Emergence and Applications of RNA InterferenceOmar Memon, Vandana Sekhar, Varnika Roy, Yizhou Yin, Alison Heffer

University of Maryland, College Park

More than a decade ago, a surprising observation was made in petunias. While trying to deepen the purple color of these flowers, Rich Jorgensen and colleagues introduced a pigment-producing gene under the control of a powerful promoter. Instead of the expected deep purple color, many of the flowers appeared variegated or even white. This phenomenon was considered to be post-transcriptional gene silencing (PTGS), since the expression of both the introduced gene and the homologous endogenous gene was suppressed.

Years later, experiments in Caenorhabditis elegans by Andrew Fire and Craig Mello revealed that injection of either “sense” or “anti-sense” mRNA molecules encoding muscle protein, led to no behavioral changes in the worms. But when they injected sense and antisense RNA together, they observed that the worms displayed peculiar, twitching movements. Similar movements were seen in worms that completely lacked a functioning gene for the muscle protein.

Fire and Mello tested the hypothesis that injection of sense and antisense RNA molecules resulted in the formation of double-stranded RNA (dsRNA). In every experiment, injection of double-stranded RNA carrying a genetic code led to silencing of the gene containing that particular code. From this, they deduced that dsRNA can silence genes and that this RNA interference is specific for the gene whose code matches that of the injected RNA molecule, and that RNA interference can spread between cells and even be inherited.

Fire and Mello published their findings in the journal Nature on February 19, 1998. Their discovery clarified many confusing and contradictory experimental observations and revealed a natural mechanism for controlling the flow of genetic information. This research awarded them a Nobel prize and heralded the start of a new research field.

HISTORY

Table 1. Advantages and disadvantages of using RNAi in two applications

CRITIQUES

The biggest problem with the use of RNAi is its successful delivery to the target. RNAi must be stable in a cell for prolonged activity without getting degraded. Non-specific interactions can occur because, though siRNA can be designed to target a specific sequence, a difference in one or two base-pairs is sufficient to cause off-target binding.

Table 2. Different delivery methods of RNAi and the advantages and disadvantages of each

The research conducted over past few years has shown the promising potential of RNAi. This powerful genetic tool has been used to a certain level of success in both proteomics and drug therapy. Though both will continue to be active fields of scientific research, the drawbacks must also be considered (Table 1).

RNAi Advantages RNAi Disadvantages

Functional genomics

Finding potential therapy targets

Easy study of gene function

Elucidating cellular mechanisms

Analyze many genes at once; RNAi libraries

Knock-downs can show a variety of phenotypes

Knock-downs do not completely inhibit gene expression or activity

Number of genes targeted at one time limited; RNAi overload

RNAi doesn’t always work

Therapeutics

More specific than most other therapies

Can be used to target many cell and tissue types

Efficiently transfers the gene to its target

Difficult to deliver siRNA to the target

Side effect of activating the interferon response (IR)

siRNA stability is a concern for effective therapy

Concern that the siRNA might interfere with natural RNAi mechanisms in the cell

May bind non-specifically to some tissues

Delivery system

Major characteristics

Advantages Disadvantages

Retrovirus

Promising results in cancer therapy through in vitro cell studies

Very efficient Genes are passed on

during mitosis

Gene is integrated into the genome; risk of mutagenesis

Only for dividing cells

Lentivirus

Related to retroviruses; eliminate some disadvantages

Effective in targeting genes in the brains of Alzheimer’s patients

Applies to non-dividing cells

Virus can be specific for recognizing one type of tissue

Good for carrying larger genes

Can only target specific areas; no systemic applications

Risk of mutagenesis Viral

Adenovirus

Vector based on adeno-associated viruses

Can target tumors For transient

expression

Small risk of host genome integration since replication occurs outside of the nucleus

Since the DNA is outside of the nucleus, it is less stable and can be lost after many cell divisions

Chemically modified

siRNA

Balances stability of siRNA without influencing RNAi mechanisms

Modifications: locked nucleic acids (LNA), phosphothioates (PS), 2’ modifications to ribose

