Therapeutic siRNAs

doi:10.1016/j.tips.2003.11.006

Trends in Pharmacological Sciences

Volume 25, Issue 1, January 2004, Pages 22-28

https://doi.org/10.1016/j.tips.2003.11.006 Get rights and content

Abstract

The ability of small-interfering RNAs (siRNAs) to silence gene expression in somatic mammalian cells has provided researchers with a novel tool to block the expression of disease-causing genes, provided that their mRNA sequences are known. siRNAs can be delivered to cells either exogenously as synthetic agents or endogenously as gene-encoding siRNAs. Recent studies demonstrate the general application of siRNAs to silence gene expression in a range of cell types and in whole mammals. Beyond their value for dissecting gene functions and target validation, siRNAs also hold great potential as gene-specific therapeutic agents.

Section snippets

Mechanism of RNAi

In many cell types long dsRNAs are processed by a host RNase III, termed Dicer, to form siRNAs that contain 21–23 nucleotides [3]. Dicer-processed siRNAs and synthetic siRNAs undergo an ATP-dependent unwinding step before being incorporated into a high-molecular-weight protein complex termed RISC (RNA-induced silencing complex) that contains single-stranded siRNAs 8, 9. However, it is not clear whether the unwinding step takes place before or immediately after the incorporation of the siRNA

Sequence specificity

For siRNA to be a useful drug, it is crucial that it must not cause any effects other than those related to the knockdown of the target gene. This issue is particularly important in therapeutic applications where unwanted side-effects would be undesirable. Although the actual substrate specificity of individual siRNAs appears to be very high 6, 10, recent studies indicate that siRNAs can tolerate single mutations located in the centre of the molecule, and up to four mutations are necessary for

Delivery

Similar to ribozymes, delivery of siRNAs can be achieved by exogenous application of synthetic siRNAs or via a gene therapy approach that relies on the endogenous expression of siRNA from plasmid or viral vectors.

Cancer

siRNA technology can be applied to a wide range of cancers and other proliferative disorders in which aberrant gene expression occurs. Oncogenic and mutant tumour suppressor genes might represent potential targets for the RNAi approach. For example, p53 protein is mutated in 50% of human malignancies. In most cases, this abolishes the function of p53 [33]. Furthermore, mutant p53 trans-dominantly impairs the function of wild-type p53 [33] Interestingly, specific elimination of p53 mutant

siRNA activity in rodent models of human disease

To translate the RNAi technology to medical use, an immediate challenge is to determine the efficacy of siRNAs in living animals. Approximately a year after the description of siRNA technology [6], the first demonstration of RNAi action in whole mammals was demonstrated by McCaffrey and colleagues [51]. They delivered siRNAs by hydrodynamic injection into mice, silencing luciferase gene expression. Similarly, Lewis and colleagues showed that gene expression could be silenced in postnatal mice

Potential regulation of siRNA activity

Advances in molecular engineering have made it possible to create allosteric ribozymes that function as RNA ‘molecular switches’ whose catalytic activities can be controlled by specific effector molecules [63]. These RNA constructs would make excellent candidates for applications that require highly responsive genetic switches. This approach of rational design could be used to create allosteric siRNAs. In certain circumstances, constitutive siRNA expression in cells or tissues is not desirable

Concluding remarks

Taken together, published studies have firmly established the efficacy of siRNA in vitro and in vivo. Although the recent preclinical studies present compelling data on the utility of siRNA in disease models, we will not know the exact potential that RNAi affords for treating human disease until suitable delivery methods are established to enable the performance of clinical trials. A second key issue is the specificity of siRNAs, and the impact that this might have on their safety in humans.

Acknowledgements

The present work is supported in part by the Norwegian Cancer Society.

