JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice
Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV <i>env</i> gene, has been previously described. Herein we provide further evidence that the JE functions as a transcr...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-11-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/12/11/1266 |
| _version_ | 1797548645400182784 |
|---|---|
| author | Darrick L. Yu Natalie Chow Sarah K. Wootton |
| author_facet | Darrick L. Yu Natalie Chow Sarah K. Wootton |
| author_sort | Darrick L. Yu |
| collection | DOAJ |
| description | Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV <i>env</i> gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart. |
| first_indexed | 2024-03-10T15:03:33Z |
| format | Article |
| id | doaj.art-e8c530e103754abdb9486b6e8515f115 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T15:03:33Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-e8c530e103754abdb9486b6e8515f1152023-11-20T20:02:43ZengMDPI AGViruses1999-49152020-11-011211126610.3390/v12111266JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of MiceDarrick L. Yu0Natalie Chow1Sarah K. Wootton2Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, CanadaDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, CanadaDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, CanadaJaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV <i>env</i> gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.https://www.mdpi.com/1999-4915/12/11/1266adeno-associated virus (AAV) vectorjaagsiekte sheep retrovirus (JSRV)LTRenhancertransduction |
| spellingShingle | Darrick L. Yu Natalie Chow Sarah K. Wootton JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice Viruses adeno-associated virus (AAV) vector jaagsiekte sheep retrovirus (JSRV) LTR enhancer transduction |
| title | JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice |
| title_full | JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice |
| title_fullStr | JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice |
| title_full_unstemmed | JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice |
| title_short | JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice |
| title_sort | jsrv intragenic enhancer element increases expression from a heterologous promoter and promotes high level aav mediated transgene expression in the lung and liver of mice |
| topic | adeno-associated virus (AAV) vector jaagsiekte sheep retrovirus (JSRV) LTR enhancer transduction |
| url | https://www.mdpi.com/1999-4915/12/11/1266 |
| work_keys_str_mv | AT darricklyu jsrvintragenicenhancerelementincreasesexpressionfromaheterologouspromoterandpromoteshighlevelaavmediatedtransgeneexpressioninthelungandliverofmice AT nataliechow jsrvintragenicenhancerelementincreasesexpressionfromaheterologouspromoterandpromoteshighlevelaavmediatedtransgeneexpressioninthelungandliverofmice AT sarahkwootton jsrvintragenicenhancerelementincreasesexpressionfromaheterologouspromoterandpromoteshighlevelaavmediatedtransgeneexpressioninthelungandliverofmice |