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Nucleic Acids Research, 2003, Vol. 31, No. 17 e100
© 2003 Oxford University Press

An enhanced U6 promoter for synthesis of short hairpin RNA

Xu Gang Xia1, Hongxia Zhou1, Hongliu Ding1, El Bashir Affar4, Yang Shi4 and Zuoshang Xu*,1,2,3

1 Department of Biochemistry and Molecular Pharmacology, 2 Department of Cell Biology and 3 Neuroscience Program, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA and 4 Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

*To whom correspondence should be addressed. Tel: +1 508 856 3309; Fax: +1 508 856 8390; Email: zuoshang.xu{at}umassmed.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1G93A) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.


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