References (65)

R Jorgensen
Altered gene expression in plants due to trans interaction between homologous genes
Trends Biotechnol.
(1990)
A Nykanen
ATP requirements and small interfering RNA structure in the RNA interference pathway
Cell
(2001)
D.S Schwarz
Evidence that siRNA function as guides, not primers, in the Drosophila and human RNAi pathways
Mol. Cell
(2002)
T.R Brummelkamp
Stable suppression of tumorigenicity by virus-mediated RNA interference
Cancer Cell
(2002)
M Leirdal et al.
Gene silencing in mammalian cells by preformed small RNA duplexes
Biochem. Biophys. Res. Commun.
(2002)
C.R Safinya
Structures of lipid-DNA complexes: supramolecular assembly and gene delivery
Curr. Opin. Struct. Biol.
(2001)
Y Zeng
Both natural and designed microRNAs can inhibit the expression of cognate mRNAs when expressed in human cells
Mol. Cell
(2002)
C Shen
Gene silencing by adenovirus-delivered siRNA
FEBS Lett.
(2003)
M Scherr
Specific inhibition of brc-abl gene expression by small interfering RNA
Blood
(2003)
L Zhang
Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer
Biochem. Biophys. Res. Commun.
(2003)

M Kramer

Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma

Blood

(1998)

D.R Sørensen

Gene silencing by systemic delivery of synthetic siRNAs in adult mice

J. Mol. Biol.

(2003)

M.W.N Deininger

The molecular biology of chronic myeloid leukemia

Blood

(2000)

A.N Houghton

Immunity against cancer: lessons learned from melanoma

Curr. Opin. Immunol.

(2001)

G.A Soukup et al.

Nucleic acid molecular switches

Trends Biotechnol.

(1999)

A Fire

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

(1998)

G.J Hannon

RNA interference

Nature

(2002)

M Montgomery

RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans

Proc. Natl. Acad. Sci. U. S. A.

(1998)

G.C Sen

Viruses and interferons

Annu. Rev. Microbiol.

(2001)

S.M Elbashir

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

(2001)

N.J Caplan

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

Proc. Natl. Acad. Sci. U. S. A.

(2001)

T Holen

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor

Nucleic Acids Res.

(2002)

J.M Jacque

Modulation of HIV-1 replication by RNA interference

Nature

(2002)

Z Mourelatos

miRNAPs: a novel class of ribonucleoproteins containing numerous microRNAs

Genes Dev.

(2002)

A.L Jackson

Expression profiling reveals off-target gene regulation by RNAi

Nat. Biotechnol.

(2003)

T Golub

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring

Science

(1999)

D Semizarov

Specificity of short interfering RNA determined through gene expression signatures

Proc. Natl. Acad. Sci. U. S. A.

(2003)

M Sioud et al.

Systemic delivery of siRNAs

Methods Mol. Biol.

(2003)

F Wincott

Synthesis, deprotection, analysis and purification of RNA and ribozymes

Nucleic Acids Res.

(1995)

J Harborth

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Antisense Nucleic Acid Drug Dev.

(2003)

N.S Templeton

Cationic liposomes as in vivo delivery vehicles

Curr. Med. Chem.

(2003)

T.R Brummelkamp

A system for stable expression of short interfering RNAs in mammalian cells

Science

(2002)

Cited by (129)

