Journal
CELLS
Volume 9, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/cells9112474
Keywords
liver-specific glucose-responsive promoter; adeno-associated virus serotype 8 (AAV8); diabetes gene therapy; long-term basal insulin expression; glucose responsive element (GlRE); albumin enhancer (3′ iALB)
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Funding
- Kidney Dialysis Foundation (KDF, Singapore)
- Lollipop Fund (UK)
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We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3 ' albumin enhancer (3 ' iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 x 10(9) vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 x 10(10) vg/mouse. The further inclusion of an 860 base-pairs 3 ' iALB enhancer component in the 3 ' untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.
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