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Therapeutic siRNA: principles, challenges, and strategies.

Gavrilov K, Saltzman WM - Yale J Biol Med (2012)

Bottom Line: The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo.An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response.This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA. kseniya.gavrilov@yale.edu

ABSTRACT
RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

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Related in: MedlinePlus

Small interfering RNAs (siRNAs) mediate silencing of target genes by guiding sequence dependent slicing of their target mRNAs. These non-coding, silencing RNAs begin as long double-stranded RNA (dsRNA) molecules, which are processed by endonuclease Dicer into short, active ~21-25 nt constructs. Once generated, a siRNA duplex is loaded by Dicer, with the help of RNA-binding protein TRBP, onto Argonaute (AGO2), the heart of the RNA-induced silencing complex (which here is represented just by AGO2). Upon loading, AGO2 selects the siRNA guide strand, then cleaves and ejects the passenger strand. While tethered to AGO2, the guide strand subsequently pairs with its complementary target mRNAs long enough for AGO2 to slice the target. After slicing, the cleaved target mRNA is released and RISC is recycled, using the same loaded guide strand for another few rounds of slicing [12].
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Figure 1: Small interfering RNAs (siRNAs) mediate silencing of target genes by guiding sequence dependent slicing of their target mRNAs. These non-coding, silencing RNAs begin as long double-stranded RNA (dsRNA) molecules, which are processed by endonuclease Dicer into short, active ~21-25 nt constructs. Once generated, a siRNA duplex is loaded by Dicer, with the help of RNA-binding protein TRBP, onto Argonaute (AGO2), the heart of the RNA-induced silencing complex (which here is represented just by AGO2). Upon loading, AGO2 selects the siRNA guide strand, then cleaves and ejects the passenger strand. While tethered to AGO2, the guide strand subsequently pairs with its complementary target mRNAs long enough for AGO2 to slice the target. After slicing, the cleaved target mRNA is released and RISC is recycled, using the same loaded guide strand for another few rounds of slicing [12].

Mentions: In mammalian cells, siRNAs are produced via endonucleolytic processing by the ribonuclease Dicer of exogenously introduced long, double-stranded RNA [12]. Dicer is an endonuclease of the RNase III family (Figure 1); it acts as a “molecular ruler” and precisely produces RNA duplexes ~21-25 nucleotides in length with characteristic termini. The 3’ end carries a dinucleotide overhang, while the 5’ end terminates in a monophosphate group [6,7,8,9,12]. The siRNA duplex length and distinctive ends are necessary features for efficient recognition by and integration into the RISC. Furthermore, recent biochemical studies show that Dicer processing itself is coupled with RISC loading through the tight association of Dicer with TRBP (the human immunodeficiency trans activating response RNA-binding protein) [10,11].


Therapeutic siRNA: principles, challenges, and strategies.

Gavrilov K, Saltzman WM - Yale J Biol Med (2012)

Small interfering RNAs (siRNAs) mediate silencing of target genes by guiding sequence dependent slicing of their target mRNAs. These non-coding, silencing RNAs begin as long double-stranded RNA (dsRNA) molecules, which are processed by endonuclease Dicer into short, active ~21-25 nt constructs. Once generated, a siRNA duplex is loaded by Dicer, with the help of RNA-binding protein TRBP, onto Argonaute (AGO2), the heart of the RNA-induced silencing complex (which here is represented just by AGO2). Upon loading, AGO2 selects the siRNA guide strand, then cleaves and ejects the passenger strand. While tethered to AGO2, the guide strand subsequently pairs with its complementary target mRNAs long enough for AGO2 to slice the target. After slicing, the cleaved target mRNA is released and RISC is recycled, using the same loaded guide strand for another few rounds of slicing [12].
© Copyright Policy - open access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375670&req=5

Figure 1: Small interfering RNAs (siRNAs) mediate silencing of target genes by guiding sequence dependent slicing of their target mRNAs. These non-coding, silencing RNAs begin as long double-stranded RNA (dsRNA) molecules, which are processed by endonuclease Dicer into short, active ~21-25 nt constructs. Once generated, a siRNA duplex is loaded by Dicer, with the help of RNA-binding protein TRBP, onto Argonaute (AGO2), the heart of the RNA-induced silencing complex (which here is represented just by AGO2). Upon loading, AGO2 selects the siRNA guide strand, then cleaves and ejects the passenger strand. While tethered to AGO2, the guide strand subsequently pairs with its complementary target mRNAs long enough for AGO2 to slice the target. After slicing, the cleaved target mRNA is released and RISC is recycled, using the same loaded guide strand for another few rounds of slicing [12].
Mentions: In mammalian cells, siRNAs are produced via endonucleolytic processing by the ribonuclease Dicer of exogenously introduced long, double-stranded RNA [12]. Dicer is an endonuclease of the RNase III family (Figure 1); it acts as a “molecular ruler” and precisely produces RNA duplexes ~21-25 nucleotides in length with characteristic termini. The 3’ end carries a dinucleotide overhang, while the 5’ end terminates in a monophosphate group [6,7,8,9,12]. The siRNA duplex length and distinctive ends are necessary features for efficient recognition by and integration into the RISC. Furthermore, recent biochemical studies show that Dicer processing itself is coupled with RISC loading through the tight association of Dicer with TRBP (the human immunodeficiency trans activating response RNA-binding protein) [10,11].

Bottom Line: The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo.An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response.This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA. kseniya.gavrilov@yale.edu

ABSTRACT
RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

Show MeSH
Related in: MedlinePlus