Gene Knock-Down Technologies

Element 3BThe Future of Molecular Medicine Seminar 14

steve.white@bris.ac.uk

Dr Stephen White

Why would you want to suppress gene expression?

ToolTo determine gene functionAssess interactions with other proteinsOften in association with overexpression studies

TherapyReduce expression of defective geneLimit replication e.g. oncogenes, hyperproliferativedisordersPrevent viral replication

Gene Knock-Down Technologies

Antisense oligonucleotidesRibozymesDecoy oligonucleotidesRNA interference(Intrabody)

Gene Knock Down Technologies

Gene Knock Down Technologies

Basic factsApplication(s)Summary

Antisense oligodeoxynucleotides (ODN)

Antisense used to block expression of target genesShort stretches of DNA 12-30 bp longComplimentary to mRNA of target geneSelectively hybridise to complimentary mRNA by Watson-Crick base pairing rulesBlocks translation:

a) Passively - prevents ribosomal progressionb) Actively - provides target for RNaseH binding and

mRNA destruction1976 first paper on inhibiting RSV replication

Antisense ODN

Only small stretches of mRNA devoid of interchain hybridisation are available for hybridisation

Model secondary structure to aid target selection

Try multiple antisense ODNs

Target sequence selection

cytoplasm

Nucleus

Antisense ODN

Protected target

Unprotected target

RNaseH

Antisenserecycled

Antisensedegradation

Passive - inhibitionof translation

Active - RNaseH digestion of mRNA

Modifications of ODNs

More resistant to nuclease digestion

Can modify RNaseH activity

Delivery of ODNs

Cellular internalisation of ODNs is inefficient

Cationic lipids frequently used to enhance cell uptake and protect from extracellular degradation

(Possible to also deliver using gene transfer vectors expressing antisense RNA)

Specific Watson and Crick interaction with an intended targetSpecific Watson and Crick interaction with an unintended targetA non-antisense interaction with a nucleic acidAn interaction with a proteinA non-specific effect on proliferation or metabolism (toxicity)

NEED TO HAVE ADAQUATE CONTROLS FOR ALL OF THE ABOVE OFF-TARGET EFFECTS

BIOLOGICAL EFFECTS OF ANTISENSE

Tool to assess gene function

Infectious disease e.g. AIDS

Anti-proliferative therapies

a) Cancer

b) Cardiovascular disease

Application of Antisense

Damage causes:

Inflammation

Smooth muscle cell proliferation

Re-narrowing of arterial lumen

Restenosis

Endothelium

(Neo)Intima

Media

Adventitia

Limiting smooth muscle proliferation prevents restenosis

And c-myb

A, Transverse histological section of control unangioplastied pig coronary artery immunostained for c-Myb. Note the minimal positive staining. l indicates lumen; m, media; and a, adventitia. Original magnification 20. B, Seven days after angioplasty. Numerous c-Myb–positive cells can be seen within the media (m, arrowhead) and are also present within the intima (i, brown). Arrow indicates internal elastic lamina.

Gunn, J. et al. Circ Res 1997;80:520-531

Effect of sense- and AS-ODN-c-myb upon VSMC proliferation in vitro

Figure 3. Effect of sense- and AS-ODN–c-mybupon VSMC proliferation in vitro. Porcine aortic SMCs were cultured in FCS (10%). After 24 hours, they were quiesced with FCS (0.5%) for 48 hours. Proliferation was then stimulated with FCS (10%) in the presence of 0.05 to 10 µmol/L sense- and AS-ODN–c-myb. After 24 hours, [3H]thymidine was added. After a further 24 hours, [3H]thymidine incorporation was assessed by scintillation spectroscopy. Each experiment was performed in triplicate and repeated up to nine times with cells from different animals. Control cells were those not exposed to ODNs. Results of a highly representative experiment are shown in which inhibition of VSMC proliferation, as assessed by [3H]thymidine incorporation, is plotted against concentration of sense- and AS-ODN–c-myb. Half-maximal inhibition is seen at 0.13 µmol/L AS-ODN–c-myb. Ninety percent inhibition was achieved with 5 µmol/L AS-ODN–c-myb. At the same concentration, sense-ODN–c-myb produced 38% inhibition in porcine cells.

