At A Glance
Name: Mark Lee
Position: Assistant professor, biology, Spelman College
Background: Research instructor, Winship Cancer Institute, Emory University — 1999-2003; Postdoc, Emory University — 1996-1999; PhD, biochemistry, Clark Atlanta University — 1996; MS, biochemistry, Clark Atlanta University — 1995; BS, chemistry, Morris Brown College — 1990
Though his work is not specifically related to RNA interference, Mark Lee saw the potential for the gene-silencing technology in his work and is exploring other applications on later projects.
Lee recently spoke to RNAi News from his Atlanta, Ga.-based laboratory about how he began using RNAi and where he wants to go with it.
The first question I always start these [interviews] off with is: How did you start getting involved with RNA interference?
Basically, I was given the challenge to develop an antibody to a T-cell receptor called CD45 — it’s a human leukocyte antigen. My work was in a rhesus macaque model of AIDS where antigens are used to distinguish between cells that are considered naïve — have never seen a certain antigen — and cells that are memory cells that would recognize an antigen.
In the macaque model, antibodies were not available for the memory cells because they actually lack certain exons that are expressed. So, any evaluation of a memory response, which would be relevant to a vaccine development or anything that would attest to the immune system’s ability to respond to an antigen, were indirectly monitored by looking at the disappearance of what is termed the RA phenotype. [The RA phenotype] is the expression of all of these genes.
The goal was to figure out a way to generate antibodies to that antigen without, say, three specific genes that were being expressed as extracellular proteins. At the time, all approaches by everyone had come up negative.
I ran across a paper that talked about RNAi as a way to do gene-specific silencing, and it dawned on me that if I could silence each of these exons then I would be able to basically induce the RO phenotype in the cell, which is the isoform O without any of the particular exons.
Let me give a brief explanation: CD45 has about 33 exons in all. Through multiple splicing, it generates different species of CD45 that can either express these glycoproteins termed A, B, and C. If you express all of them, that would be considered the RA. If you express none of them, that would be considered the RO. Again, RA is the naïve phenotype and RO is the memory phenotype.
The goal was to generate antibodies to RO that would recognize rhesus macaque T-cells.
Other people had done sequencing work on CD45 in humans, but none had been done in rhesus macaques. So, my goal was to use siRNA to generate the RO phenotype in the rhesus macaques and then use that as an antigen to synthesize or make antibodies.
Prior to this you hadn’t had any experience with RNAi?
Have you actually started this work?
Yes. The biggest challenge for us was actually getting the sequences.
The sequences, like I said before, were not published. There’s enough variability in the RO between rhesus macaques and humans that the antibody doesn’t recognize the rhesus macaque, which isn’t usually the case but there are certain situations [where it is].
There wasn’t a lot of difference in the nucleotide sequence, but a lot of the antigen specificity for these antibodies is due to glycoprotein processing. So, we also thought about trying to express them in E. coli or CHO cells, but we had concerns about having a differential glycoprotein expression that would also not mimic the native response in the animal.
This was about a year ago when RNAi was much less popular and the companies weren’t developing kits that you could buy to do it. So, the challenge has been to sequence exons 4, 5, and 6, which code for glycoproteins A, B, and C, respectively.
I have actually been using my students at Spelman to do that, and we’ve just gotten the sequences for exons 4 and 6. Exon 5 is giving us a little trouble. We isolated RNA from activated T-cells and use primers from the literature that had been shown to work in the human model to hopefully amplify sequences that would be analogous in the rhesus macaque model.
They did not work. We thought it was the PCR, and I thought it was my undergraduate students that was the problem. Then it finally dawned on me that I might want to redesign primers, so I redesigned the primers specifically for each exon. Then, like magic, each of the bands showed up, except for exon 5. Because exon 5 is between exons 4 and 6, we basically just used primers forward for exon 4 and reverse for exon 6, and vice versa. Now, we have new products that we are about to sequence, and hopefully they will give us something related to a human exon 5.
Now, with those sequences, it should be relatively easy to use [a kit], probably the Ambion kit, to generate double-stranded DNA first. We’re taking the DNA and making double-stranded RNA from that, and then having that sliced by the Dicer enzyme, then hopefully transfecting those species into rhesus macaque T-cells.
