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Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.
Luo W, Galvan DL, Woodard LE, Dorset D, Levy S, Wilson MH
(2017) Nucleic Acids Res 45: 8411-8422
MeSH Terms: Bacterial Proteins, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cell Line, Tumor, DNA Transposable Elements, Endonucleases, Gene Knockout Techniques, Gene Targeting, Gene Transfer Techniques, Humans, Hypoxanthine Phosphoribosyltransferase, Mutagenesis, Insertional, Recombinant Fusion Proteins, Reproducibility of Results, Transcription Activator-Like Effector Nucleases, Transcription Activator-Like Effectors, Transposases, Zinc Fingers
Show Abstract · Added September 11, 2017
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.
Published by Oxford University Press on behalf of Nucleic Acids Research 2017.
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18 MeSH Terms
Blocking Zebrafish MicroRNAs with Morpholinos.
Flynt AS, Rao M, Patton JG
(2017) Methods Mol Biol 1565: 59-78
MeSH Terms: Animals, Electroporation, Gene Expression Regulation, Gene Silencing, Gene Transfer Techniques, MicroRNAs, Microinjections, Morpholinos, Oligonucleotides, Antisense, Retina, Zebrafish
Show Abstract · Added August 4, 2017
Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy. This redundancy, plus their small size which can limit targeting by CRISPR/Cas9, makes morpholino-based strategies particularly attractive for inhibition of miRNA function. We provide the rationale, background, and methods to inhibit miRNA function with antisense morpholinos during early development and in the adult retina in zebrafish.
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11 MeSH Terms
Kidney-specific transposon-mediated gene transfer in vivo.
Woodard LE, Cheng J, Welch RC, Williams FM, Luo W, Gewin LS, Wilson MH
(2017) Sci Rep 7: 44904
MeSH Terms: Acute Kidney Injury, Animals, DNA Transposable Elements, Erythropoietin, Gene Expression, Gene Expression Regulation, Gene Transfer Techniques, Genes, Reporter, Genetic Vectors, Hydrodynamics, Immunosuppressive Agents, Kidney, Male, Mice, Organ Specificity, Promoter Regions, Genetic, Transfection
Show Abstract · Added September 11, 2017
Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.
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17 MeSH Terms
Erythropoietin Slows Photoreceptor Cell Death in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa.
Rex TS, Kasmala L, Bond WS, de Lucas Cerrillo AM, Wynn K, Lewin AS
(2016) PLoS One 11: e0157411
MeSH Terms: Animals, Cell Death, Dependovirus, Disease Models, Animal, Erythropoietin, Gene Transfer Techniques, Genetic Therapy, Humans, Mice, Opsins, Point Mutation, Retinal Cone Photoreceptor Cells, Retinitis Pigmentosa, Vision, Ocular
Show Abstract · Added April 2, 2019
PURPOSE - To test the efficacy of systemic gene delivery of a mutant form of erythropoietin (EPO-R76E) that has attenuated erythropoietic activity, in a mouse model of autosomal dominant retinitis pigmentosa.
METHODS - Ten-day old mice carrying one copy of human rhodopsin with the P23H mutation and both copies of wild-type mouse rhodopsin (hP23H RHO+/-,mRHO+/+) were injected into the quadriceps with recombinant adeno-associated virus (rAAV) carrying either enhanced green fluorescent protein (eGFP) or EpoR76E. Visual function (electroretinogram) and retina structure (optical coherence tomography, histology, and immunohistochemistry) were assessed at 7 and 12 months of age.
RESULTS - The outer nuclear layer thickness decreased over time at a slower rate in rAAV.EpoR76E treated as compared to the rAAV.eGFP injected mice. There was a statistically significant preservation of the electroretinogram at 7, but not 12 months of age.
CONCLUSIONS - Systemic EPO-R76E slows death of the photoreceptors and vision loss in hP23H RHO+/-,mRHO+/+ mice. Treatment with EPO-R76E may widen the therapeutic window for retinal degeneration patients by increasing the number of viable cells. Future studies might investigate if co-treatment with EPO-R76E and gene replacement therapy is more effective than gene replacement therapy alone.
