Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons
Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction st...
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Series: | Stem Cells International |
Online Access: | http://dx.doi.org/10.1155/2019/7281912 |
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doaj-81d0b03f3f4247b2a10601923f3480b42020-11-24T23:07:44ZengHindawi LimitedStem Cells International1687-966X1687-96782019-01-01201910.1155/2019/72819127281912Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical NeuronsThu T. Duong0James Lim1Vidyullatha Vasireddy2Tyler Papp3Hung Nguyen4Lanfranco Leo5Jieyan Pan6Shangzhen Zhou7H. Isaac Chen8Jean Bennett9Jason A. Mills10F.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USADepartment of Neurosurgery, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USADepartment of Computer and Information Science, University of Pennsylvania, PA 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USADepartment of Neurosurgery, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USAF.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Pennsylvania, PA 19104, USARecombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.http://dx.doi.org/10.1155/2019/7281912 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Thu T. Duong James Lim Vidyullatha Vasireddy Tyler Papp Hung Nguyen Lanfranco Leo Jieyan Pan Shangzhen Zhou H. Isaac Chen Jean Bennett Jason A. Mills |
spellingShingle |
Thu T. Duong James Lim Vidyullatha Vasireddy Tyler Papp Hung Nguyen Lanfranco Leo Jieyan Pan Shangzhen Zhou H. Isaac Chen Jean Bennett Jason A. Mills Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons Stem Cells International |
author_facet |
Thu T. Duong James Lim Vidyullatha Vasireddy Tyler Papp Hung Nguyen Lanfranco Leo Jieyan Pan Shangzhen Zhou H. Isaac Chen Jean Bennett Jason A. Mills |
author_sort |
Thu T. Duong |
title |
Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons |
title_short |
Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons |
title_full |
Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons |
title_fullStr |
Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons |
title_full_unstemmed |
Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons |
title_sort |
comparative aav-egfp transgene expression using vector serotypes 1–9, 7m8, and 8b in human pluripotent stem cells, rpes, and human and rat cortical neurons |
publisher |
Hindawi Limited |
series |
Stem Cells International |
issn |
1687-966X 1687-9678 |
publishDate |
2019-01-01 |
description |
Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics. |
url |
http://dx.doi.org/10.1155/2019/7281912 |
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