Perspectives of using microRNA-loaded nanocarriers for epigenetic reprogramming of drug resistant colorectal cancers
2022, Seminars in Cancer Biology
Epigenetic regulation by microRNAs (miRs) demonstrated a promising therapeutic potential of these molecules to regulate genetic activity in different cancers, including colorectal cancers (CRCs). The RNA-based therapy does not change genetic codes in tumor cells but can silence oncogenes and/or reactivate inhibited tumor suppressor genes. In many cancers, specific miRs were shown to promote or stop tumor progression. Among confirmed and powerful epigenetic regulators of colon carcinogenesis and development of resistance are onco-miRs, which include let-7, miR-21, miR-22, miR-23a, miR-27a, miR-34, miR-92, miR-96, miR-125b, miR-135b, miR-182, miR-200c, miR-203, miR-221, miR-421, miR-451, and others. Moreover, various tumor-suppressor miRs (miR-15b-5b, miR-18a, miR-20b, miR-22, miR-96, miR-139–5p, miR-145, miR-149, miR-197, miR-199b, miR-203, miR-214, miR-218, miR-320, miR-375–3p, miR-409–3p, miR-450b-5p, miR-494, miR-577, miR-874, and others) were found silenced in drug-resistant CRCs. Re-expression of tumor suppressor miR is complicated by the chemical nature of miRs that are not long-lasting compounds and require protection from the enzymatic degradation. Several recent studies explored application of miRs using nanocarrier complexes. This study critically describes the most successfully tested nanoparticle complexes used for intracellular delivery of nuclear acids and miRs, including micelles, liposomes, inorganic and polymeric NPs, dendrimers, and aptamers. Nanocarriers shield incorporated miRs and improve the agent stability in circulation. Attachment of antibodies and/or specific peptide or ligands facilitates cell-targeted miR delivery. Addressing in vivo challenges, a broad spectrum of non-toxic materials has been tested and indicated reliable advantages of lipid-based (lipoplexes) and polymer-based liposomes. Recent cutting-edge developments indicated that lipid-based complexes with multiple cargo, including several miRs, are the most effective approach to eradicate drug-resistant tumors. Focusing on CRC-specific miRs, this review provides a guidance and insights towards the most promising direction to achieve dramatic reduction in tumor growth and metastasis using miR-nanocarrier complexes.
Comparison of effects of dsRNA and siRNA RNA interference on insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii
2020, Gene
RNA interference (RNAi), which employs double-strand RNA (dsRNA) or small interference RNA (siRNA), is a popular reverse genetic manipulation tool to study gene function. Presently, there is few reports on the implementation of RNAi on the insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii. In this study, the effective sequence of siRNA and optimal injection dose were determined, and the effects of RNAi using dsRNA, siRNA, and long-term RNAi were investigated. The results showed that the doses of 0.5 and 1 µg/g of body weight of IAG-siRNA3 produced significantly better inhibition than 0.1 µg/g. qPCR assays showed that both dsRNA and siRNA silenced the IAG expression in five tissues (brain, ventral nerve cord, androgenic gland, testis, and vas deferens) in adult P. clarkii, with the effectiveness decreasing over time, inhibiting the production of spermatid. dsRNA exhibited a longer interference effect than siRNA in adults. For long-term interference (P. clarkii juveniles were injected 7 times with 1 µg/g of body weight of IAG-dsRNA), and found that the secondary sexual characteristics of juveniles were affected, while the control group developed normally. The results of this study could lay the foundation for crayfish sex reversal with IAG RNAi, and provide the reference for those studies in which the technique of RNAi was used.
Design, mechanism, delivery and therapeutics of canonical and Dicer-substrate siRNA
2019, Asian Journal of Pharmaceutical Sciences
Citation Excerpt :
siRNA is a 21–25 nucleotide long dsRNA [23]. Its potential to silence expression of target gene in the somatic tissues of mammals has offered many researchers a new approach to treat genetic-based diseases [5]. siRNA has also been widely deployed as an investigational tool in the validation of useful gene targets.
Upon the discovery of RNA interference (RNAi), canonical small interfering RNA (siRNA) has been recognized to trigger sequence-specific gene silencing. Despite the benefits of siRNAs as potential new drugs, there are obstacles still to be overcome, including off-target effects and immune stimulation. More recently, Dicer substrate siRNA (DsiRNA) has been introduced as an alternative to siRNA. Similarly, it also is proving to be potent and target-specific, while rendering less immune stimulation. DsiRNA is 25–30 nucleotides in length, and is further cleaved and processed by the Dicer enzyme. As with siRNA, it is crucial to design and develop a stable, safe, and efficient system for the delivery of DsiRNA into the cytoplasm of targeted cells. Several polymeric nanoparticle systems have been well established to load DsiRNA for in vitro and in vivo delivery, thereby overcoming a major hurdle in the therapeutic uses of DsiRNA. The present review focuses on a comparison of siRNA and DsiRNA on the basis of their design, mechanism, in vitro and in vivo delivery, and therapeutics.
Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events
2015, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Synthetic nucleic acid carriers – whetherlipid-, dendrimer- or polymer-based – are promising candidates for the treatment of various diseases [1–14].
Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome–NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5–GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5–GFP, we found that at early time points (t < 1 h), only a fraction (≈ 35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5–Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5–GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5–Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes.
Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging
2014, Biomaterials
Steric stabilization of cationic liposome–DNA (CL–DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL–DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL–DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL–DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL–DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σ_M; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σ_M. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σ_M. We present a model describing the interactions between PEGylated CL–DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.
HSP47 siRNA conjugated with cationized gelatin microspheres suppresses peritoneal fibrosis in mice
2012, Acta Biomaterialia
Citation Excerpt :
Their gene-silencing effect is more specific and powerful than either antisense oligonucleotides [11] or dominant negative mutant genes. Although several feasibility studies have been conducted on the therapeutic efficacy of siRNA using animal models for various diseases [12–16], obstacles to siRNA use remain, such as its short period of gene-silencing effect and its low transfection efficiency. Several viral and non-viral vectors have so far been explored for the enhancement of gene transfection efficiency.
Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for the biosynthesis and secretion of collagen and is expressed in the fibrotic peritoneum. In the present study, we evaluated the efficacy of HSP47 small interfering RNA (siRNA) to suppress the development of peritoneal fibrosis induced by chlorhexidine gluconate in mice. We initially confirmed that biodegradable cationized gelatin microspheres (CGMs) containing HSP47 siRNA could continuously release siRNA over 21 days as a result of microsphere degradation. We then determined that a single injection of CGMs incorporating HSP47 siRNA suppressed collagen expression and macrophage infiltration, thereby preventing peritoneal fibrosis. Therefore, we suggest that this controlled-release technology using HSP47 siRNA is a potential treatment for peritoneal fibrosis. Additionally, RNA interference combined with CGMs as a drug-delivery system may lead to new strategies for knocking down specific genes in vivo.