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MeSH Terms
Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin.
de Lucas Cerrillo AM, Bond WS, Rex TS
(2015) Gene Ther 22: 365-73
MeSH Terms: Animals, CHO Cells, Cells, Cultured, Cricetinae, Cricetulus, Endothelium, Vascular, Erythropoiesis, Erythropoietin, Gene Transfer Techniques, Humans, Mice, Mice, Inbred C57BL, Mutation, Missense, Neovascularization, Physiologic, Retinal Vessels
Show Abstract · Added April 2, 2019
Erythropoietin (EPO) is critical for red blood cell production and is also an effective neuroprotective agent. However, it may contribute to pathological angiogenesis. Here we investigate the angiogenic potential of EPO and a mutant form with attenuated erythropoietic activity, EPO-R76E, on primary human retinal microvascular endothelial cells (HRMECs) and in the adult retina. Assays of death, proliferation and tube formation were performed on HRMECs exposed to EPO, EPO-R76E or media alone. Postnatal day-9 wild-type mice were injected intramuscularly with adeno-associated virus vectors expressing either enhanced green fluorescent protein or EpoR76E. At 3 months, levels of EPO-R76E in the eye were quantified, and the health of the retinal vasculature was assessed by fluorescein angiography and isolectin immunolabeling. Immunohistochemistry, histology and electroretinogram (ERG) assessments were performed as measures of retinal health. Neither EPO nor EPO-R76E induced proliferation or tube formation in HRMECs under the conditions used. EPO-R76E decreased HRMEC death in a dose-dependent manner. Long-term systemic gene delivery of EPO-R76E was safe in terms of retinal vasculature, histology and the ERG in vivo. Our results show that EPO-R76E can block HRMEC death, consistent with its role in erythropoiesis and neuroprotection. In addition, long-term gene delivery of EPO-R76E is safe in the adult retina.
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MeSH Terms
Dual MMP7-proximity-activated and folate receptor-targeted nanoparticles for siRNA delivery.
Li H, Miteva M, Kirkbride KC, Cheng MJ, Nelson CE, Simpson EM, Gupta MK, Duvall CL, Giorgio TD
(2015) Biomacromolecules 16: 192-201
MeSH Terms: Drug Delivery Systems, Folate Receptors, GPI-Anchored, Gene Transfer Techniques, Humans, MCF-7 Cells, Matrix Metalloproteinase 7, Nanoparticles, RNA, Small Interfering
Show Abstract · Added March 14, 2018
A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity.
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8 MeSH Terms
Lipid nanoparticles as ideal delivery modules for siRNA.
Friedman H, Holt AT, Pham W
(2013) Nanomedicine (Lond) 8: 1910-1
MeSH Terms: Animals, Endocytosis, Gene Transfer Techniques, Humans, Lipids, RNA, Small Interfering
Added May 27, 2014
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6 MeSH Terms
Intracellular neutralization of a virus using a cell-penetrating molecular transporter.
Sapparapu G, Sims AL, Aiyegbo MS, Shaikh FY, Harth EM, Crowe JE
(2014) Nanomedicine (Lond) 9: 1613-24
MeSH Terms: Animals, Antibodies, Antibodies, Monoclonal, Biological Transport, Capsid Proteins, Cytoplasm, Dendrimers, Enzyme-Linked Immunosorbent Assay, Gene Transfer Techniques, HIV-1, Humans, Immunoglobulin Fragments, Kidney, Macaca mulatta, Magnetic Resonance Spectroscopy, Microscopy, Confocal, Nanomedicine, Neutralization Tests, Peptides, RNA, Small Interfering, Rotavirus, Virion, Viruses
Show Abstract · Added March 7, 2014
AIMS - Antibodies are the principal mediator of immunity against reinfection with viruses. Antibodies typically neutralize viruses by binding to virion particles in solution prior to attachment to susceptible cells. Once viruses enter cells, conventional antibodies cannot inhibit virus infection or replication. It is desirable to develop an efficient and nontoxic method for the introduction of virus-inhibiting antibodies into cells.
MATERIALS & METHODS - In this article, we report a new method for the delivery of small recombinant antibody fragments into virus-infected cells using a dendrimer-based molecular transporter.
RESULTS & CONCLUSION - The construct penetrated virus-infected cells efficiently and inhibited virus replication. This method provides a novel approach for the immediate delivery of inhibitory antibodies directed to virus proteins that are exposed only in the intracellular environment. This approach circumvents the current and rather complicated expression of inhibitory antibodies in cells following gene transfer.
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23 MeSH Terms
Identification of a therapeutic dose of continuously delivered erythropoietin in the eye using an inducible promoter system.
Hines-Beard J, Desai S, Haag R, Esumi N, D'Surney L, Parker S, Richardson C, Rex TS
(2013) Curr Gene Ther 13: 275-81
MeSH Terms: Animals, Drug Delivery Systems, Erythropoietin, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors, Humans, Mice, Promoter Regions, Genetic, Retina, Retinal Degeneration
Show Abstract · Added January 20, 2015
Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 μm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.
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11 MeSH Terms
An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression.
Doherty JE, Woodard LE, Bear AS, Foster AE, Wilson MH
(2013) FASEB J 27: 3753-62
MeSH Terms: Animals, DNA Transposable Elements, Fluorescent Antibody Technique, Gene Transfer Techniques, HeLa Cells, Humans, Immunoblotting, Mice, Transgenes, Transposases
Show Abstract · Added August 22, 2013
Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.
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10 MeSH Terms