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Two Publications Raise Questions About In Vivo Efficacy, Safety of siRNA Drugs

Two papers raising questions about the efficacy and safety of RNAi-based therapeutics were published last month, adding to a growing body of literature that cautions all may not be what it seems when it comes to drugs based on the gene-silencing technology.
In the first, which appeared in the online edition of Human Gene Therapy, Tekmira Pharmaceuticals Executive Vice President and CSO Ian MacLachlan and colleagues describe how previously demonstrated antiviral effects of siRNAs targeting influenza in a mouse model were actually due to immune responses triggered by the duplexes, rather than by the RNAi mechanism.
“The potential influence of siRNA-mediated immune responses on key readouts of therapeutic efficacy is a critical consideration when designing and interpreting in vivo RNAi studies,” the Tekmira team wrote. “However, surprisingly few of the reported studies have adequately tested or controlled for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.”
In the second paper, which appeared in the online edition of The New England Journal of Medicine, an international research team reported data indicating that siRNA treatments for wet age-related macular degeneration, including two currently in clinical trials, may accelerate the development of a related condition in individuals with a certain genetic makeup.
This paper, written by the University of California, San Diego’s Kang Zhang and Johns Hopkins University’s Nicholas Katsanis, comes about five months after another group published data indicating that all siRNAs suppress neovascularization, regardless of their sequences or targets, due to the activation of a cellular immune response (see RNAi News, 3/27/2008).
‘Common Deficiencies’
“Over the past five years, many groups have reported on the therapeutic effects of synthetic short interfering RNA in a wide array of animal models for human diseases,” MacLachlan and his colleagues wrote in Human Gene Therapy.
More recently, researchers have shown that siRNAs can trigger the mammalian immune response, primarily through the activation of the Toll-like receptor-7/8 and TLR3 pathways, he noted. But while investigators are well aware of the potential for immune stimulation, oftentimes in vivo RNAi studies fail to appropriately address this issue.
“Common deficiencies in study design include the use of non-responsive cell lines that cannot recapitulate the immune response elicited by profession immune cells; assay time points that likely miss the rapid and transient induction of cytokines; testing for only one class of cytokine to conclude a lack of response; and the selective testing of only one siRNA sequence — either active or control, not both — for immunostimulatory potential,” the researchers wrote.
To demonstrate their point, the Tekmira team refers to demonstrations of the in vivo anti-influenza activity of siRNAs conducted independently by two groups. The first, published by Massachusetts Institute of Technology researcher Jianzhu Chen in The Proceedings of the National Academy of Sciences in 2004, served as the basis for an influenza program initiated by startup Galena (see RNAi News, 10/15/2004), which was later acquired by MDRNA predecessor Nastech Pharmaceutical (see RNAi News, 2/23/2006).

“Surprisingly few of the reported studies have adequately tested or controlled for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.”

The second, which also appeared in The Proceedings of the National Academy of Sciences in 2004, was published by researchers from the US Food and Drug Administration’s Center for Biologics Evaluation and Research.
Using the murine influenza A infection model, MacLachlan’s group was able to replicate the antiviral effects of siRNAs originally reported in PNAS, according to the Human Gene Therapy paper.
“However, through the use of non-immunostimulatory [2’-0-methyl-modified] active siRNA variants and additional non-targeting immunostimulatory control duplexes, we draw the conclusion that the in vivo therapeutic activity of these anti-influenza [siRNAs] is due primarily to immune stimulation and not to specific RNAi in this model,” the team wrote.
Notably, both MDRNA and Alnylam Pharmaceuticals have recently put their respective influenza programs on hold. In 2007, Alnylam CEO John Maraganore said the company had shelved its once-promising flu effort indefinitely amid concerns that “the level of in vivo efficacy we’re seeing [is not] related to specific RNAi effects” (see RNAi News, 8/16/2007).
Last month, MDRNA said that it would not pursue its flu drug “for strategic business reasons” unless it could find a partner for the program (see RNAi News, 8/7/2008).
According to the Tekmira researchers, the misinterpretation of the efficacy data from the two PNAS studies is likely in large part due to the use of a control siRNA sequence targeting green fluorescent protein that has an unusually low capacity to activate immune responses compared with all other formulated siRNAs studied in the experiments detailed in Human Gene Therapy.
Importantly, they added, this GFP siRNA has been used as a control in a number of in vivo RNAi studies.
“It is not clear at present why the GFP siRNA sequence has such unusually low immunostimulatory capacity, or indeed how it has come to be used as a negative control in such a wide variety of in vivo RNAi studies” that do not address the potential for immune stimulation to impact biological readouts, MacLachlan and colleagues wrote.
The Tekmira group concluded that their findings “emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and [call] into question the interpretation of numerous published reports for therapeutic RNAi in vivo.
“The use of chemically modified siRNA with minimal immunostimulatory capacity will help to more accurately delineate the mechanism of action underlying such studies,” they added.
‘Potential Toxic Effects’
Wet AMD has been one of the most attractive indications for RNAi-based therapeutics, in part because of the ease with which drugs can be administered into the eye.
Currently, there are three siRNAs in human trials for the disease: Opko Health’s phase III compound bevasiranib (see RNAi News, 6/28/2007), Allergan’s phase II agent AGN211745, formerly Sirna-027 (see RNAi News, 10/7/2005), and Pfizer’s phase I/II drug PF-4523655, which was licensed from Quark Pharmaceuticals (see RNAi News, 9/28/2006).
All three drugs work, at least in part, by suppressing neovascularization, the hallmark of the disease. However, a research team led by the University of Kentucky’s Jayakrishna Ambati recently published a paper in Nature indicating that all siRNAs, including bevasiranib and AGN211745, are anti-angiogenic due to their stimulation of TRL3 and not because of an RNAi-specific effect.
Now, in NEJM, investigators led by UCSD’s Zhang and Johns Hopkins’ Katsanis and including Ambati have reported that a variant of TLR3 was found in certain individuals to protect against the dry form of AMD, also known as geographic atrophy, which is characterized by extensive atrophy of the retinal pigment epithelium and overlying photoreceptors.
“Since double-stranded RNA can activate TLR3-mediated apoptosis, our results suggest a role of viral dsRNA in the development of geographic atrophy and point to the potential toxic effects of short interfering RNA therapies in the eye,” they wrote.
According to the investigators, it is increasingly believed that perturbed inflammatory cascades cause susceptibility to AMD. “Because of the speculation that microbial and viral entities may provoke the pathologic inflammation that drives [AMD] … we tested for associations between polymorphisms in the [TLR3] gene, which encodes a viral sensor that supports innate immunity and host defense, and the manifestations” of the ocular disease, they wrote.
In cell-culture and animal experiments, the researchers found that one variant of TLR3 is associated with protection against geographic atrophy and that this effect “is probably mediated by a reduction of dsRNA-induced cell death in retinal pigment epithelial cells in vitro and in vivo,” they stated in NEJM.
There was no association found between TRL3 variants and the choroidal neovascularization, suggesting that TLR3 “seems to affect the geographic atrophy phenotype in particular,” they noted.
Speculating that TLR3 activation might promote the progression of an individual already predisposed to AMD toward geographic atrophy, and given the TLR3-stimulatory effects of siRNAs, the investigators caution that patients receiving RNAi-based therapy for wet AMD may be at risk for developing the dry form of the disease as a result of the treatment.
“The problem is that if you happen to be an individual who has the 'wrong' genetic code in TLR3, you might inadvertently trigger a detrimental effect in your retina," Katsanis said in a statement. "You might cure the individual of one thing and increase their risk in something else.”

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