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Intronn s Colette Cote on Combining RNAi and Pre-Trans-Splicing Molecules

Colette Cote
Program director

Name: Colette Cote

Position: Program director, Intronn

Background: Postdoc, National Institutes of Health — 1998-2002

PhD, molecular biology, Brown University — 1999

BA, biology, Rhode Island College — 1992

Intronn is a company developing RNA trans-splicing technology for use in therapeutics and molecular imaging. Recently, the company began exploring the use of its technology in conjunction with RNA interference.

Collete Cote, a program director at Intronn, spoke with RNAi News this week about the company and how RNAi has become part of its research and development efforts.

Could you give an overview of Intronn?

Intronn has a proprietary technology called SMaRT, or spliceosome-mediated RNA trans-splicing. The concept of that is to go in and essentially reprogram genes, but at the RNA level. We build these pre-trans-splicing molecules — we call them PTMs — that have an upstream binding domain containing sequences complementary to the targeted pre-mRNA, followed by splicing elements including a branch point and polypyrimidine tract, and a downstream coding domain. We can pretty much target any RNA or pre-mRNA that has an intron in it, and we can trans-splice in our cargo of choice — for gene therapy, it can be [used] to replace defective exons, for instance. Basically, we can trans-splice to a target molecule and change the coding region of that to produce chimeric molecules for therapeutics or other applications.

Our basis for the company is to develop PTMs for different applications. [This includes] gene therapy, [in which] we have a few different programs, [but] we try to [develop] some alternative uses for our technology and … we have another program in genomics that we're looking at right now for uses of our PTMs to annotate splicing junctions — intron/exon junctions. So that's the platform, and everything we build is built off of a PTM and how we use that to reprogram something.

How long has the company been around?

The company has been around for quite awhile — since 1996. It had a sort of earlier life in Raleigh/Durham, North Carolina. It was then moved up here [to Gaithersburg, Md.] in 2002.

Is the primary focus the gene therapy work?

There is a large focus on it right now. We have a gene therapy program for dyslipidemia, one for hemophilia A, and we have one for alpha 1 anti-trypsin, but that's sort of in the background. Then we have these secondary programs, like the genomics program and the SMaRT RNAi program.

So we are an RNA therapeutics company.

The RNAi stuff. Can you give an overview of that?

It started with our alpha 1 anti-trypsin program. Just to give you a little background, alpha 1 anti-trypsin [deficiency] is a disease with the frequency similar to cystic fibrosis in the population. The most severe patients — we call these PiZ homozygotes — have a defective alpha 1 anti-trypsin protein. Basically, there's a single amino acid change that misfolds the protein. Alpha 1 anti-trypsin is synthesized in the liver, secreted into the bloodstream, and then it works in the lungs as a protease inhibitor that will keep neutrophil elastase, which will degrade your lung tissues, in check.

In alpha 1 anti-trypsin deficient patients, there are a number of different genotypes, but the basic phenotype is that the AAT protein is misfolded, [and] it aggregates in the [endoplasmic reticulum] of the hepatocytes, so not enough gets into the bloodstream and not enough gets into the lungs. So these patients develop emphysema and other chronic obstructive pulmonary diseases. But there's also an alternative phenotype — because this protein gets built up in the liver, you start seeing carcinomas and hepatic injuries.

We had thought … we could use our PTMs to do the double whammy for alpha 1 anti-trypsin; that is, for every defective pre-mRNA that we could re-program to the corrected form, not only will we help get more out into the bloodstream, but we'll also prevent the aggregation in the liver.

That was the basis for that, but then we thought we could take things one step further. Is there another way that we can help accentuate the amount of reduction in the defective protein that we see? That's where the SMaRT RNAi came in — can we develop a PTM that not only reprograms the PiZ to the PiM form, which is the wild-type form of this, but also carries an RNAi molecule that targets the Z form? Would that help knock that down so you have even less [AAT protein] accumulating in the liver? That was the first thinking that got us into the concept of melding SMaRT technology with RNAi technology.

One of the biggest challenges that is out there for the siRNA/miRNA world is to try to keep these molecules in check so you don't have off-target effects. The more we started thinking about this, the more we realized that we can actually use our PTMs to do conditional expression. We would actually design [RNAi molecules] into our PTM so that they'd be more of a microRNA-type molecule that would then get recognized and processed out to form a functional microRNA that would then hit a target.

So if we design these in such a way that our PTM, upon trans-splicing to the correct target, would form, for instance, a miR-30-like structure that would then be recognized by the RNAi machinery to form a functional microRNA, that would be great. If our PTM trans-splices to the wrong target, the potential base-pairing interaction (within the final trans-spliced product) to form that structure should be very minimal, so in essence you'd keep your microRNA silent in the absence of trans-splicing to the correct molecule. Because both the target being trans-spliced into and the PTM contribute sequences to the formation of the final miR-30 structure, the expression of the miRNA is made absolutely dependent upon the PTM trans-splicing to the correct target.

So you're trying to create molecules that mimic microRNAs?

Correct. And we're trying to do it in a conditional way. The nice thing about PTMs is that, in and of themselves, they are not necessarily functional in the context of gene expression. They pick up all of the endogenous regulation of the target they trans-splice to. So a PTM floating around by itself would be innocuous. So [with SMaRT RNAi], not only can we express a microRNA that hits a certain target, but we can keep it quiet until it's in the right cell that is expressing the right target for it.

The nice thing about it is you can start getting creative about what you want to target for trans-splicing versus what you want to target for silencing. So, in essence, you can use this almost as a suicide gene therapy. For instance, if you have — and I'm just using this as an example — an HPV-infected cell … and an adjacent cell that's not infected, if you carry in a PTM that's going to trans-splice specifically to HPV, even if that PTM trans-splices [to an off-target] in the non-infected cell the final trans-spliced product should not form the correct sequence combination/structure to produce a functional microRNA. In an HPV-infected cell, with that target there [the trans-spliced product] forms a very specific hairpin that would then be recognized by the RNAi machinery.

You could have that microRNA target HPV, or you could have that microRNA target an essential gene in the cell … one that would cause cell death. Then you could have targeted cell death.

Specifically, what sort of applications are you looking at this for?

This is more a proof of principle for us to see if this will work as a mechanism to conditionally produce microRNAs. We're sort of leaving it out there. Suicide gene therapy is one thing, but … it all depends on how you want to apply it. I think there's a wide application for this, but in our hands right now it's proof of principle.

Do you have an idea when you'd expect to know if this works or not?

We've been working on it for quite awhile, and the design phase is a huge chunk of the work because, unlike the RNAi field where you can library screen to find the optimal siRNA or miRNA sequences, we're defined by the targets that we trans-splice that sequence into. We're doing a lot of basic QC to make sure that … if you hit this exon, you'll form the right structure and that sort of thing. So I can't give you a timeframe — we have a lot more trial and error to pick out optimal sequences than someone in the RNAi field.

And the work at this point is in vitro?

Yeah. We're at an early phase of this project.

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