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Colorado State University's Ramesh Akkina on Silencing CCR5

Name: Ramesh Akkina
Position: Professor, microbiology/immunology/pathology, Colorado State University
Visiting professor, hematology/oncology, UCLA School of Medicine — 1993-1994
Associate professor, pathology, Colorado State University — 1992-1998
Assistant professor, microbiology, Colorado State University — 1986-1992
Postdoc, UCLA School of Medicine — 1982-1985
PhD, veterinary microbiology, University of Minnesota, Minneapolis — 1982
MS, microbiology, University of Agricultural Sciences (India) — 1975
DVM, veterinary medicine, Agricultural University (India) — 1972

This week, researchers from Colorado State University published data showing that they have developed siRNAs that can completely knock down CCR5, a key co-receptor in HIV transmission.
“These siRNAs conferred strong antiviral protection during viral challenge,” the researchers wrote in a paper appearing in the online version of Gene Therapy. “To translate these results to a stem-cell gene therapy-setting, CD34 hematopoietic progenitor cells were transduced with lentiviral
vectors to derive transgenic macrophages. The transgenic cells also exhibited high levels of CCR5 down-regulation and viral resistance.”
RNAi News spoke with Ramesh Akkina, one of the paper’s authors, about the findings.
Let’s start with a little background on your lab and its research focus.
One of our main goals is to [develop] a long-range HIV treatment, and our focus is on … stem-cell gene therapy for HIV. We would like to program blood-forming bone-marrow stem cells with anti-HIV genes such that viral-resistant and immunocompetent T cells are continuously produced in the body, thus counteracting HIV infection. We’ve been working with RNAi since 2002.
In Gene Therapy, you noted that up until now, knocking down CCR5 with RNAi has been tough. So better silencing was the goal of your experiments?
Exactly. … CCR5 is a co-receptor of HIV, and people who … have a deletion of this particular gene are a lot more resistant to HIV infection and [if infected, their disease] progresses very slowly. So people have known CCR5 to be a good target [for HIV] and have been working on [developing drugs targeting the co-receptor], but no one has been able to knock it down [using RNAi] to the level we have achieved.
Can you walk me through the experiments you did and the findings?
CCR5 is a co-receptor that is present on the cells which HIV infects — for example, macrophages and monocytes. … When HIV is transmitted, the viruses that are tropic for CCR5 receptor seem to be more common during the initial stage of infection.
If one can knock down CCR5 on the cells, you have a better chance of inhibiting infection in the beginning stages and prevent transmission.
What we did was put these potent siRNAs [found to be highly effective in silencing CCR5] into a lentivirus vector ... and transduced them into CD34 [hematopoietic progenitor cells]. Then we derived, in vitro, macrophages and monocytes. We found that these particular macrophages are highly resistant to HIV infection.
In summary, we are able to get into stem cells and have particular siRNAs retained throughout the differentiation steps of these particular cells ... while still maintaining the down-regulation of CCR5 on the cell surface. That means the siRNAs are still working as the cell has gone into its final stage as a macrophage.
Then, when we challenge these cells, they are highly resistant to HIV infection.
And you designed the siRNAs in collaborating with Dharmacon?
We did a lot of work on our own, but recently got some help from Dharmacon. We have been collaborating with them for about a year and a half or two. We got their assistance in designing a better siRNA sequence using their new algorithm. However, these needed further refinements as we described in the paper.
Can you describe the siRNAs that were so effective for you, and comment on why you were able to get this significant knockdown compared with other groups?
The design of siRNAs is still kind of an imperfect science — you think you might have a good one, but when you throw it in the cell it may not act as well as you’d expect. So the trial-and-error method is still the way to improve and confirm the efficacy of several candidates generated by algorithms.
People have developed good algorithms to design good siRNAs, and Dharmacon has done a good job of doing this kind of analysis. … But at the same time, I want to caution that [the process isn’t straightforward]. Let’s say you made four siRNAs — even with any design [program], some of them may not work out that well.
In other words, you really have to test out whether they are working properly.
So do you have an idea of what it is about the siRNAs you designed that made them so effective?
Although the current algorithms have become much better in siRNA design, the real intricacies of those things are not yet fully understood. One key aspect in siRNA design is the RNA/protein interaction between the siRNA and the cellular machinery involved in RNAi. 
One of the things we did was make some siRNAs longer and paid attention to the intervening loop structure of the short hairpin. On the other hand, we had some that are kind of short but also worked well. In addition, we experimented with different Pol-III promoters to express the siRNAs. 
We found that these siRNAs, unlike compared to others described previously, were not toxic to the cells even when expressed at higher levels.   
Is that an area of research for you now — better characterizing why these are so effective?
Yes. We are in much better shape now than, say, three years ago, in designing the siRNAs. We also have better insights into the structure of the target, and the nature of siRNAs [in regard to] sequence, target sequence, and so on. Our future goal is to bring these highly effective siRNAs into the clinic. 
The best siRNAs you designed in these experiments, what level of CCR5 knockdown were you getting?
We were getting more than 99 percent knockdown.
And those were the 28-mers described in the paper?
Yeah. One of [the very effective ones] was a shorter one, too.
What’s the next step? In vivo experimentation?
The next step is to really do this stuff in an in vivo system. … We have a [humanized] animal model called a SCID-hu system ... [but] also have a brand new system called a RAG-hu mouse system … that we adapted to do HIV gene therapy.
Recently, we published a paper in Retrovirology saying that this type of model works very well and has the advantage of … multi-lineage human hematopoiesis, which means that we can create T cells, B cells, monocytes, macrophages — all the human white blood cells — in a particular mouse body. We’re in the process of testing some of the siRNA constructs [detailed in Gene Therapy] using both SCID-hu mice and RAG-hu mice.
[Ultimately, we’d like to] go beyond that to clinical trials [wherein] we’d put an siRNA for CCR5 that is highly effective [like the ones described in Gene Therapy] into bone marrow cells from [HIV patients] and put the cells back into the individual so they would differentiate into monocytes and T cells with a lower or absent CCR5.
And you think this kind of therapy would need to be done in conjunction with other HIV treatments?
I think so … [at least] in the beginning. … If things are working well, it may be possible to wean patients off of an HIV drug or reduce the dosage to prevent drug-related toxicities in the long run. But that needs to be tested and evaluated.

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