Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays
Vectors based on adeno-associated viruses (AAVs) are promising therapeutic modalities used in gene therapy. Robust cell-based assays that demonstrate and quantify the potency of AAV vectors in expressing their transgene are needed for clinical development. However, many AAV clinical serotypes poorly...
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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/S2329050125000117 |
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| author | James Zengel Emma S. Esterman Anitha Ponnuswami Nicholas R. Wall Jan E. Carette |
| author_facet | James Zengel Emma S. Esterman Anitha Ponnuswami Nicholas R. Wall Jan E. Carette |
| author_sort | James Zengel |
| collection | DOAJ |
| description | Vectors based on adeno-associated viruses (AAVs) are promising therapeutic modalities used in gene therapy. Robust cell-based assays that demonstrate and quantify the potency of AAV vectors in expressing their transgene are needed for clinical development. However, many AAV clinical serotypes poorly transduce cells in vitro and often contain cell-type-specific promoters inactive in commonly used cell lines. Here, we enhance the efficiency of in vitro AAV transduction by overexpressing the AAV receptor (AAVR/KIAA0319L), preventing transcriptional silencing by the HUSH complex, and using CRISPR activation (CRISPRa) to drive transgene expression. For the latter, we utilized guide RNAs targeting the conserved AAV2 inverted terminal repeat (ITR) sequence present in most AAV transfer vectors. Using this strategy, we engineered cell lines that showed marked increases in transduction by AAV vectors across a wide range of clinically relevant serotypes and containing cell-type-specific promoters. These improvements enabled the efficient determination of AAV functional titers (also referred to as transducing titers), which can be used to robustly monitor potency across diverse AAV preparations. The strongly enhanced susceptibility of these cell lines to transduction by a variety of divergent AAV vectors could facilitate the development of standardized in vitro quantitative assays for AAV-based gene therapy products. |
| format | Article |
| id | doaj-art-635a163a5917484cb8472676db7ccce9 |
| 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-635a163a5917484cb8472676db7ccce92025-02-08T05:00:33ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-03-01331101416Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assaysJames Zengel0Emma S. Esterman1Anitha Ponnuswami2Nicholas R. Wall3Jan E. Carette4Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USADepartment of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USAWu Tsai Neurosciences Institute, Stanford University, Stanford, CA 94305, USAWu Tsai Neurosciences Institute, Stanford University, Stanford, CA 94305, USADepartment of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA; Corresponding author: Jan E. Carette, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.Vectors based on adeno-associated viruses (AAVs) are promising therapeutic modalities used in gene therapy. Robust cell-based assays that demonstrate and quantify the potency of AAV vectors in expressing their transgene are needed for clinical development. However, many AAV clinical serotypes poorly transduce cells in vitro and often contain cell-type-specific promoters inactive in commonly used cell lines. Here, we enhance the efficiency of in vitro AAV transduction by overexpressing the AAV receptor (AAVR/KIAA0319L), preventing transcriptional silencing by the HUSH complex, and using CRISPR activation (CRISPRa) to drive transgene expression. For the latter, we utilized guide RNAs targeting the conserved AAV2 inverted terminal repeat (ITR) sequence present in most AAV transfer vectors. Using this strategy, we engineered cell lines that showed marked increases in transduction by AAV vectors across a wide range of clinically relevant serotypes and containing cell-type-specific promoters. These improvements enabled the efficient determination of AAV functional titers (also referred to as transducing titers), which can be used to robustly monitor potency across diverse AAV preparations. The strongly enhanced susceptibility of these cell lines to transduction by a variety of divergent AAV vectors could facilitate the development of standardized in vitro quantitative assays for AAV-based gene therapy products.http://www.sciencedirect.com/science/article/pii/S2329050125000117adeno-associated virusAAVAAVRserotypetransductioncell line development |
| spellingShingle | James Zengel Emma S. Esterman Anitha Ponnuswami Nicholas R. Wall Jan E. Carette Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays Molecular Therapy: Methods & Clinical Development adeno-associated virus AAV AAVR serotype transduction cell line development |
| title | Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays |
| title_full | Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays |
| title_fullStr | Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays |
| title_full_unstemmed | Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays |
| title_short | Development of cell lines with increased susceptibility to diverse adeno-associated viral vectors to enable in vitro potency assays |
| title_sort | development of cell lines with increased susceptibility to diverse adeno associated viral vectors to enable in vitro potency assays |
| topic | adeno-associated virus AAV AAVR serotype transduction cell line development |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000117 |
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