Stability of siRNA is increased

More specific targeting

No in vivo studies on many modified RNAs

5’ modifications might interfere with silencing

Bulky modifications may hinder RNA unwinding

Liposomes

siRNA packaged into an envelope with a signal for target cells

Easy to obtain Targets many different

types of organs

Non-specific targeting Liposome electric

charge on may interfere with tissue uptake

Non-viral

Naked siRNA

siRNA is injected directly into the organism

Accomplished with relatively little work

Encounters RNAses in serum

Delivery to non-specific sites

Taking RNAi from Bench to Bedside- First Trial Treating Age Related Macular Degeneration

MECHANISM1. Introduction of ds RNA in the

cell by viral infection or by artificial means using vectors based short hairpin RNA (shRNA)

2. Recognition and processing of long dsRNA by Dicer, an RNase III enzyme

3. Duplexes of siRNA of 21-24 nucleotides length formed by Dicer

4. miRNA are naturally synthesized long ds RNA in the nucleus, which are processed by Drosha enzyme into small pre-miRNA and exported to cytoplasm.

5. Incorporation of both synthetic siRNA or endogenously expressed miRNA into RNA-induced silencing complex(RISC)

6. Unwinding of duplex siRNA by a helicase in RISC and removal of passenger strand (RISC activation)

7. Recruitment of RISC along with antisense strand to target mRNA

8. Cleavage of target mRNA by an unidentified RNase (Slicer) within RISC. Degrades mRNA at sites not bound by siRNA

ds RNA virus

shRNA

ADP + Pi

RISC

Target mRNA

miRNA

RISC activation

Degraded target mRNA

ds DNA

1

2

34

5

6

7

8

DICER

ATP

ATPADP + Pi

Disease Therapy

APPLICATIONS

Local intravitreal injection of siRNA (100-800µg) per eye diluted in phosphate buffer saline)

siRNA duplex

RISC activation

VEGF target recognized

Target cleaved

Ocular angiogenesis Reduced

SIRNA-027 Target = VEGFR-1

PHENOTYPIC EFFECT

Dose dependent Improvement of Vision

IN VIVO MECHANISMDELIVERY OF DRUG

Common RNAi Targeted Diseases

ONCOGENESIS

•siRNA drugs directly target cancer promoting genes

•Chemotherapeutic avoidance of tumors is decreased by targeting clusterin (antiapoptotic gene).

•Ex:-Imatinib drug for Philadelphia chromosome target BCR-ABL fusion protein causes chronic myelogenous leukemia

NEURODEGENERATIVE DISEASES

•RNAi is an important process in normal neuronal function

•Its manipulation is important for treating many untreatable neurological disorders

• Ex:-Mouse models for Alzheimer's disease, DYT1 dystonia, and polyglutamine disease in progress

VIRAL DISEASES

•Targets are viral and host genes that are essential for entry of the virus

•Hepatitis B and C, Influenza and HIV are common targets

•Ex:- Silencing of the HIV chemokine receptor (CCR5) by RNAi therapy is under trial by Benetic and City of Hope company

Different from classical forward genetics, RNAi is a very powerful technique to investigate gene function in the reverse genetics way. Because of its convenience, high efficiency and economy, it is ideal for analyzing the functions of large numbers of genes and whole genome-wide screens. Based on the completion of sequencing of several organisms and the development of techniques such as cell microarrays, high-throughput RNAi screen is an invaluable tool for functional genomics in a wide range of different species.

