Dispatches from ARVO: Day 3

May 4, 2011

By Jennifer Phillips, Ph.D.

Today's cool Usher science story comes from Kate McCaffrey and colleagues at Rosalind Franklin University, who are making some interesting discoveries about a new potential therapy for Usher type 1C. 

In the past when we've talked about gene replacement therapy, the focus has been to find ways of replacing a gene product that is absent or faulty in some way. The exact nature of the mutation causing the absence or faultiness doesn't usually matter-as long as you know which gene is affected, substituting a good copy should solve the problem.

Be that as it may, Usher gene replacement has barely gotten going in animal models yet, let alone human patients. And, as we've discussed, some Usher molecules will likely be very difficult to work with, due to their large size or other constraints. 

Another approach to this that has met with some preliminary success in the hands of McCaffrey et al. is to target a particular mutation for correction, rather to replace the whole gene. The mutation in question is the USH1C G216A mutation, which is prevalent in Acadian populations as well as in people with Ashkenazi Jewish ancestry[this is only common in Acardian populations as far as we can tell]. This is a mutation that alters the normal splicing of the USH1C gene. 

So what is 'normal splicing' exactly? Briefly, splicing occurs in the transitional molecule between the DNA code housed in the nucleus and the protein product that goes out into the cell and does stuff. The transitional molecule is called messenger RNA, and its job is to faithfully copy the genetic code from a particular address on the chromosome and then serve as the blueprint for assembling the protein. But before it can provide a coherent recipe, the content has to be condensed. 

The information on the chromosome that codes for the protein, called exon sequences, is interspersed with other DNA sequence that isn't part of the instructions, called intron sequences. When the messenger RNA is first synthesized, it contains both exonic and intronic bits, which need to be excised before the message will make any sense as protein building instructions. Think about an article on how to build a gazebo in a Home Improvement magazine. If the instructions were spread out on consecutive pages, it would be straightforward to build. But if after every page or two there were instructions for a different project, or page after page of advertising, you would probably either include extra, incorrect steps in your building project or perhaps abandon it altogether, leaving it unfinished and non-functional. 

Fortunately, the cellular machinery that processes messenger RNA is pretty clever about locating the parts that need to be deleted (or, if you will, the pages that need to be torn out) and then splicing together the ends of the exons so that the instructions are arranged in one continuous string of code. How does this machinery know how to tell exons from introns, you might ask? It's all in the code! DNA base sequences indicate where the junctions are between the parts to keep and the parts to delete. 

So, sorry for the protracted explanation (I'm not doing to well at 'short and pithy', am I? ) but if you followed it, it now shouldn't come as much of a surprise that while mutations in the protein coding, exonic sequence of Usher genes can and do lead to Usher syndrome, so can mutations affecting the splice sites of these genes. The G216A mutation is an example of this. It creates an extra splice site in the middle of the code, which leads to some of the exon sequence being lost and the protein being incomplete, which in turn leads to a severe form of Usher syndrome. 

As it happens, we molecular biologists have several tools for inhibiting splicing at a particular site on a messenger RNA molecule. We can generate small molecules that physically interfere with the splicing machinery, blocking a particular part of the code that indicates 'splice here' such that it will skipped over while the splicing machinery moves on to the next splice site. Sometimes we do this when we want to interfere with normal splicing and create a poorly constructed protein, but in this case, McCaffrey and colleagues used it to block that mutated extra splice site, G216A. They tested their interfering molecule in cell cultures containing the human USH1C G216A mutation and in a mouse model containing the same mutation. When they did this, they found that splicing at the wrong site caused by the mutation was effectively blocked, and both the human cells in the dish and the tissues they examined from the mouse were able to revert back to making the normal USH1C protein again. 

The research presented was mostly a test of whether or not this technique would work at the molecular level, and it did, rather impressively. More work is needed to decide how and when to deliver a dose of this molecule to the affected tissues in the mouse model, and then to examine the effects on the observed hearing and vision defects, but it's a great start.

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