Identifying adeno‐associated virus (AAV) vectors that efficiently target high grade glioma cells, for in vitro monitoring of temporal cell responses

To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient‐derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standa...

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Published inFEBS open bio Vol. 14; no. 11; pp. 1914 - 1925
Main Authors Sarker, Farhana A., Chen, Yuyan, Westhaus, Adrian, Lisowski, Leszek, O'Neill, Geraldine M.
Format Journal Article
LanguageEnglish
Published England John Wiley & Sons, Inc 01.11.2024
John Wiley and Sons Inc
Wiley
Subjects
AAV
Bar codes
barcoded
Biotechnology
Brain Cancer
Brain Neoplasms - genetics
Brain Neoplasms - pathology
Brain tumors
Cell culture
Cell Line, Tumor
Cells
Dependovirus - genetics
Expression vectors
Genetic Vectors - genetics
Genomes
Genomics
Glioblastoma
Glioma
Glioma - genetics
Glioma - pathology
Glioma cells
High-Throughput Nucleotide Sequencing - methods
Humans
Invoices
Medical research
Next Generation Sequencing
Pediatrics
Phenotypes
Proteins
Transcription activation
Transcription factors
Tropism
Tumors
Viral Vector
Viruses
YAP/TAZ
Yes-associated protein
England
United Kingdom > UK
Australia
United States > US
Germany
glioma
barcoded
YAP/TAZ
AAV
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Abstract To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient‐derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno‐associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next‐generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high‐grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co‐activators yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ). Transcriptional activation was monitored by promoter‐driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV‐mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS‐screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells. Primary patient‐derived cancer cells better emulate human physiology, improving research outcomes. However, delivery of recombinant DNA is a challenge using standard transduction methods. We report a high‐throughput screen to identify an optimal adeno‐associated virus (AAV) vector. We then provide proof‐of‐principle evidence with AAV transduction of primary high‐grade glioma brain cancer cells encoding a novel reporter for measuring YAP/TAZ dynamic signalling.
AbstractList To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.
To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient‐derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno‐associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next‐generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high‐grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co‐activators yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ). Transcriptional activation was monitored by promoter‐driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV‐mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS‐screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells. Primary patient‐derived cancer cells better emulate human physiology, improving research outcomes. However, delivery of recombinant DNA is a challenge using standard transduction methods. We report a high‐throughput screen to identify an optimal adeno‐associated virus (AAV) vector. We then provide proof‐of‐principle evidence with AAV transduction of primary high‐grade glioma brain cancer cells encoding a novel reporter for measuring YAP/TAZ dynamic signalling.
To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.
To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient‐derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno‐associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next‐generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high‐grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co‐activators yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ). Transcriptional activation was monitored by promoter‐driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV‐mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS‐screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.
Author Westhaus, Adrian
Chen, Yuyan
O'Neill, Geraldine M.
Sarker, Farhana A.
Lisowski, Leszek
AuthorAffiliation 3 Translational Vectorology Research Unit, Faculty of Medicine and Health, Children's Medical Research Institute The University of Sydney Westmead Australia
4 Australian Genome Therapeutics Centre Children's Medical Research Institute and Sydney Children's Hospitals Network Westmead Australia
5 Laboratory of Molecular Oncology and Innovative Therapies Military Institute of Medicine Warsaw Poland
1 Children's Hospital Clinical School, Faculty of Medicine and Health University of Sydney Australia
2 Children's Cancer Research Unit The Children's Hospital at Westmead Sydney Australia
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SubjectTerms AAV
Bar codes
barcoded
Biotechnology
Brain Cancer
Brain Neoplasms - genetics
Brain Neoplasms - pathology
Brain tumors
Cell culture
Cell Line, Tumor
Cells
Dependovirus - genetics
Expression vectors
Genetic Vectors - genetics
Genomes
Genomics
Glioblastoma
Glioma
Glioma - genetics
Glioma - pathology
Glioma cells
High-Throughput Nucleotide Sequencing - methods
Humans
Invoices
Medical research
Next Generation Sequencing
Pediatrics
Phenotypes
Proteins
Transcription activation
Transcription factors
Tropism
Tumors
Viral Vector
Viruses
YAP/TAZ
Yes-associated protein
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Title Identifying adeno‐associated virus (AAV) vectors that efficiently target high grade glioma cells, for in vitro monitoring of temporal cell responses
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Volume 14
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