Abstract
BackgroundDuchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible.ResultsWe have previously shown that the zinc finger-based artificial transcriptional factor “Jazz” corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named “UtroUp”, engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus.ConclusionsThis novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.
Highlights
Duchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin
We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, and its low dissociation constant (Kd) possesses a strong affinity/specificity toward its DNA-binding site
To be able to test the therapeutic potential of our artificial transcription factor in a dystrophic mouse model, we chose a DNA target sequence in a region that was completely conserved in both the human and the mouse utrophin promoter “A” (Figure 1A)
Summary
Duchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. To obtain upregulation of utrophin, we have engineered artificial zinc finger-based transcription factors that are capable of binding and activating transcription from promoter “A” of both the human and mouse utrophin genes [18,19,20,21,22,23]. UtroUp, coupled with its activation domain that was derived from the adaptor protein Che-1/AATF [21], efficiently accesses the active chromatin in the utrophin promoter locus and strongly activates utrophin transcription This novel artificial molecule represents an improved platform for the development of future applications in the DMD gene therapy field
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