Disease- and cell-type-specific transcriptional targeting of vectors for osteoarthritis gene therapy: further development of a clinical canine model.
OBJECTIVE
The potential for undesirable systemic effects related to constitutive expression of certain therapeutic transgenes may be limited through the development of transcriptionally targeted disease- and cell-type-specific vectors. The objective of this study was to analyse the canine matrix metalloproteinase-9 (MMP-9) promoter and deletion constructs for its ability to drive expression in response to pro-inflammatory cytokines (interleukin-1beta and tumour necrosis factor-alpha).


METHODS
Initial analysis of MMP-9 deletion constructs was made using a luciferase reporter system. The promoter was subsequently engineered to incorporate multiple NF-kappaB sites. In parallel experiments we used the mouse collagen type XI promoter to study cell-type-specific promoter activity in chondrocyte-specific cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5).


RESULTS
Incorporation of multiple NF-kappaB sites into the MMP-9 promoter enhanced activity while maintaining disease specificity. Further, manipulation of the mouse collagen type XI (mColXI) promoter by the incorporation of SOX9 enhancer sites downstream of a reporter gene, increased gene activity while maintaining cell type specificity.


CONCLUSIONS
Manipulation of promoter and enhancer regions can improve transcriptionally targeted genes. A combination of these systems, in the context of the canine model, has the potential to improve the safety of osteoarthritis gene therapy vectors.
Campbell
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SE
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706-1102, USA.
Bennett
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D
Nasir
L
L
Gault
E A
EA
Argyle
D J
DJ
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D024161:Collagen Type XI / Q000235:genetics; D004195:Disease Models, Animal; D004285:Dogs; D017353:Gene Deletion; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006609:High Mobility Group Proteins / Q000235:genetics; D007375:Interleukin-1 / Q000235:genetics; D007438:Introns / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D051379:Mice; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics; D010003:Osteoarthritis / Q000235:genetics* / Q000628:therapy; D011401:Promoter Regions, Genetic / Q000235:genetics; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D024161:Collagen Type XI; D006609:High Mobility Group Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D020780:Matrix Metalloproteinase 9