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Q&A: TJU Team Publishes Data on Controllable shRNA-Expression System

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Name: Noreen Hickok

Position: Associate professor, orthopedic surgery, Thomas Jefferson University

Background:

• Assistant professor, orthopedic surgery, Thomas Jefferson University — 1996-2006
• Assistant professor, dermatology, Thomas Jefferson University — 1988-1996
• Postdoc, endocrinology/molecular biology, The Population Council — 1984-1985
• PhD, chemistry, Brandeis University — 1981
• BS, chemistry, Massachusetts Institute of Technology — 1975

Name: James Gilbert

Position: Post-doctoral fellow

Background:

• Postdoc, gene therapy, Louisiana State University Health Sciences — 2002-2005
• PhD, biomedical sciences, University of California, San Diego
• MS, biology, University of New Orleans — 1994
• BS, microbiology, University of Alabama — 1990

Last month, researchers in the lab of Thomas Jefferson University researcher Noreen Hickok published data on the development of a plasmid-based shRNA-expression system with the goal of creating a way of temporally controlling the expression of target genes for tissue engineering applications.

According to the paper, which appeared in the online edition of Plasmid, the expression system was designed using the human pol III H1 promoter, which was supplemented with DNA binding sites for the cartilage-specific transcription factor Sox9.

"The resulting shRNA expression system displays robust, Sox9-dependent gene silencing," the paper's abstract states. "This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression."

Last week, RNAi News spoke with Hickok and the paper's lead author, James Gilbert, about the work.

Let's start with an overview of the lab.

NH: The lab is interested in questions of [therapeutic] implant-associated infections, but when I saw that James needed a home [following Hurricane Katrina] and that he is skilled in human gene therapy, I thought, "The field of musculoskeletal research really needs somebody with the level of sophistication he brings, and that perhaps he could come up with something for cartilage formation that relies on a transient, rather than a permanent, expression of genes."

That's the problem I set for James.

James, where were you coming from when you joined [the lab at] Thomas Jefferson?

JG: I was coming from the Louisiana State University gene therapy program, located in New Orleans. … [The hurricane] basically shut down the gene therapy program there and it destroyed everything I owned. We were evacuated, and I had to begin the process of looking for a new career.

I was very fortunate to end up in Dr. Hickok's laboratory. It's been exciting trying to translate the gene-therapy experience into orthopedic applications.

What were the specific questions you were looking to address [with the work detailed in Plasmid] and how did RNAi come into play there?

JG:
In orthopedics, there is a definite need to facilitate bone repair and soft-tissue repair. And in looking at the developmental pathways involved in these processes, it was evident that we were going to need not only to induce gene expression, but also to silence it at appropriate time points.

[The goal was to develop] a system of controlled [gene] expression and silencing.

Can you give an overview of the system you developed?

JG: The idea … was to replace come of the basal transcriptional elements within the native [human pol III] H1 promoter … with binding elements from cartilage-specific transcription factors.

NH: And you could use the cartilage's own developmental program to tell this [construct] when to turn on or to tamp down the expression of your transfected genes.

What are some of the other specific applications for this system in the lab?

NH: The idea [is] that you have to not only just [drive gene expression], but you also need expression to drop. Many developmental processes are not only dependent on transcription factor induction, but also on the ultimate lack of their presence.

So even though James is focused on cartilage, we could also see this being applicable in many tissue-engineering situations where you'd want to grow a new tissue, and you have to have an initial set of factors that are turned on for induction of the tissue, but then need those turned off in order for normal development to happen.

The way most people in the orthopedics field have approached this is by using a viral vector to turn on gene X, which in bone is usually [a bone morphogenic protein gene], and just let it keep expressing. There are all sorts of deleterious consequences to that. The same applies to cartilage, and nobody has come up with a good cartilage-formation program.

We really thought that … [given] the tool box James was walking in with, if he could actually get tissue-specific, controlled expression of an shRNA, he would be way ahead of the game — we could use this [system] for anything that required some sort of timing of an on-and-off signal.

People [with osteoarthritis] desperately need new cartilage … so it seemed like a logical place to start in a lab like mine, which is very translationally oriented.

Can you talk a little about Sox9, its role, and why that was a focus for the paper?

JG: Sox9 is considered one of the master transcriptional regulators of cartilage development. Its expression is induced by a number of growth factors, including bone morphogenic proteins. This induction of Sox9 expression is what fundamentally drives the development of mesenchymal stem cells into dividing, pre-hypertrophic chondrocytes.

Within this chondrogenic development, there is a precise [moment], which is referred to as the pre-hypertrophic to hypertrophic border … where Sox9 has to be silenced for progression beyond that border to occur. If Sox9 is over-expressed and is not subsequently silenced at that particular phase of development, you end up with an arrest in chondrogenic development.

Obviously, this system could be used for various research purposes, but what about the therapeutic applications?

JG: Endochondral [bone] repair comes immediately to mind. There are a number of long-bone breaks [for which] current therapies are limited to [a surgical reconstruction process called] distraction therapy, which is a very painful process for the patient, and is very time-consuming ... [requiring] a great deal of effort from the patient and the medical team.

[This system] could be applied toward facilitating the repair of these breaks without requiring distraction osteogenesis.

NH: With distraction osteogenesis, you have a bone defect that is too large for the bone to fill it in. But there are also cases where the two ends of the bone, for whatever underlying disease reason, just don't knit back together again — [a situation] called non-union. Both of these systems require formation of cartilage that is eventually replaced by bone. James' system could be used to facilitate union in cases where new bone formation or where non-unions occur.

In theory, would the system be used directly within a patient, or would this be something done ex vivo?

NH: We've discussed both [approaches] and [are currently] viewing it as an ex vivo stem-cell transfection. Let me emphasize that we're not looking for an integration phenomenon; we're looking [at patients] carrying it as an episomal plasmid until it gets diluted out of the system.

What are the next steps?

NH: The paper that is in Plasmid has to do with the model system, which are 293t cells. James, at the moment, has been [testing the system] in a chondrogenic cell line to make sure that, under the real chondrogenic stimuli, you get the sort of regulation you need.

JG: We have some preliminary data, which is hopefully going to be published in the near future, looking at the effects of introducing this expression system into a chondrogenic cell site. Our data at this point suggest that, at the very least, the introduction of this plasmid into these cells seems to be pushing them along this chondrogenic pathway.

NH: Of course, this has got to be nailed down, characterized, and if everything works the way [Gilbert] expects it to work, he would go on and do it in an animal model to see if these transplanted cells would work under those conditions.

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