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A status report on RNAi therapeutics.

Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, Meyers R, de Fougerolles T, Maraganore J - Silence (2010)

Bottom Line: More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics.The implications were profound.One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alnylam Pharmaceuticals Inc,, 300 Third Street, Cambridge, MA 02142, USA. avaishnaw@alnylam.com.

ABSTRACT
Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.

No MeSH data available.


Related in: MedlinePlus

Profiling immunostimulatory small interfering (si)RNAs. (a) A panel of siRNAs including negative and positive controls were evaluated in an in vitro human peripheral blood mononuclear cell (PBMC) assay as described previously [10] with supernatants examined for tumor necrosis factor (TNF) (left panel) and interferon-α (right panel) levels. The right-hand side of each panel has a chemically unmodified siRNA, which is compared with the same sequence after incorporation of a combination of phosphorothioate and 2'-O-methyl chemical modifications. (b) The left-hand panels show a series of immunostimulatory (A-D) and non- immunostimulatory (X-Z) siRNAs evaluated in an in vitro PBMC assay. The right-hand panel shows plasma cytokine profiles in mice injected intravenously with the same siRNAs formulated in LNP01 [26]. Common immunostimulatory siRNAs are identified by the in vitro and in vivo assays.
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Figure 4: Profiling immunostimulatory small interfering (si)RNAs. (a) A panel of siRNAs including negative and positive controls were evaluated in an in vitro human peripheral blood mononuclear cell (PBMC) assay as described previously [10] with supernatants examined for tumor necrosis factor (TNF) (left panel) and interferon-α (right panel) levels. The right-hand side of each panel has a chemically unmodified siRNA, which is compared with the same sequence after incorporation of a combination of phosphorothioate and 2'-O-methyl chemical modifications. (b) The left-hand panels show a series of immunostimulatory (A-D) and non- immunostimulatory (X-Z) siRNAs evaluated in an in vitro PBMC assay. The right-hand panel shows plasma cytokine profiles in mice injected intravenously with the same siRNAs formulated in LNP01 [26]. Common immunostimulatory siRNAs are identified by the in vitro and in vivo assays.

Mentions: Sequence-independent off-target effects refer to either saturation of the endogenous RISC machinery [38,39] or to the immunostimulatory potential of siRNAs [48]. Single-stranded and double-stranded RNAs, particularly chemically unmodified sequences, can stimulate the innate immune system via Toll-like receptor (TLR)-3 [14], TLR-7/8 [40-42] and non-TLR pathways, such as retinoic acid inducible gene (RIG)-I [49] or PKR [50]. Cytokine induction can contribute to target suppression via an RNAi-independent mechanism [14,51]. Eliminating this proinflammatory liability is therefore crucial from both a safety and efficacy perspective. Tractable in vitro [10,40] and in vivo [41,52] preclinical assays exist, which can assess the proinflammatory potential of an siRNA. Similar approaches were validated when proinflammatory DNA oligonucleotides acting as TLR-9 agonists were studied preclinically and then in clinical practice [53,54]. From a practical viewpoint, because there is no a priori knowledge as to which pathway might be engaged for any candidate siRNA, a wide range of inflammatory markers should be evaluated during lead selection, including type I and II interferons, and cytokines and chemokines induced by TLR-3, TLR-7 and TLR-8 agonists. The use of appropriate control siRNAs is crucial in immunostimulatory screening assays, and a widely used, chemically modified green fluorescent protein (GFP) sequence has served as an important negative control for many groups [51]. In certain cases, a given sequence with proinflammatory potential might be very desirable for potency or other considerations. Fortunately, both increased stability and reduced pro-inflammatory liabilities can be achieved simultaneously by chemical modifications (Figure 4). Again, the process of modification is empiric and requires subsequent confirmation that potency has been maintained. It is to be hoped that greater knowledge of TLR recognition will enhance bioinformatic exclusion of proinflammatory sequences and guide chemical modification strategies to specific motifs [40,41,55], increasing overall screening efficiency and throughput.


