Partial genome content within rAAVs impacts performance in a cell assay-dependent manner
Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlle...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-09-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050123001092 |
| _version_ | 1797752001304461312 |
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| author | Bryan Troxell Sarah L. Jaslow I-Wei Tsai Chelsea Sullivan Benjamin E. Draper Martin F. Jarrold Kate Lindsey Levi Blue |
| author_facet | Bryan Troxell Sarah L. Jaslow I-Wei Tsai Chelsea Sullivan Benjamin E. Draper Martin F. Jarrold Kate Lindsey Levi Blue |
| author_sort | Bryan Troxell |
| collection | DOAJ |
| description | Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance. |
| first_indexed | 2024-03-12T16:56:53Z |
| format | Article |
| id | doaj.art-8a4925de91024196b5902ddf22890b66 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-03-12T16:56:53Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-8a4925de91024196b5902ddf22890b662023-08-08T04:05:44ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012023-09-0130288302Partial genome content within rAAVs impacts performance in a cell assay-dependent mannerBryan Troxell0Sarah L. Jaslow1I-Wei Tsai2Chelsea Sullivan3Benjamin E. Draper4Martin F. Jarrold5Kate Lindsey6Levi Blue7StrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USA; AjaxBio, LLC, Holly Springs, NC 27540, USA; Corresponding author: Bryan Troxell, StrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USA.StrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USAStrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USAStrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USAMegadalton Solutions, Inc., 3750 E. Bluebird Ln., Bloomington, IN 47401, USAChemistry Department, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN 47405, USAStrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USAStrideBio Analytical Development and Quality Control, 5 Laboratory Drive, Suite 1200, Research Triangle Park, NC 27709, USARecombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance.http://www.sciencedirect.com/science/article/pii/S2329050123001092CD-MSSEC-MALSlive-cell imagingtransductionNGSddPCR |
| spellingShingle | Bryan Troxell Sarah L. Jaslow I-Wei Tsai Chelsea Sullivan Benjamin E. Draper Martin F. Jarrold Kate Lindsey Levi Blue Partial genome content within rAAVs impacts performance in a cell assay-dependent manner Molecular Therapy: Methods & Clinical Development CD-MS SEC-MALS live-cell imaging transduction NGS ddPCR |
| title | Partial genome content within rAAVs impacts performance in a cell assay-dependent manner |
| title_full | Partial genome content within rAAVs impacts performance in a cell assay-dependent manner |
| title_fullStr | Partial genome content within rAAVs impacts performance in a cell assay-dependent manner |
| title_full_unstemmed | Partial genome content within rAAVs impacts performance in a cell assay-dependent manner |
| title_short | Partial genome content within rAAVs impacts performance in a cell assay-dependent manner |
| title_sort | partial genome content within raavs impacts performance in a cell assay dependent manner |
| topic | CD-MS SEC-MALS live-cell imaging transduction NGS ddPCR |
| url | http://www.sciencedirect.com/science/article/pii/S2329050123001092 |
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