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Transposon-modified antigen-specific T lymphocytes for sustained therapeutic protein delivery in vivo.
O'Neil RT, Saha S, Veach RA, Welch RC, Woodard LE, Rooney CM, Wilson MH
(2018) Nat Commun 9: 1325
MeSH Terms: Adoptive Transfer, Animals, Cell Engineering, Cell- and Tissue-Based Therapy, DNA Transposable Elements, Erythropoietin, Gene Expression, Genetic Vectors, Hematopoiesis, Herpesvirus 4, Human, Humans, Mice, Ovalbumin, Receptors, Antigen, T-Cell, T-Lymphocytes, Transgenes, Vaccination
Show Abstract · Added September 24, 2018
A cell therapy platform permitting long-term delivery of peptide hormones in vivo would be a significant advance for patients with hormonal deficiencies. Here we report the utility of antigen-specific T lymphocytes as a regulatable peptide delivery platform for in vivo therapy. piggyBac transposon modification of murine cells with luciferase allows us to visualize T cells after adoptive transfer. Vaccination stimulates long-term T-cell engraftment, persistence, and transgene expression enabling detection of modified cells up to 300 days after adoptive transfer. We demonstrate adoptive transfer of antigen-specific T cells expressing erythropoietin (EPO) elevating the hematocrit in mice for more than 20 weeks. We extend our observations to human T cells demonstrating inducible EPO production from Epstein-Barr virus (EBV) antigen-specific T lymphocytes. Our results reveal antigen-specific T lymphocytes to be an effective delivery platform for therapeutic molecules such as EPO in vivo, with important implications for other diseases that require peptide therapy.
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17 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 either Prevents or Exacerbates Retinal Damage from Eye Trauma Depending on Treatment Timing.
Bricker-Anthony C, D'Surney L, Lunn B, Hines-Beard J, Jo M, Bernardo-Colon A, Rex TS
(2017) Optom Vis Sci 94: 20-32
MeSH Terms: Animals, Blast Injuries, Cell Survival, Dependovirus, Disease Models, Animal, Erythropoietin, Eye Injuries, Ferritins, Genetic Therapy, Genetic Vectors, Green Fluorescent Proteins, In Situ Nick-End Labeling, Injections, Intramuscular, Injections, Intraperitoneal, Mice, Mice, Inbred BALB C, Mice, Inbred DBA, NADPH Oxidases, Oxidative Stress, Polymerase Chain Reaction, Retina, Retinal Diseases, Time Factors, Vision Disorders, Wounds, Nonpenetrating
Show Abstract · Added April 2, 2019
PURPOSE - Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective in traumatic eye injury.
METHODS - The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi overpressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7 days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre- or post-blast and analyses were performed at 1 month post-blast.
RESULTS - EPO had a bimodal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3 days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. The gene therapy approach protected against axon degeneration, cell death, and oxidative stress when given after blast, but not before.
CONCLUSIONS - Systemic, exogenous EPO and EPO-R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3 weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron levels from erythropoiesis and, thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an increase in levels of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious.
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MeSH Terms
Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma.
Bond WS, Hines-Beard J, GoldenMerry YL, Davis M, Farooque A, Sappington RM, Calkins DJ, Rex TS
(2016) Mol Ther 24: 230-239
MeSH Terms: Animals, Axons, Dependovirus, Disease Models, Animal, Erythropoietin, Genetic Therapy, Genetic Vectors, Glaucoma, Humans, Intraocular Pressure, Mice, Mutation, Optic Nerve
Show Abstract · Added February 4, 2016
Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.
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3 Members
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13 MeSH Terms
piggyBac-ing models and new therapeutic strategies.
Woodard LE, Wilson MH
(2015) Trends Biotechnol 33: 525-33
MeSH Terms: Animals, Baculoviridae, Genetic Engineering, Genetic Vectors, Genome, Humans, Mice
Show Abstract · Added July 28, 2015
DNA transposons offer an efficient nonviral method of permanently modifying the genomes of mammalian cells. The piggyBac transposon system has proven effective in genomic engineering of mammalian cells for preclinical applications, including gene discovery, simultaneous multiplexed genome modification, animal transgenesis, gene transfer in vivo achieving long-term gene expression in animals, and the genetic modification of clinically relevant cell types, such as induced pluripotent stem cells and human T lymphocytes. piggyBac has many desirable features, including seamless excision of transposons from the genomic DNA and the potential to target integration events to desired DNA sequences. In this review, we explore these recent applications and also highlight the unique advantages of using piggyBac for developing new molecular therapeutic strategies.
Published by Elsevier Ltd.
