ABSTRACT

The interest in integrase-defective lentiviral vectors (IDLVs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV) to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector.Molecular Therapy - Nucleic Acids (2012) 1, e60; doi:10.1038/mtna.2012.53; published online 11 December 2012.

8.
Hum Gene Ther ; 23(9): 980-91, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22716662

ABSTRACT

RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.


Subject(s)
Genetic Therapy/methods , Genetic Vectors , Infectious Anemia Virus, Equine , Macular Degeneration/metabolism , Macular Degeneration/therapy , Vitreous Body/metabolism , Angiostatins/biosynthesis , Angiostatins/genetics , Animals , Endostatins/biosynthesis , Endostatins/genetics , Humans , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Macaca mulatta , Macular Degeneration/pathology , Rabbits , Time Factors , Vitreous Body/pathology , Vitreous Body/virology
9.
Int J Mol Med ; 27(2): 173-80, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21165551

ABSTRACT

This study describes a combined gene and cell therapy based on the genetic modification of primary human macrophages, as a treatment for cancer. Here, we have utilised the tumour-infiltrating properties of macrophages as vehicles to deliver a gene encoding a prodrug-activating enzyme such as human cytochrome P450 2B6 (CYP2B6) inside tumours followed by killing the tumour cells with the prodrug cyclophosphamide (CPA). Macrophages were transduced with an adenoviral vector that expresses human cytochrome CYP2B6 via a synthetic hypoxia responsive promoter (OBHRE) and with human P450 reductase (P450R), via the CMV promoter. In the presence of CPA, these genetically modified macrophages showed increased cytotoxicity against various tumour cell lines compared to untransduced macrophages or macrophages transduced with CYP2B6 alone. In human ovarian carcinoma xenograft models, the median survival of mice treated with genetically modified macrophages plus CPA increased up to two-fold compared to the survival of mice treated with untransduced macrophages and CPA. Genetically modified autologous macrophages may be a feasible therapeutic option for the treatment of some solid tumours, such as ovarian cancer.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Genetic Therapy/methods , Hypoxia , Macrophages/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Neoplasms/therapy , Adenoviridae/genetics , Animals , Cell Line, Tumor , Cytochrome P-450 Enzyme System/genetics , Female , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Humans , Mice , Mice, Nude , NADPH-Ferrihemoprotein Reductase/genetics , Xenograft Model Antitumor Assays
10.
Hum Gene Ther ; 20(1): 31-9, 2009 Jan.
Article in English | MEDLINE | ID: mdl-20377369

ABSTRACT

Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruch's membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.


Subject(s)
Angiostatins/genetics , Choroidal Neovascularization/therapy , Endostatins/genetics , Genetic Vectors/administration & dosage , Infectious Anemia Virus, Equine/genetics , Retinal Pigment Epithelium/metabolism , Angiostatins/metabolism , Animals , Bestrophins , Disease Models, Animal , Endostatins/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Female , Gene Expression Regulation , Genetic Therapy/methods , Genetic Vectors/genetics , Humans , Infectious Anemia Virus, Equine/physiology , Ion Channels , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Retinal Pigment Epithelium/virology , Treatment Outcome
11.
J Gene Med ; 9(12): 1015-23, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17963276

ABSTRACT

BACKGROUND: We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. METHODS: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. RESULTS: All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. CONCLUSIONS: It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.


Subject(s)
Gene Transfer Techniques , Genetic Vectors , Infectious Anemia Virus, Equine/genetics , Photoreceptor Cells, Vertebrate/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Cell Line, Tumor , DNA Primers , Enhancer Elements, Genetic , Humans , Mice , Retina/metabolism
12.
Invest Ophthalmol Vis Sci ; 48(7): 3056-61, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17591873

ABSTRACT

PURPOSE: To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). METHODS: Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti-collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. RESULTS: Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. CONCLUSIONS: Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.


Subject(s)
Cryopreservation , Endothelium, Corneal/cytology , Gene Expression/physiology , Genetic Vectors , Infectious Anemia Virus, Equine/genetics , Organ Preservation , Adult , Cell Proliferation , Cells, Cultured , Child, Preschool , Collagen Type VIII/metabolism , Endothelium, Corneal/metabolism , Flow Cytometry , Green Fluorescent Proteins/genetics , Humans , Membrane Proteins/metabolism , Microscopy, Fluorescence , Phosphoproteins/metabolism , Tissue Donors , Zonula Occludens-1 Protein
13.
Clin Sci (Lond) ; 110(1): 37-46, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16336203

ABSTRACT

Gene therapy holds great promise for the treatment of a wide range of inherited and acquired disorders. The development of viral vector systems to mediate safe and long-lasting expression of therapeutic transgenes in specific target cell populations is continually advancing. Gene therapy for the nervous system is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses provide particularly attractive vehicles for delivery of therapeutic genes to treat neurological and ocular diseases, since they efficiently transduce non-dividing cells and mediate sustained transgene expression. Furthermore, novel routes of vector delivery to the nervous system have recently been elucidated and these have increased further the scope of lentiviruses for gene therapy application. Several studies have demonstrated convincing therapeutic efficacy of lentiviral-based gene therapies in animal models of severe neurological disorders and the push for progressing such vectors to the clinic is ongoing. This review describes the key features of lentiviral vectors that make them such useful tools for gene therapy to the nervous system and outlines the major breakthroughs in the potential use of such vectors for treating neurodegenerative and ocular diseases.


