Thursday, 24 July 2008

Uncommon ways of looking at common problems- gene therapy for mitochondrial DNA diseases

ResearchBlogging.orgI’m always very interested in less conventional approaches to conventional problems and a recent review found while looking for a paper for a journal club provided a good example.

As you know, gene therapy aims at correcting a genetic deficiency by the introduction and/or substitution of the mutated gene by a functional copy into relevant cells. The classic examples for gene therapy are normally diseases such as Cystic Fibrosis- monogenic, recessive, autosomal diseases, as these are, for obvious reasons, those that theoretically offer higher chances of success. There are, however, other types of genetic diseases that offer more space for original, unconventional gene therapy approaches.

Mitochondrial DNA diseases are a good example. The human mitochondrial genome is small (16kb) and is housed in the mitochondrial matrix. As most of its genes encode proteins involved in oxidative phosphorylation necessary for cellular respiration and ATP production, is not too surprising that mutations in such genes can cause a variety of diseases, many of which with symptoms associated with progressive defects in muscle and nerve systems.

There are two obvious strategies to tackle this problem using gene therapy. One is to introduce the functional copy of the mutated gene into the nucleus. The protein, with an appropriate mitochondrial targeting sequence, can then be transported to the mitochondria. This is the so-called allotropic expression of the gene, and has been shown to produce promising results in culture. Other option is to target the plasmid containing the functional gene into the mitochondria itself, as these organelles contain all the ribosomal RNA and tRNAs necessary for intra-mitochondrial protein synthesis (assuming that these are not the mutated genes). These strategies have been tried out with some success both using a wild type sequence of the mutated gene, or a gene from another species, known to perform its function in the pathway more efficiently (xenotopic expression).

However, as you might have already noticed, a mitochondrial DNA disease is characterised by the fact that not all the mitochondrial genome copies in a cell are necessarily mutated (the presence of different genome types within the same cell is known as heteroplasmy). This has two main implications: firstly, mtDNA diseases are threshold diseases: only when around 60-95% of copies in a cell are mutated will symptoms appear. This threshold will obviously vary depending on the disease. Secondly, rather than trying to introduce a functional copy into the mutated mitochondria, a much more interesting approach can be to just manipulate the relative amounts of the mutated and the wild type populations of mitochondria.

One approach is to promote specific types of exercise training to promote mitochondrial biogenesis. However, as the geneticist that I am, the alternative strategy- to manipulate the mutated mt genome- is much more interesting. A brilliant idea, in my point of view, is to take advantage of the fact that some mutation types introduce restriction sites. That way, rather than trying to express a functional copy of the mutated gene, it may be more successful to express a specific restriction enzyme that will degrade the mutated genome altogether. This is limited, obviously, to those diseases in which a unique new site is introduced, or in those where the number of restriction sites is higher enough in the mutated population to have a significant effect.

Another clever idea is to methylate the mutated mt genome so that it is not expressed, allowing the wild type population to proliferate and overcome the mutated one with time. The use of mitochondrial targeted PNAs (peptide nucleic acids) were a potential method, but were shown not to cross the inner membrane of the mitochondria. However, the use of zinc-finger peptides with DNA sequence specific binding capacities have been used to successfully methylate differently the mutated mitochondrial genome. If these domains are linked to a nuclease, it may be possible to degrade the mutated genome as well.

These are all new strategies, only attempted in cell culture and their usefulness in in vivo models and ultimately in patients is yet to be determined. Nevertheless, I think these novel approaches just show that even standard problems can be approached in innovative ways.


Kyriakouli, D., Boesch, P., Taylor, R., & Lightowlers, R. (2008). Progress and prospects: gene therapy for mitochondrial DNA disease Gene Therapy, 15 (14), 1017-1023 DOI: 10.1038/gt.2008.91

Alexeyev, M., Venediktova, N., Pastukh, V., Shokolenko, I., Bonilla, G., & Wilson, G. (2008). Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes Gene Therapy, 15 (7), 516-523 DOI: 10.1038/gt.2008.11

(the most recent paper I could find in this field, using the restriction enzyme approach in culture)

Wednesday, 16 July 2008

Yet another twist in the world of gene expression - transcription factories

ResearchBlogging.orgWhen I went to a conference on post translational modification and chromatin, one talk really grabbed me, ironically it was the one that didn’t relate to the topic of the conference. I was going to facebook people about it and then remembered this existed.

