A versatile cell line for establishing potency of cell type-specific AAV transgenes
Potency assays are essential for adeno-associated virus (AAV) therapy development, allowing simple and quantitative assessments of therapeutic activity, target engagement, vector stability, and activity per batch. However, cell culture-based AAV potency assays are generally hampered by two limitatio...
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Elsevier
2025-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050124002171 |
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| author | Refugio A. Martinez Yemeserach M. Bishaw Meagan A. Quinlan Erin L. Groce Bryan B. Gore Ed S. Lein John K. Mich Boaz P. Levi |
| author_facet | Refugio A. Martinez Yemeserach M. Bishaw Meagan A. Quinlan Erin L. Groce Bryan B. Gore Ed S. Lein John K. Mich Boaz P. Levi |
| author_sort | Refugio A. Martinez |
| collection | DOAJ |
| description | Potency assays are essential for adeno-associated virus (AAV) therapy development, allowing simple and quantitative assessments of therapeutic activity, target engagement, vector stability, and activity per batch. However, cell culture-based AAV potency assays are generally hampered by two limitations: (1) commonly used cell lines have low AAV transduction efficiency due to their lack of appropriate surface receptors, and (2) cell type-specific AAV transgenes are often not expressed because their regulatory elements (enhancers, promoters, etc.) may not be active in the cell lines used for testing. Here, we present the CRISPR activation-AAV receptor (CRAAVR) cell line, a stable HEK293T-derived line that overcomes both problems. CRAAVR cells constitutively express the AAV receptor (AAVR) and an AAV-targeted CRISPR transactivator, and, as a result, exhibit high transduction efficiency and drive transgene expression from AAVs with cell type-specific regulatory elements. Furthermore, CRAAVR cells can assess AAV transgene potency in a quantitative manner, as demonstrated by gamma-aminobutyric acid (GABA) uptake of an AAV-delivered GABA transporter (GAT-3). Due to their greater transduction efficiency, regulatory element-agnostic expression, and utility as a potency assay tool, CRAAVR cells will help streamline the development and validation of AAV-based reagents and therapeutics. |
| format | Article |
| id | doaj-art-7d555b0c57cc444ca5d3706781b5a5ca |
| institution | Kabale University |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-7d555b0c57cc444ca5d3706781b5a5ca2025-01-30T05:14:22ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-03-01331101401A versatile cell line for establishing potency of cell type-specific AAV transgenesRefugio A. Martinez0Yemeserach M. Bishaw1Meagan A. Quinlan2Erin L. Groce3Bryan B. Gore4Ed S. Lein5John K. Mich6Boaz P. Levi7Allen Institute for Brain Science, Seattle, WA 98109, USA; Corresponding author: Refugio A. Martinez, Allen Institute for Brain Science, Seattle, WA 98109, USA.Allen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USAAllen Institute for Brain Science, Seattle, WA 98109, USA; Corresponding author: Boaz P. Levi, Allen Institute for Brain Science, Seattle, WA 98109, USA.Potency assays are essential for adeno-associated virus (AAV) therapy development, allowing simple and quantitative assessments of therapeutic activity, target engagement, vector stability, and activity per batch. However, cell culture-based AAV potency assays are generally hampered by two limitations: (1) commonly used cell lines have low AAV transduction efficiency due to their lack of appropriate surface receptors, and (2) cell type-specific AAV transgenes are often not expressed because their regulatory elements (enhancers, promoters, etc.) may not be active in the cell lines used for testing. Here, we present the CRISPR activation-AAV receptor (CRAAVR) cell line, a stable HEK293T-derived line that overcomes both problems. CRAAVR cells constitutively express the AAV receptor (AAVR) and an AAV-targeted CRISPR transactivator, and, as a result, exhibit high transduction efficiency and drive transgene expression from AAVs with cell type-specific regulatory elements. Furthermore, CRAAVR cells can assess AAV transgene potency in a quantitative manner, as demonstrated by gamma-aminobutyric acid (GABA) uptake of an AAV-delivered GABA transporter (GAT-3). Due to their greater transduction efficiency, regulatory element-agnostic expression, and utility as a potency assay tool, CRAAVR cells will help streamline the development and validation of AAV-based reagents and therapeutics.http://www.sciencedirect.com/science/article/pii/S2329050124002171AAVAAVRCRISPRain vitro potency assayHEK293T cells |
| spellingShingle | Refugio A. Martinez Yemeserach M. Bishaw Meagan A. Quinlan Erin L. Groce Bryan B. Gore Ed S. Lein John K. Mich Boaz P. Levi A versatile cell line for establishing potency of cell type-specific AAV transgenes Molecular Therapy: Methods & Clinical Development AAV AAVR CRISPRa in vitro potency assay HEK293T cells |
| title | A versatile cell line for establishing potency of cell type-specific AAV transgenes |
| title_full | A versatile cell line for establishing potency of cell type-specific AAV transgenes |
| title_fullStr | A versatile cell line for establishing potency of cell type-specific AAV transgenes |
| title_full_unstemmed | A versatile cell line for establishing potency of cell type-specific AAV transgenes |
| title_short | A versatile cell line for establishing potency of cell type-specific AAV transgenes |
| title_sort | versatile cell line for establishing potency of cell type specific aav transgenes |
| topic | AAV AAVR CRISPRa in vitro potency assay HEK293T cells |
| url | http://www.sciencedirect.com/science/article/pii/S2329050124002171 |
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