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

University of Maryland, College Park

HISTORY

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.

MECHANISM

ds RNA virus 1. Introduction of ds RNA in the cell by viral infection or by artificial means using vectors based short hairpin RNA (shRNA) 2 4 miRNA ds DNA 1 8 2. Recognition and processing of long dsRNA by Dicer, an RNase III enzyme shRNA 3 6 7 DICER 3. Duplexes of siRNA of 21-24 nucleotides length formed by Dicer ATP ADP + Pi 5 RISC 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.

ATP 5. Incorporation of both ADP + Pi synthetic siRNA or RISC activation endogenously expressed miRNA into RNA-induced silencing complex(RISC) Target mRNA 6. Unwinding of duplex siRNA by a helicase in RISC and removal of passenger strand (RISC activation) Degraded target mRNA 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

APPLICATIONS

Functional Genomics

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.

Step 1 Choose organisms or cell lines

N.benthamiana C.elegans D.melagonaster

Use of RNAi in genome-wide screening

Step 2 Choose RNAi reagents: Long dsRNA, synthetic siRNA, plasmid or viruses based shRNA Step 3 Screening with some specific paradigm and format

CRITIQUES

Functional genomics Therapeutics RNAi Advantages



Finding potential therapy targets

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Easy study of gene function

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Elucidating cellular mechanisms

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Analyze many genes at once; RNAi libraries

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Knock-downs can show a variety of phenotypes



More specific than most other therapies



Can be used to target many cell and tissue types



Efficiently transfers the gene to its target RNAi Disadvantages

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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

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Difficult to deliver siRNA to the target

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Side effect of activating the interferon response (IR)

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siRNA stability is a concern for effective therapy

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

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May bind non-specifically to some tissues 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).

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

Arabidopsis Mouse Human 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,

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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

Disease Therapy

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.

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

DELIVERY OF DRUG Local intravitreal injection of siRNA (100 800µg) per eye diluted in phosphate buffer saline) siRNA duplex IN VIVO MECHANISM RISC activation VEGF target recognized Target cleaved PHENOTYPIC EFFECT SIRNA-027 Target = VEGFR-1 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

Viral Non viral

Delivery system Retrovirus Lentivirus Adenovirus Chemically modified siRNA Liposomes Naked siRNA Major characteristics



Promising results in cancer therapy through in vitro cell studies

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Related to retroviruses; eliminate some disadvantages



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



Vector based on adeno associated viruses



Can target tumors



For transient expression



Balances stability of siRNA without influencing RNAi mechanisms

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



siRNA packaged into an envelope with a signal for target cells



Applies to non-dividing cells



Virus can be specific for recognizing one type of tissue



Good for carrying larger genes



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



Stability of siRNA is increased



More specific targeting



Easy to obtain



Targets many different types of organs



siRNA is injected directly into the organism

  

Advantages Very efficient Genes are passed on during mitosis Accomplished with relatively little work

  

Disadvantages



Gene is integrated into the genome; risk of mutagenesis



Only for dividing cells



Can only target specific areas; no systemic applications



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



No in vivo studies on



many modified RNAs 5’ modifications might interfere with silencing



Bulky modifications may hinder RNA unwinding Non-specific targeting Liposome electric charge on may interfere with tissue uptake



Encounters RNAses in serum



Delivery to non-specific sites

SUMMARY

RNAi 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.

Ocular angiogenesis Reduced Dose dependent Improvement of Vision ONCOGENESIS

•

siRNA drugs directly target cancer promoting genes

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

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

Common RNAi Targeted Diseases

NEURODEGENERATIVE DISEASES

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RNAi is an important process in normal neuronal function

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Its manipulation is important for treating many untreatable neurological disorders

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

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

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Hepatitis B and C, Influenza and HIV are common targets

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

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