Peter - my perspective on the article:
[I have used some language directly from the article to avoid 'translational errors']
Sangamo's goal is to use synthetic transcription factors containing a zinc finger DNA binding domain (Zinc Finger Protein, ZFP) to activate promoters at endogenous chromosomal loci (in this case the human EPO gene). Thus their proprietary product and platform is the design and implementation of ZFPs which can specifically interact with promoters to modulate (induce, suppress, potentiate, attenuate) transcriptional activity at countless loci.
The paper describes their latest efforts using EPO-activating, ZFP constructs. They tested the efficacy of EPO ZPFs in 2 experimental scenarios: a) in cells transiently transfected with ('artificial') constructs containing sequences within the promoter of the EPO gene (linked to a Luciferase gene for readout of activity from the promoter) and b) in the endogenous EPO gene nestled within its normal chromosomal context. The ZFPs did well in activating the artificial constructs within the cells - the ability to activate the EPO construct was directly correlated to the strength the ZFP bound to the promoter. Activation of the endogenous gene - the scenario confronting therapeutic approaches - was more problematic. Only a subset of the ZFPs were able to activate the endogenous gene and their efficacy is dependent upon - and perhaps greatly limited by - their ability to penetrate the complex and often 'closed' structure of chromatin. Promoter activation of the endogenous gene was unrelated to the strength of DNA binding of the ZFP constructs.
A brief review of chromatin may give some perspective on Sangamo’s future: chromatin is the supercoiled, compressed form of DNA - the DNA is wrapped around histones to form nucleosomes and then further compacted into filamentous strands. This is the structure of chromosomes found in cells. When the product of a gene is 'needed', the chromatin **generally** needs to be in an 'active' form to allow the transcriptional machinery access to the DNA. The transition to active chromatin involves, in part, a partial relaxation of the compaction of the DNA. The mechanisms that form active chromatin are not well understood and, as this paper appears to indicate, may be one of the more significant roadblocks for Sangamo in achieving activation of endogenous genes with their ZFPs.
To quote the paper, ‘These studies indicate that chromosome and chromatin organization is a determinant of zinc finger transcription factor function within endogenous chromosomal loci.’. If Sangamo had found that activation of endogenous chromosomes by their ZFP’s was a direct function of the strength of DNA binding, then they could easily design countless activation factors and their platform would be a multi-, multi-billion dollar approach. The fact that access to endogenous DNA appears to be primarily correlated with the vagaries of chromosome and chromatin structure indicates that the therapeutic use of ZFPs to regulate endogenous genes remains very, very early in development. |