View all citing articles on Scopus

View full text

Trends in Pharmacological Sciences

Therapeutic siRNAs

Abstract

Section snippets

Mechanism of RNAi

Sequence specificity

Delivery

Cancer

siRNA activity in rodent models of human disease

Potential regulation of siRNA activity

Concluding remarks

Acknowledgements

Trends Biotechnol.

Cell

Mol. Cell

Cancer Cell

Biochem. Biophys. Res. Commun.

Curr. Opin. Struct. Biol.

Mol. Cell

FEBS Lett.

Blood

Biochem. Biophys. Res. Commun.

Blood

J. Mol. Biol.

Blood

Curr. Opin. Immunol.

Trends Biotechnol.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

RNA interference

Nature

RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans

Proc. Natl. Acad. Sci. U. S. A.

Viruses and interferons

Annu. Rev. Microbiol.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

Proc. Natl. Acad. Sci. U. S. A.

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor

Nucleic Acids Res.

Modulation of HIV-1 replication by RNA interference

Nature

miRNAPs: a novel class of ribonucleoproteins containing numerous microRNAs

Genes Dev.

Expression profiling reveals off-target gene regulation by RNAi

Nat. Biotechnol.

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring

Science

Specificity of short interfering RNA determined through gene expression signatures

Proc. Natl. Acad. Sci. U. S. A.

Systemic delivery of siRNAs

Methods Mol. Biol.

Synthesis, deprotection, analysis and purification of RNA and ribozymes

Nucleic Acids Res.

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Antisense Nucleic Acid Drug Dev.

Cationic liposomes as in vivo delivery vehicles

Curr. Med. Chem.

A system for stable expression of short interfering RNAs in mammalian cells

Science