(slightly OT, & apologies for not posting to the gene therapy thread...)
In view of the forgoing chromatin discussion, would anyone qualified like to comment on the following ? (MTIA) -
cobrat.com
Cobra has developed new gene expression technology which, through chromatin remodelling, gives more productive integration events and more predictable high level expression in a sustained fashion.
One of our founding technologies was Locus Control Regions (LCRs), which confer sustained gene expression in a tissue specific fashion. LCRs have already been identified which give expression restricted to erythroid cells, T cells, antigen presenting cells, pigment cells, liver, brain, pituitary, muscle or lung, as indicated in Figure 1, and it is likely that LCRs with many other useful specificities will be discovered in the next few years....
In transgenic mice LCRs give tissue specific expression of linked transgenes at levels similar to that of the endogenous LCR’s structural gene in its natural chromosomal location. This occurs irrespective of the integration site in the genome. For some LCRs it has been demonstrated that endogenous expression level and appropriate tissue specificity are still evident even with low copy number integrations in (highly condensed) heterochromatin. Transcription enhancers represent the other main class of regulatory element giving expression restricted to particular cell types or tissues. Unlike LCRs, however, utility of these elements is limited by a lower frequency of productive integration events, variable and low level expression resulting from local differences in the chromosome environment, and short-lived expression due to transcriptional silencing. The capacity of LCRs to confer reliable, tissue specific transgene expression in a sustained fashion will allow many important applications, notably in human gene therapy and the use of transgenic animals for disease models, safety studies and functional genomics.
Cobra has been granted patents on LCRs in all major territories. These are available for license for research purposes. We are also able to supply details on sources of LCR fragments and vectors, and a comprehensive information package.
Our recent work on novel transgene expression technology has focused on identifying regulatory elements which act in a ubiquitous rather than tissue specific fashion but which share with LCRs the capacity to affect the structure of chromatin. We have characterised elements associated with two ubiquitously expressed human loci which confer physiological levels of transgene expression in a wide range of tissues in transgenic mice. These tissues include heart, kidney, lung, liver, guts, muscle, gonads, spleen, thymus and blood. The elements, which we term Ubiquitous Chromatin Opening Elements (UCOEs), show structural features that are distinct from those of LCRs. However they share with LCRs the ability to confer endogenous levels of expression following integration of a single transgene copy, and correspondingly higher expression following multicopy integration.
We have also demonstrated the advantages of these UCOEs for expressing transgenes in mammalian cell lines in tissue culture. We have shown that a DNA fragment, CET7.5, of 7.5Kb carrying one of these elements confers at least a tenfold elevation in expression level in CHO cells, with the transgene expressed either from a relatively weak endogenous promoter or from the strong, heterologous CMV promoter. Furthermore the element increases the number of transfectant colonies observed and substantially improves the stability of expression.
The likely mode of action of CET7.5 and LCR elements is illustrated in Figure 2 below. It is widely believed that chromatin adopts one of two forms : "open" or "closed". In closed chromatin the DNA is in a condensed state and genes within it are transcriptionally silent. In open chromatin the DNA is decondensed and genes within these domains are transcribed. We have shown that CET7.5 and related structures are capable of giving endogenous expression levels even when integrated in highly condensed heterochromatin.
Cobra has constructed vectors incorporating CET7.5 which allow convenient evaluation of the technology for a wide range of applications. These include functional genomics, therapeutic protein manufacture, transgenic animals and gene therapy. CET7.5 vectors are available for evaluation under a research license. We are also minimising the size of UCOEs in order to identify fragments small enough for delivery by integrating viral vectors for gene therapy purposes... |