First of all I will ask you, did you know that transcription only happened at a few sites within the nucleus? In mouse cells from the animal there are between 100-300 of these but in cultured cells such as HeLa cells there are many more. Transcription factories also known as RNAPII foci are where most, if not all mRNA is produced. This amazed me and raises the obvious questions of why and how. The why may be obvious. It is a good idea to keep the nucleus as I see it ‘tidy’ but in more technical terms it is a way of keeps gene expression organised and regulating it (see below). The how this work I don’t think has been addressed! All I can say is watch this space.

Another interesting feature of mammalian gene expression that has come out of these studies is that most ‘active genes’ are not on all the time like classically thought. Most genes appear to show a pulse fashion of expression. Travelling to transcription factories and being expressed then returning to a dormant stage until they travel back to transcription foci for another round of expression and so on. However, some genes show the classical continuous expression pattern and are seen at these sites all the time. One example is Beta-globin. Genes like these are relatively rare compared to those showing the pulse type expression but both are continuously on in there cell type. If elements of the beta-globin promoter is deleted then not only is overall expreesion decreased but it shows the pulse type expression.

One of the most interesting things is that some genes will co-localize to the same transcription factory more often than you expect by pure chance. What is more interesting is these genes can be separated by several megabases! or even be on different chromosome. Using the 3C and 4C assay you can join different pieces of DNA together from different parts of the genome that have transcribed within the same transcription foci. These have shown along with RNA FISH the physical movement of genes to the same place to be transcribed. One of the two Beta-globin alleles within a cell was found to enter the same factory with some other genes at a rate of ~25% with a background rate expected to be less than 2%. These gene’s promoters were searched and a simple motif was found: CCACC. This is actually a binding site for a regulator of Haem biosynthesis and haemoglobhin production called Eklf. When Eklf is knocked out the association between these genes stops! This transcription factor must recruit to these genes to the transcription factories it is located at. I can see two mutually exclusive models here (tell me what you think or extra ones to consider). Either 1) something, perhaps Eklf recruits genes to these sites and physically moves them there or 2) genes with this motif move to all transcription foci and only stay to be transcribed at ones that have Eklf present. I would guess the second is more likely but I see no evidence for either model at this time. Another question I would like answered is whether other or maybe all transcription factors work in this way. I think it is unlikely but perhaps that is the case!

One more interesting thing related to this is the story in B cells of mice. It has been shown that the proto-oncogene Myc is expressed in the same foci as immunoglobulin heavy chain (IgH) a very high proportion of the time. The interesting part is that these two genes have been show in many blood cancers to be translocated to be on the same chromosome so perhaps there is a link between transcription factories and cancer. Well that is what the people doing the research on it want to think. After all related to cancer equal more funding as I told a friend of mine earlier. A new lab has been set up by the post-doc who led most of the described research to explore the cancer link.

And I now realise one very important thing, I should have been writing my result section now I have a deadline as well as lab work, S#£!!%$! Hell!

Cameron S. Osborne, Lyubomira Chakalova, Jennifer A. Mitchell, Alice Horton, Andrew L. Wood, Daniel J. Bolland, Anne E. Corcoran, Peter Fraser (2007). Myc Dynamically and Preferentially Relocates to a Transcription Factory Occupied by Igh PLoS Biology, 5 (8) DOI: 10.1371/journal.pbio.0050192

Cameron S Osborne, Lyubomira Chakalova, Karen E Brown, David Carter, Alice Horton, Emmanuel Debrand, Beatriz Goyenechea, Jennifer A Mitchell, Susana Lopes, Wolf Reik, Peter Fraser (2004). Active genes dynamically colocalize to shared sites of ongoing transcription Nature Genetics, 36 (10), 1065-1071 DOI: 10.1038/ng1423