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2 Members
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7 MeSH Terms
Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.
Chiou SH, Winters IP, Wang J, Naranjo S, Dudgeon C, Tamburini FB, Brady JJ, Yang D, Grüner BM, Chuang CH, Caswell DR, Zeng H, Chu P, Kim GE, Carpizo DR, Kim SK, Winslow MM
(2015) Genes Dev 29: 1576-85
MeSH Terms: Adenocarcinoma, Animals, Carcinoma, Pancreatic Ductal, Clustered Regularly Interspaced Short Palindromic Repeats, Disease Models, Animal, Gene Expression Regulation, Neoplastic, Genetic Vectors, Genome, Humans, Lentivirus, Mice, Mice, Inbred C57BL, Mice, Transgenic
Show Abstract · Added September 7, 2016
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.
© 2015 Chiou et al.; Published by Cold Spring Harbor Laboratory Press.
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13 MeSH Terms
Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.
Yin L, Maddison LA, Li M, Kara N, LaFave MC, Varshney GK, Burgess SM, Patton JG, Chen W
(2015) Genetics 200: 431-41
MeSH Terms: Animals, Animals, Genetically Modified, CRISPR-Cas Systems, Gene Expression, Gene Order, Gene Silencing, Gene Targeting, Genetic Vectors, Glucose, Hypopigmentation, Mutagenesis, Phenotype, RNA, Guide, Transgenes, Zebrafish
Show Abstract · Added July 23, 2015
Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.
Copyright © 2015 by the Genetics Society of America.
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2 Members
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15 MeSH Terms
MiRNA inhibition in tissue engineering and regenerative medicine.
Beavers KR, Nelson CE, Duvall CL
(2015) Adv Drug Deliv Rev 88: 123-37
MeSH Terms: Bone and Bones, Cicatrix, Genetic Vectors, Humans, Inflammation, Kidney, Liver, MicroRNAs, Muscle, Skeletal, Myocardium, Neovascularization, Pathologic, RNA, Messenger, Regeneration, Regenerative Medicine, Tissue Engineering, Tissue Scaffolds, Wound Healing
Show Abstract · Added March 14, 2018
MicroRNAs (miRNAs) are noncoding RNAs that provide an endogenous negative feedback mechanism for translation of messenger RNA (mRNA) into protein. Single miRNAs can regulate hundreds of mRNAs, enabling miRNAs to orchestrate robust biological responses by simultaneously impacting multiple gene networks. MiRNAs can act as master regulators of normal and pathological tissue development, homeostasis, and repair, which has motivated expanding efforts toward the development of technologies for therapeutically modulating miRNA activity for regenerative medicine and tissue engineering applications. This review highlights the tools currently available for miRNA inhibition and their recent therapeutic applications for improving tissue repair.
Copyright © 2014 Elsevier B.V. All rights reserved.
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17 MeSH Terms
CRISPR-Cas9 knockin mice for genome editing and cancer modeling.
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F
(2014) Cell 159: 440-55
MeSH Terms: Adenocarcinoma, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Dendritic Cells, Disease Models, Animal, Gene Knock-In Techniques, Genes, Tumor Suppressor, Genetic Engineering, Genetic Vectors, Lentivirus, Lung Neoplasms, Mice, Mice, Transgenic, Oncogenes
Show Abstract · Added September 7, 2016
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
Copyright © 2014 Elsevier Inc. All rights reserved.
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14 MeSH Terms
Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.
Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, Maeda Y, Sato Y, Okabe S, Inukai T, Sugita K, Wilson MH, Rooney CM, Koike K
(2014) Cytotherapy 16: 1257-69
MeSH Terms: Antigens, CD19, Cancer Vaccines, Cell Line, Tumor, Cell Proliferation, Culture Media, Serum-Free, Cytotoxicity, Immunologic, DNA Transposable Elements, Drug Resistance, Neoplasm, Genetic Engineering, Genetic Vectors, Humans, Immunotherapy, Adoptive, Interleukin-15, Interleukin-2, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Mutation, Protein Kinase Inhibitors, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, T-Lymphocytes, TNF-Related Apoptosis-Inducing Ligand, Up-Regulation
Show Abstract · Added October 28, 2014
BACKGROUND AIMS - To develop a treatment option for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR).
METHODS - A CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15-containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph(+)ALL cell lines including three TKI-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines.
RESULTS - We obtained ∼1.3 × 10(8) CAR T cells (CD4(+), 25.4%; CD8(+), 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor-related apoptosis-inducing ligand and interleukin-2.
CONCLUSIONS - We generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph(+)ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph(+)ALL.
Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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22 MeSH Terms