A status report on RNAi therapeutics.

Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, Meyers R, de Fougerolles T, Maraganore J - Silence (2010)

Profiling immunostimulatory small interfering (si)RNAs. (a) A panel of siRNAs including negative and positive controls were evaluated in an in vitro human peripheral blood mononuclear cell (PBMC) assay as described previously [10] with supernatants examined for tumor necrosis factor (TNF) (left panel) and interferon-α (right panel) levels. The right-hand side of each panel has a chemically unmodified siRNA, which is compared with the same sequence after incorporation of a combination of phosphorothioate and 2'-O-methyl chemical modifications. (b) The left-hand panels show a series of immunostimulatory (A-D) and non- immunostimulatory (X-Z) siRNAs evaluated in an in vitro PBMC assay. The right-hand panel shows plasma cytokine profiles in mice injected intravenously with the same siRNAs formulated in LNP01 [26]. Common immunostimulatory siRNAs are identified by the in vitro and in vivo assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908561&req=5

Figure 4: Profiling immunostimulatory small interfering (si)RNAs. (a) A panel of siRNAs including negative and positive controls were evaluated in an in vitro human peripheral blood mononuclear cell (PBMC) assay as described previously [10] with supernatants examined for tumor necrosis factor (TNF) (left panel) and interferon-α (right panel) levels. The right-hand side of each panel has a chemically unmodified siRNA, which is compared with the same sequence after incorporation of a combination of phosphorothioate and 2'-O-methyl chemical modifications. (b) The left-hand panels show a series of immunostimulatory (A-D) and non- immunostimulatory (X-Z) siRNAs evaluated in an in vitro PBMC assay. The right-hand panel shows plasma cytokine profiles in mice injected intravenously with the same siRNAs formulated in LNP01 [26]. Common immunostimulatory siRNAs are identified by the in vitro and in vivo assays.
Mentions: Sequence-independent off-target effects refer to either saturation of the endogenous RISC machinery [38,39] or to the immunostimulatory potential of siRNAs [48]. Single-stranded and double-stranded RNAs, particularly chemically unmodified sequences, can stimulate the innate immune system via Toll-like receptor (TLR)-3 [14], TLR-7/8 [40-42] and non-TLR pathways, such as retinoic acid inducible gene (RIG)-I [49] or PKR [50]. Cytokine induction can contribute to target suppression via an RNAi-independent mechanism [14,51]. Eliminating this proinflammatory liability is therefore crucial from both a safety and efficacy perspective. Tractable in vitro [10,40] and in vivo [41,52] preclinical assays exist, which can assess the proinflammatory potential of an siRNA. Similar approaches were validated when proinflammatory DNA oligonucleotides acting as TLR-9 agonists were studied preclinically and then in clinical practice [53,54]. From a practical viewpoint, because there is no a priori knowledge as to which pathway might be engaged for any candidate siRNA, a wide range of inflammatory markers should be evaluated during lead selection, including type I and II interferons, and cytokines and chemokines induced by TLR-3, TLR-7 and TLR-8 agonists. The use of appropriate control siRNAs is crucial in immunostimulatory screening assays, and a widely used, chemically modified green fluorescent protein (GFP) sequence has served as an important negative control for many groups [51]. In certain cases, a given sequence with proinflammatory potential might be very desirable for potency or other considerations. Fortunately, both increased stability and reduced pro-inflammatory liabilities can be achieved simultaneously by chemical modifications (Figure 4). Again, the process of modification is empiric and requires subsequent confirmation that potency has been maintained. It is to be hoped that greater knowledge of TLR recognition will enhance bioinformatic exclusion of proinflammatory sequences and guide chemical modification strategies to specific motifs [40,41,55], increasing overall screening efficiency and throughput.

Bottom Line: More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics.The implications were profound.One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alnylam Pharmaceuticals Inc,, 300 Third Street, Cambridge, MA 02142, USA. avaishnaw@alnylam.com.

ABSTRACT
Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.

No MeSH data available.


Related in: MedlinePlus