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UMN s Andrew Huang on RNAi and Ophthalmic Disease

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At A Glance

Name: Andrew Huang

Position: Associate professor, director of cornea and refractive surgery service, University of Minnesota

Education: MPH, Johns Hopkins University, School of Public Health — 1982

MD, National Taiwan University — 1981


After completing his ophthalmic residency and clinical corneal fellowship at the Bascom Palmer Eye Institute at the Unvirsity of Miami, Andrew Huang made his way to the University of Minnesota, where he splits his time as a member of the faculty and as a surgeon at the school's Refractive Surgery Center. His research focuses on ocular surface diseases, corneal stem cells, and corneal new vessel formation, and has recently expanded to include the investigation of whether RNAi can be used to treat ophthalmic diseases.

Huang recently spoke to RNAi News about his work.

Could we start with what you do in your lab?

Basically, I'm a clinician scientist. I'm a clinical ophthalmologist. My specialty is cornea and refractive surgery, and my research interest has been on cornea ocular surface disease. In the past, I have done some research on epithelial wound healing, and more recently I have been interested in trying to use gene therapy to manage the conditions [known] as corneal dystrophies — those are hereditary corneal diseases caused by abnormal protein deposits.

What diseases [are considered] corneal dystrophies?

Diseases such as lattice, granular, and avellino dystrophies. It's a big group of diseases, and recently it has been found to be caused by different point mutations on the same gene, [which is] located in chromosome 5 — [the] 5231 [gene].

As it stands now, are there any treatments for these conditions?

At this moment, everything has been geared towards palliative treatment. [For example], you remove the corneal opacities [that] can cause decreased vision and sometimes [lead to] … painful epithelial breakdown. Severe cases need corneal transplantation to restore eyesight. That's the current status.

I'm trying to see if I can combine my knowledge in epithelial biology with gene therapy to interfere with the production of those undesirable protein deposits [associated] with the corneal dystrophies.

Do you have any background in gene therapy?

No. I don't have any formal gene-therapy training, but my research has directed me towards using RNA interference. I [have also] found that the cornea is a very good disease model for knocking down undesirable genes because the cornea is readily accessible for the application of any therapeutic agent. You can devise a solution or drug, and immediately apply it to the cornea. As a result, you can probably ameliorate the corneal dystrophies [with RNAi] while avoiding any systemic complications or toxicities.

Have you worked with RNAi before?

We have some previous experience, but [nothing] extensive. Basically, we design our own … small interfering RNAs, and then we use that to test our hypotheses. The 5231 gene has been well characterized — we were able to use the existing knowledge [about RNAi] to devise several potent small interfering RNAs to knock down the gene. In our earlier corneal dystrophy studies, we were able to identify [and] purify proteins. So for us, [to begin looking at therapeutic uses of siRNAs] is a natural extension of using the small interfering RNAs as a tool.

What kind of work have you done [in this area] at this point?

Primarily in vitro work. We haven't done any clinical work yet.

What about [preclinical] in vivo work?

We haven't done in vivo work because there is no animal model for these dystrophies. Basically, what we've been doing is using various corneal cell lines, and we transfect the cells with a plasmid that will generate either wild-type or mutant proteins … responsible for the corneal dystrophies. We're trying to use our in-house-designed siRNAs to knock down genes to see if we can down-regulate the expression of the proteins.

Are you looking at all these dystrophies, or are you focusing on a particular one first?

All these dystrophies are [associated] with the same gene, so we are using a shotgun approach. We want to see if we can identify one or several unique siRNAs that can [be used as] a universal [therapy]. Even though these are different conditions, they all are classified under one big umbrella, so we're trying to see if we can use one specific or several specific bullets to knock down all undesirable [gene] expressions.

How far along is this work?

We are very comfortable with our protein expression, which is somewhat different that in previous reports. We have a steady expression of proteins, whether they're mutant or wild-type, by our constructed plasmids. Also, we have a fairly stable expression of those proteins in mammalian cells, not [just] in E. coli or prokaryotic cells. The siRNAs that we use are very, very effective in the suppressing the genes in both wild-type and mutant proteins.

What would be the next step? Would you try to develop an animal model?

Several other investigators have tried to either knock out the gene or knock in the mutant genes to see if the phenotype can be expressed. Unfortunately, at this moment, we don't have those kinds of animals to test our siRNAs. But we're trying to [develop] an in vitro model that can duplicate the normal protein deposits in the corneal tissues in an organ-culture system.

When do you think you'll know if that in vitro model is successful?

We envision that in the next six months we'll have a conclusive answer. We've had some preliminary success of depositing the proteins on the corneal stroma. But when you do the organ culture, you want to maintain long-term survival of the cells and stable expression of the proteins before we introduce the siRNAs to knock them down. So, at this moment we are comfortable that in the short-term expression of the protein [we can achieve] short-term knockdown with siRNAs. But we don't really know how frequently we have to [introduce] siRNAs to [achieve long-term] knockdown and how long the cell line will survive. Only time will tell. In a few months, we should have more conclusive evidence.

Do you expect that, in treating these kinds of diseases with siRNAs, a transitory knockdown would be effective? Or would you need continuous, long-term knockdown?

I think, especially for corneal dystrophies, because the [protein] deposits can be observed clinically … that we won't need long-term, continuous, steady suppression. For example, we may be able to knock down expression for a few weeks and then stop the medication. If the conditions improve, we don't need to use the siRNAs [right away]. If, after a few weeks or a few months, the deposits continue to increase, then we would put [the treatment] back. I don't think we would be using these siRNAs to eradicate disease, but as a long-term therapy.

You designed the siRNAs yourself. Do you make yourself or purchase them from a provider?

We use the [oligonucleotide and peptide] synthesis facility at the University of Minnesota. We give them the sequence we want, and the synthesize it for use. We confirm the sequence and insert them into various plasmids to generate hairpin siRNAs.

Are you doing this all within your lab or are you collaborating with anybody?

Primarily, this is within our laboratory. We also [get some help from] a nanoparticle specialist within the university. It's a core facility, so we ask them for help to design a delivery system for us to deliver the siRNAs into the cell in a specific way.

What's this delivery vehicle like?

It's really a nanoparticle, but because we are submitting this in a patent application, I won't be able to disclose too much. Basically, it's a protein-incorporated siRNA delivery [method]. The protein will be delivered intracellularly and fused with the nuclear membrane.

Is there any other RNAi work you are doing?

There is a protein in the eye called myocilin. There is some evidence suggesting that the misfolding of the myocilin can cause an obstruction of the outflow mechanism of intraocular fluid. As a result, the accumulation of intraocular fluid can cause glaucoma.

We are trying to use the same approach [as with the corneal dystrophies] using siRNA to knock down myocilian genes.

Has that work started yet?

We have some preliminary results. It's not as established as the corneal dystrophy work, but we have some results that seem to be fairly promising in the sense that we have several potent siRNAs, and those can effectively knock down the expression of the wild-type myocilin.

Those data are in vitro?

Yes. Everything is in vitro at this stage.

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