Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo.
BACKGROUND
Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.


RESULTS
We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC1D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. In vivo studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC1D promoters.


CONCLUSIONS
This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.
Liu
Beihui
B
Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK. bh.liu@bristol.ac.uk
Paton
Julian F
JF
Kasparov
Sergey
S
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D006801:Humans; D007002:Hypoglossal Nerve / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008526:Medulla Oblongata / Q000378:metabolism; D008845:Microinjections; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D016704:Synapsins / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics
D005904:Glial Fibrillary Acidic Protein; D016704:Synapsins