Dispatches from ARVO 2013: Day 3 - Stem Cells, Knock-ins, and Why Failure Isn't Always a Bad Thing

May 8, 2013

by Jennifer Phillips, Ph.D. 

Day 3 of the ARVO meeting was filled with a lot of presentations on retinal cell biology—nitty gritty details about how tiny molecules are transported hither and yon inside the cell, and what goes wrong if the transportation system breaks down. Mother’s milk for me, but probably not for most of our readers. There were, however, a number of poster presentations that might be of general interest here:

Two stories about the gene CEP290 particularly caught my attention. CEP290 isn’t an Usher gene, but certain mutations in it can cause the non-syndromic retinal degeneration of Leber’s Congenital Amaurosus (LCA). It’s a large-ish gene that encodes a pretty big protein. So, like some of the larger Usher genes, CEP290 presents some challenges for researchers trying to design gene therapies. The first study I learned about, from the Tucker Stem Cell Laboratory at the University of Iowa, created a cell culture from the skin biopsy of an LCA patient with mutations in CEP290. The cultured skin cells were then fed a gene cocktail to induce them to differentiate into photoreceptor-like cells. Once this cultured cells had a molecular profile similar to that of photoreceptors, the researchers used them to test a gene replacement therapy they’d developed. They used a viral vector delivery system that was big enough to hold the complete coding information of the CEP290 gene, as well as some sequence that would tell the cells to activate the gene once the virus had been introduced. From the first experiments designed to get the viral vector + cargo inside the cells, it became apparent that too much of the virus + cargo was toxic to the cells. The researchers have spent a lot of time and effort figuring out the magic number of virus packages that will deliver detectable amounts of the healthy gene products without killing the cells, and testing modified activation sequences that might help the process along. It might not sound like earth shattering news, but it’s another example of using patient-derived cells, which contain the exact genetic mutation in need of therapy, to generate stem cell cultures for this type of study. Furthermore, it’s another reminder of how much time and effort it takes to vet potential therapies before they’re anywhere close to trials in animal models, let alone people. The good news is that, although it’s pretty cutting edge research today, there are more and more people using these techniques every year, which means multiple research groups working the kinks out of the system .

The other story that caught my eye was on the development of another type of therapy for the LCA caused byCEP290. The researchers from Radboud University in the Netherlands are targeting a particular CEP290 mutation that causes abnormal gene splicing. They are taking essentially the same approach as was previously, successfully undertaken by Jennifer Lentz and colleagues with the USH1C 216G>A gene therapy, attempting to reduce the amount of incorrectly spliced gene product with a small molecule designed to interfere with the improper splicing at that precise location. In this, they have succeeded, finding an oligonucleotide that effectively reduces the amount of incorrectly spliced product in cultured cells taken from the skin of an LCA patient with this particular CEP290 mutation (in this case they didn’t induce the skin cells to become any other cell type. Cultured skin cells were sufficient to test this targeted therapy).

So, another potentially therapeutic breakthrough, tailored to the specific nature of the genetic mutation, is on the horizon. The only hindrance at this point is that the second part of the successful research produced by Lentz and colleagues, the creation of a ‘knock-in’ mouse bearing the portion of the human CEP290 gene sequence containing this improperly coded splice site, has proved elusive for the Radboud team. They have indeed succeeded in creating mice with human sequence—and even went the extra step of making two mouse lines—one bearing the healthy human sequence, and one with the sequence containing the erroneous splice instructions. However, both of these mice produced several unexpected splice forms with the human DNA. The mouse bearing the human mutation did indeed make the expected, improperly spliced mRNA, along with some additional splice variants. The oligonucleotide designed to interfere with the mutated splice site would presumably still work on some of thse splice forms, but not all of them. Moreover, the Knock-in mouse with the mutation does not appear to have any retinal defects. Thus, unlike the Ush1c Knock-in mouse story, it will be very difficult to show any effect of the treatment in the Cep290 knock-in mice. 

I guess the take home message from all this is that biology is full of surprises. Experiments take unexpected turns. Presumably simple techniques become unimaginably complex with the addition of a single new variable. It’s hard work, and sometimes, despite the most diligent efforts, experiments fail. Take heart, though. Most scientists learn far more from their failures than they do from their successes.

My itinerary for the penultimate day of the conference is filled with immensely interesting presentations, including one from the Tucker Stem Cell lab on USH2A. Come back tomorrow and I’ll tell you all about it. 

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