Subject(s)
Eye Diseases/therapy , Genetic Therapy/methods , Genetic Vectors , Lentivirus/genetics , Neurodegenerative Diseases/therapy , Animals , Gene Transfer Techniques , Humans , Retinal Degeneration/therapy
14.
Genetics ; 166(2): 707-19, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15020461

ABSTRACT

Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants.


Subject(s)
Actins/genetics , Endocytosis/genetics , Genes, Lethal , Saccharomyces cerevisiae/genetics , Actins/metabolism , Cell Cycle/genetics , Cell Cycle/physiology , Endocytosis/physiology , Fungal Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phenotype , Protein Kinase C , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion , Symporters/genetics , Symporters/metabolism
15.
Genomics ; 83(1): 1-8, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14667803

ABSTRACT

The cellular response to hypoxia involves the promotion of angiogenesis, leading to increased blood flow and oxygenation. The macrophage has been identified as an orchestrator of this response in several pathologies, through the release of angiogenic factors in response to hypoxia. We have produced the first comprehensive transcriptome analysis of hypoxic primary human macrophages with respect to the regulation of angiogenesis. There is a marked induction of genes encoding factors known to stimulate angiogenesis, rather than factors that inhibit this process. We show that overexpression of the transcription factor EPAS1 using a recombinant adenoviral vector amplifies the induction of genes encoding angiogenic proteins in response to hypoxia. This defines a new strategy for enhancing transcriptome and proteome analyses by overexpressing disease-implicated genes using viral gene transfer methodologies.


Subject(s)
Genes, Regulator/genetics , Neovascularization, Physiologic/genetics , Transcription, Genetic/genetics , Adenoviridae/genetics , Basic Helix-Loop-Helix Transcription Factors , Cell Hypoxia/physiology , Cells, Cultured , Gene Expression Regulation , Genetic Vectors/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , RNA/genetics , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/genetics , Transcription Factors/genetics
16.
Curr Opin Mol Ther ; 5(6): 650-6, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14755892

ABSTRACT

Hypoxia (low oxygen) is a defining physiological feature of a number of diseases, including cancer, cardiovascular disease and retinopathy. Hypoxia plays an active role in the pathology of these diseases through its impact on gene expression, thereby making the hypoxia-signaling pathway a key target for the development of novel molecular therapies. This review focuses on how the elucidation of this pathway has led to the development of novel therapeutic strategies, including physiologically targeted gene therapy and the identification of novel therapeutic targets within the hypoxia-signaling pathway.


Subject(s)
Gene Expression Regulation , Genetic Therapy/methods , Hypoxia/metabolism , Hypoxia/therapy , Signal Transduction/physiology , Gene Expression Profiling , Humans , Hypoxia/physiopathology , Procollagen-Proline Dioxygenase/metabolism , Promoter Regions, Genetic
17.
Blood ; 100(7): 2406-13, 2002 Oct 01.
Article in English | MEDLINE | ID: mdl-12239150

ABSTRACT

Anemia is a common clinical problem, and there is much interest in its role in promoting left ventricular hypertrophy through increasing cardiac workload. Normally, red blood cell production is adjusted through the regulation of erythropoietin (Epo) production by the kidney. One important cause of anemia is relative deficiency of Epo, which occurs in most types of renal disease. Clinically, this can be corrected by supplementation with recombinant Epo. Here we describe an oxygen-regulated gene therapy approach to treating homozygous erythropoietin-SV40 T antigen (Epo-TAg(h)) mice with relative erythropoietin deficiency. We used vectors in which murine Epo expression was directed by an Oxford Biomedica hypoxia response element (OBHRE) or a constitutive cytomegalovirus (CMV) promoter. Both corrected anemia, but CMV-Epo-treated mice acquired fatal polycythemia. In contrast, OBHRE-Epo corrected the hematocrit level in anemic mice to a normal physiologic level that stabilized without resulting in polycythemia. Importantly, the OBHRE-Epo vector had no significant effect on the hematocrit of control mice. Homozygous Epo-TAg(h) mice display cardiac hypertrophy, a common adaptive response in patients with chronic anemia. In the OBHRE-Epo-treated Epo-TAg(h) mice, we observed a significant reversal of cardiac hypertrophy. We conclude that the OBHRE promoter gives rise to physiologically regulated Epo secretion such that the hematocrit level is corrected to healthy in anemic Epo-TAg(h) mice. This establishes that a hypoxia regulatory mechanism similar to the natural mechanism can be achieved, and it makes EPO gene therapy more attractive and safer in clinical settings. We envisage that this control system will allow regulated delivery of therapeutic gene products in other ischemic settings.


Subject(s)
Anemia/therapy , Erythropoietin/genetics , Genetic Therapy , Animals , Antigens, Polyomavirus Transforming/genetics , Cell Line , Erythropoietin/analysis , Erythropoietin/therapeutic use , Gene Expression Regulation , Genetic Vectors , Hypoxia , Kidney/physiology , Lymphocyte Activation , Mice , Mice, Transgenic , Polymerase Chain Reaction , Simian virus 40/genetics , Spleen/immunology , Transfection
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