Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Bart Nieuwenhuis; Elise Laperrousaz; James R. Tribble; Joost Verhaagen; James W. Fawcett; Keith R. Martin; Pete A. Williams; Andrew Osborne

doi:10.1038/s41434-022-00380-z

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Bart Nieuwenhuis^*, Elise Laperrousaz, James R. Tribble, Joost Verhaagen, James W. Fawcett, Keith R. Martin, Pete A. Williams, Andrew Osborne^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Original language	English
Pages (from-to)	503-519
Number of pages	17
Journal	Gene Therapy
Volume	30
Issue number	6
Early online date	13 Jan 2023
DOIs	https://doi.org/10.1038/s41434-022-00380-z
Publication status	Published - Jun 2023

Bibliographical note

Funding Information:
This research was primarily funded by a seed funding project grant by Sight Research UK (SAC 041). Additional funding from the Medical Research Council (MR/V002694/1, MR/R004544/1 and MR/R004463/1), NWO (013-16-002), Fight for Sight (5065/5066, 5119/5120 and 5123/5124), Wings for Life (WFL-GB-04/19), Czech Ministry of Education (CZ.02.1.01/0.0./0.0/15_003/0000419), Cambridge Eye Trust, ERA-NET NEURON AxonRepair, International Foundation for Research in Paraplegia, onderzoekfonds KNAW instituten, Hersenstichting Nederland made this project possible. PAW is supported by Karolinska Institutet in the form of a Board of Research Faculty Funded Career Position and by St. Erik Eye Hospital philanthropic donations, Vetenskapsrådet 2018-02124.

Publisher Copyright:
© 2023, The Author(s).

Funding

This research was primarily funded by a seed funding project grant by Sight Research UK (SAC 041). Additional funding from the Medical Research Council (MR/V002694/1, MR/R004544/1 and MR/R004463/1), NWO (013-16-002), Fight for Sight (5065/5066, 5119/5120 and 5123/5124), Wings for Life (WFL-GB-04/19), Czech Ministry of Education (CZ.02.1.01/0.0./0.0/15_003/0000419), Cambridge Eye Trust, ERA-NET NEURON AxonRepair, International Foundation for Research in Paraplegia, onderzoekfonds KNAW instituten, Hersenstichting Nederland made this project possible. PAW is supported by Karolinska Institutet in the form of a Board of Research Faculty Funded Career Position and by St. Erik Eye Hospital philanthropic donations, Vetenskapsrådet 2018-02124.

Funders	Funder number
Board of Research
Karolinska Institutet
UK Research and Innovation
S:t Eriks Ögonforskningsstiftelse
NWO	013-16-002
Fight for Sight UK	5123/5124, WFL-GB-04/19, 5065/5066, 5119/5120
Vetenskapsrådet	2018-02124
Sight Research UK	SAC 041
Medical Research Council	MR/R004544/1, MR/R004463/1, MR/V002694/1
Ministerstvo Školství, Mládeže a Tělovýchovy	CZ.02.1.01/0.0

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title = "Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters",

abstract = "Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including M{\"u}ller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.",

author = "Bart Nieuwenhuis and Elise Laperrousaz and Tribble, {James R.} and Joost Verhaagen and Fawcett, {James W.} and Martin, {Keith R.} and Williams, {Pete A.} and Andrew Osborne",

note = "Funding Information: This research was primarily funded by a seed funding project grant by Sight Research UK (SAC 041). Additional funding from the Medical Research Council (MR/V002694/1, MR/R004544/1 and MR/R004463/1), NWO (013-16-002), Fight for Sight (5065/5066, 5119/5120 and 5123/5124), Wings for Life (WFL-GB-04/19), Czech Ministry of Education (CZ.02.1.01/0.0./0.0/15_003/0000419), Cambridge Eye Trust, ERA-NET NEURON AxonRepair, International Foundation for Research in Paraplegia, onderzoekfonds KNAW instituten, Hersenstichting Nederland made this project possible. PAW is supported by Karolinska Institutet in the form of a Board of Research Faculty Funded Career Position and by St. Erik Eye Hospital philanthropic donations, Vetenskapsr{\aa}det 2018-02124. Publisher Copyright: {\textcopyright} 2023, The Author(s).",

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doi = "10.1038/s41434-022-00380-z",

language = "English",

volume = "30",

pages = "503--519",

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AU - Laperrousaz, Elise

AU - Tribble, James R.

AU - Verhaagen, Joost

AU - Fawcett, James W.

AU - Martin, Keith R.

AU - Williams, Pete A.

AU - Osborne, Andrew

N1 - Funding Information: This research was primarily funded by a seed funding project grant by Sight Research UK (SAC 041). Additional funding from the Medical Research Council (MR/V002694/1, MR/R004544/1 and MR/R004463/1), NWO (013-16-002), Fight for Sight (5065/5066, 5119/5120 and 5123/5124), Wings for Life (WFL-GB-04/19), Czech Ministry of Education (CZ.02.1.01/0.0./0.0/15_003/0000419), Cambridge Eye Trust, ERA-NET NEURON AxonRepair, International Foundation for Research in Paraplegia, onderzoekfonds KNAW instituten, Hersenstichting Nederland made this project possible. PAW is supported by Karolinska Institutet in the form of a Board of Research Faculty Funded Career Position and by St. Erik Eye Hospital philanthropic donations, Vetenskapsrådet 2018-02124. Publisher Copyright: © 2023, The Author(s).

PY - 2023/6

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N2 - Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

AB - Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

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Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

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