Constructs for enhancement of gene expression in muscle
Efficient and muscle-specific gene expression can be obtained with constructs containing two or more copies of USE and/or ΔUSE fused to the minimal promoter of the TnISlow gene. USE is a small (about 160-bp) upstream enhancer of the TnISlow gene that confers slow-twitch muscle fiber specificity. ΔUSE is generated from a 100-bp deletion at the 5′ end of USE. ΔUSE confers expression in slow- and fast-twitch muscle fibers. The strength and relatively small size (less than 600-bp) of these constructs make them useful for gene therapy applications.
1. A construct comprising two or more enhancers from a slow troponin I gene fused in line and operably linked to a muscle cell specific promoter, and a nucleotide sequence of interest, wherein each enhancer comprises the nucleotide sequence as set forth in nucleotides 152 to 218 of SEQ ID NO:17 or as set forth in nucleotides 57-218 of SEQ ID NO: 17, and wherein the construct ensures tissue-specific expression of the nucleotide of interest in muscle cells.
2. The construct of claim 1 , wherein the two or more enhancers are upstream of the promoter.
3. The construct of claim 1 , wherein the enhancers are oriented in a natural orientation.
4. The construct of claim 1 , wherein there are 3 or more enhancers.
5. The construct of claim 1 , wherein there are from 2 to 5 enhancers.
6. The construct of claim 1 , wherein the promoter comprises minimal promoter from the slow troponin I gene.
7. The construct of claim 1 , further comprising an intron downstream of the promoter.
8. The construct of claim 1 , wherein the enhancers are three enhancers each comprising the nucleotide sequence as set forth in nucleotides 57 to 218 of SEQ ID NO: 17.
9. The construct of claim 1 , wherein the enhancers are three enhancers each comprising the nucleotide sequence as set forth in nucleotides 152 to 218 of SEQ ID NO: 17.
10. The construct of claim 1 , wherein the enhancers are four enhancers each comprising the nucleotide sequence as set forth in nucleotides 57 to 218 of SEQ ID NO: 17.
11. The construct of claim 1 , wherein the enhancers are four enhancers each comprising the nucleotide sequence as set forth in nucleotides 152 to 218 of SEQ ID NO: 17.
12. The construct of claim 1 , wherein the enhancers are five enhancers each comprising the nucleotide sequence as set forth in nucleotides 57 to 218 of SEQ ID NO: 17.
13. The construct of claim 1 , wherein the enhancers are five enhancers each comprising the nucleotide sequence as set forth in nucleotides 152 to 218 of SEQ ID NO: 17.
14. A method of specifically expressing a nucleotide sequence of interest in a muscle cell in vitro, comprising transfecting into the muscle cell a construct of claim 1 ; culturing said cell under suitable condition to express the nucleotide sequence of interest.
15. The method of claim 14 , wherein the muscle cell is a skeletal muscle cell.
16. The method of claim 14 , wherein the nucleotide sequence of interest codes for a protein.
17. The method of claim 16 , wherein the protein mitigates pathology associated with hemophilia, Pompe disease, Fabry's disease, anaemia, emphysema or familial hypercholesterolemia.
18. The method of claim 16 , wherein the protein mitigates pathology associated with a neuromuscular disorder.
19. A method of specifically expressing a nucleotide sequence of interest in a muscle cell in a mouse model, comprising administering the construct of claim 1 into a muscle cell of a mouse; measuring the expression of the nucleotide sequence of interest in said muscle cell.