It shouldn’t take more than about two or three weeks to do that. I’m actually sending off the sequence for this new product, hopefully, today. I’ll have that by Tuesday, and then from there it’s relatively easy to go forward with the RNA part. We’ll probably have to do some quality control on the transfection for maybe a week or two, then show that we have stable expression or stable knockdown of those genes through some Western blotting and some facts analysis. Then, we’ll hopefully be able to submit that to an antibody company to make some monoclonals.
Given what you’ve seen thus far, and everything you’ve heard about the technology, are there any other applications for it in your work down the road?
Oh, definitely. The umbrella project at the laboratory is the SIV macaque model of AIDS. The other molecule I work with is termed IL-16, [which] is another cytokine that has been shown to inhibit HIV, as well SIV replication. It’s been shown to be inversely proportional to progression towards disease state in macaques, as well as humans. It can help fight against apoptosis, which is programmed cell death. It can help expand CD4+ T-cells. It has all of the functions we would want from an endogenous factor, hopefully without any of the untoward effects of, say, drug cocktails that [HIV/AIDS] patients currently use. But we have to figure out some cell-specific consequences of its expression or its exposure.
One of the things I’d like to do is show its direct correlation in the actual cell that is infected by turning it off and seeing if that actually increases all of the things that IL-16 supposedly decreases — in other words, causes higher replication of virus, higher incidence of apoptosis, lower proliferation of those cells, and the like.
To me, siRNA is probably one of the biggest deals and, I think, one of the biggest benefits of the genome concept because, as a biochemist, I am more interested in proteins than in genes. I always tell my students: ‘You’ve got the gene, but if you don’t have the protein, then who cares?’
So, siRNA, to be, bridges the gap between genomics and proteomics so that you actually use the gene to manipulate the proteome of the cell or system — basically you have more direct analysis of the function of that cell.
Another [project I’d like to get involved in] is sickle cell [disease].
The problem with sickle cell is that you have expression of a mutated hemoglobin. So, if you could use siRNA to turn off hemoglobin S and use bone marrow transplants to turn on hemoglobin A, that would be a way to use gene therapy to cure sickle cell.
That is one I haven’t really spoken out loud about with a whole lot of people, but that’s an application of [RNAi], especially if you know that it’s simply a single nucleotide that is making the biggest difference between health and sickness for a population.
In that way, it’s autologous. You could basically take [a patient’s] bone marrow and transfect it with a vector that would express hemoglobin A, and also transfect it with a vector that would produce siRNA that would inhibit hemoglobin S. In that same model, that person would produce cells that would be expressing the positive hemoglobin and producing siRNA that would inhibit the mutant hemoglobin.
I told my students that the problem [with beginning this project] is that I haven’t been working with [sickle cells] and I would probably seek a proper collaboration. To me, that would be huge.
(Lee noted in an e-mail after the interview that a potential collaborator on this project “would be Greg Evans, Ph.D., a scientist with the sickle cell disease scientific research group of the Blood Diseases Program within NHLBI.”)
It’s easy to talk to people about why RNAi is so great, but do you have any opinion on limitations you see with the technology?
One of the basic limitations is that people are thinking of using it as a direct therapeutic agent for AIDS. But the drawback is that HIV is a highly mutable virus. It mutates very rapidly and siRNA — the good part about it is that it’s specific; the bad part about it is its specific.
If the virus mutates, and you’re directing your siRNA at a viral antigen, then chances are it will only recognize one or two species. As the virus mutates, [the siRNA] will not recognize other forms of it.
That’s a limitation, so it will probably be more advantageous for any siRNA techniques that are applicable to the AIDS model to be directed at cellular targets that the virus is exploiting, but that the cell has in a redundancy. In other words, if there’s a receptor that the cell needs to be infected — like CCR5, which is a co-receptor for HIV, or even CXCR4, which is supposedly correlated with late states of disease — and you could actually direct siRNA to that, then that would be a better target, because that’s not going to be mutable, that’s going to be pretty stable and then the cell will not be infected by late-stage viruses. The person would still be infected, but you would hopefully prevent them from advancing to an AIDS-like disease state.