Functional Genomics

Step 1 Choose organisms or cell lines

Step 2 Choose RNAi reagents: Long dsRNA, synthetic siRNA, plasmid or viruses based shRNA

Step 3 Screening with some specific paradigm and format

Step 4 Read out and analyze results, microarray can be imaged or stained

Large-scale RNAi screens have been done:

•About 90% genes on C.elegans chromosome III for several basic cellular processes,

•Screen on C.elegans chromosome I for embryonic lethal genes,

•Functional screen for RNAi itself in C.elegans

Meanwhile, high-throughput screens and RNAi libraries have proved to be very useful to therapeutic research

N.benthamiana C.elegans D.melagonaster

Arabidopsis Mouse Human

Use of RNAi in genome-wide screening

Wide therapeutic applications of siRNA are the new sensation in the biotechnology drug world. Major traditional drug targets have been proteins (enzymes and receptors), which are targeted at the post translational level. But siRNA drug selectively silences a disease causing gene, at the post transcriptional level itself. Side-effects are decreased by targeting a disease inducing gene in which genetic polymorphisms distinguish it from the RNA of wild type alleles.

Unlike the antisense approach, dsRNA employs a normal cellular process thus it is more specific and allows a cell-cell spreading of the gene silencing effect. The knockdown of the target gene by RNAi is heritable and stable.

REFERENCESBantounas I, Phylactou L, and Uney J. 2004. RNA interference and the use of small interfering RNA to study gene function in mammalian systems. J. Mol. Endo. 33: 545-57. Beal J, 2005, Silence is golden: can RNA interferance therapeutics deliver?, Drug Discovery Today, 10 (3), 169-172 Bargmann C I, 2001. High-throughput reverse genetics: RNAi screens in Caenorhabditis elegans Genome Biology, 2(2): 1005.1-1005.3Caenorhabditis elegans experimental illustration: Annika RohlEcheverri C J, Perrimon N, 2006.High-throughput RNAi screening in cultured cells: a user’s guide , Nature Reviews Genetics, (7), 373-384Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, and Mello CC. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.Jorgensen RA, Cluster PD, English J, Que Q, and Napoli CA. (1996) Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol Biol 31: 957-973.Leung R K M, Whittaker P A, 2005. RNA interference: from gene silencing to gene specific therapeutics, Pharmacology and Therapeutics, 107: 222-239Li C, Parker A, Menocal E, Xiang S, Borodyansky L, and Fruehauf J. 2006. Delivery of RNA interference. Cell Cycle. 5(18): 2103-9.Lieberman J, Song E, Lee S, and Shankar P. 2003. Interfering with disease: opportunities and roadblocks to harnessing RNA interference. Trends in Mol. Med. 9(9): 397-403.Miller V, Paulson H, and Gonzalez-Alegre P. 2005. RNA interference in neuroscience: progress and challenges. Cellular and Molecular Neurobiology. 25(8): 1195-1207.Shuey D L, Mc Callus D E and Giordano T, 2002. RNAi: gene-silencing in therapeutic intervention, Drug Discovery Today, 7(20): 1040-1046Sonnichsen B, et al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans , (2005), Nature Vol 434(24), 460-469Stevenson M, 2002. Therapeutic Potential of RNA Interference, The New England Journal of Medicine, 351(17), 1772-1777Tuschl T, 2003.Functional genomics RNA sets the standard, Nature Vol.421 16 January, 220-221Wheeler D B, Carpenter A E, Sabatini D M, 2005. Cell microarrays and RNA interference chip away at gene function, Nature Genetics Supplement, (37) 25-30Whelan Jo, 2005. First Clinical data on RNAi, Drug Discovery Today, 10(15), 1014-1015Poster: RNA Silencing, (2005) Science 309, 1518

SUMMARYRNAi is a powerful and attractive genetic approach because of the diversity of its applications. The potential uses currently in progress include the identification of specific gene functions in living systems and creation of genome wide screens. Development of antiviral and anticancer therapies are broadening the horizons of the therapeutic arena.

Another value of RNAi screens is in combining it with other functional genomic assays enabling mapping of biochemical pathways. Impact of RNAi is also being extended to the field of agriculture for example by increasing disease resistance in plants.

Many potential obstacles in the path of RNAi therapeutics can be overcome, but further insight into the non-coding functions of RNA in vivo will provide better understanding of mechanisms underlying RNAi. Future applications of RNAi technology will revolutionize genetic, genomic and proteomic aspects of biology and will take the field of medicine into new scientific realms.