Figure 1. The Transport catheter (SciMed). A, A diagrammatic longitudinal section is shown. The catheter is an over-the-wire balloon dilatation and drug-delivery device. Its profile is similar to conventional dilatation balloon catheters. There are three channels: one for passage of a coronary guidewire, a second for inflation of the inner balloon, and a third for infusion of drug via 48 pores in the outer envelope.

Gunn, J. et al. Circ Res 1997;80:520-531

Effect of oversized-balloon angioplasty on porcine coronary arteries: histological changes

Figure 8. Effect of local delivery of AS-ODN–c-myb, sense-ODN–c-myb, and saline delivered via the Transport catheter at the time of PTCA upon porcine coronary neointima formation 4 weeks later. The porcine coronary arteries were explanted. PTCA-only vessels (n=15) were compared with uninjured control vessels (n=14) and with those that had undergone PTCA and local delivery of AS-ODN–c-myb(n=14), sense-ODN–c-myb (n=10), or saline (n=9). Serial cross sections were made at 5-mm intervals. For each vessel, the section with maximum intimal thickness was identified. From this section, the thickness and area of the intima and media were measured by computerized semiautomatedquantitative histology (SeeScan). Results were expressed as intimal-to-medial ratios for CSA, to correct for the differing sizes of the vessels, and divided by the percent breach of the IEL, to correct for the varying degree of trauma. Significance was assessed with the Wilcoxon-Mann-Whitney rank sum test for nonparametric unpaired data.

Summary:

Used antisense against an essential transcription factor needed for cell cycle progression

Inhibition of c-myb induced apoptosis in treated arteries limiting slightly the re-occlusion of the artery (restensosis)

(application of control ODN or PBS caused a more aggressive regrowth response)

This group has gone on to coat stents with the antisense ODN… still awaiting results.

SummaryShort stretches of DNA 12-30 bp longComplimentary to mRNA of target geneSelectively hybridise to complimentary mRNA Blocks translation both Passively and ActivelyCan modify base linkage to increase stabilityWidely used research toolClinical application

Disadvantages:Try multiple antisense ODNs (2° structure)Non-specific effects on other genesSustained in vivo delivery technically difficult and expensiveToxicity?

Antisense ODN

Hammerhead

Consensus of hammerhead ribozyme

6 families of different autocatalytic RNAs

Hammerhead and hairpin groups most frequently utilised

Contain targeting motif capable of selectively binding to target

Once bound, catalytic domain cleaves target

RIBOZYMES

RIBOZYMES

RIBOZYMESTarget sequence cleavage efficiency affected by RNA secondary structure

Use predicted secondary structure algorithms to choose target site

Target

Applications RIBOZYMES

Integration and gene expression from HIV is inhibited by expression of the ribozymes in tissue culture.

RIBOZYMESSummary

6 families of autocatalytic RNAs identified

Capable of specific cleavage of mRNA

Efficiency affected by RNA structure

Emerging tool

Can express ribozymes from gene transfer vector e.g.

Adenoviral or lentiviral vector for extended delivery

Decoy ODNs used to block expression of target genesShort stretches of DNA 10-30 bp longComplimentary to transcription factor binding siteDecoy competes for transcription factor binding and reduces promoter activationBlocks transcriptionWorks for genes where the promoter has an absolute requirement for a particular transcription factor to induce transcription

Transcription Factor Decoys

Receptor

Receptor

Decoy oligodeoxynucleotides(ODN)

Decoy ODN

And c-myb

Dzau et. al. 2002 Nat. Med. 8 p1249

Decoy ODNs

E2F decoy down-regulates these genes essential for cell cycle progression

Decoy ODN

ApplicationE2F decoy infused into vein during bypass graftingPrevented neointimal formationImproved patency

Decoy ODNSummary

Short stretches of DNA 10-30 bp longComplimentary to transcription factor binding siteDecoy competes for transcription factor binding and reduces promoter activation blocking transcription

DisadvantagesOnly applicable for small number of genes or processes which have a specific transcription factor regulating promoter activityDelivery in vivo difficult

RNA interference

What is it?

What functions does it perform?

How can it be used?

Video…

RNA interference

What is it?

Conserved mechanism for specifically turning off gene expression

Found in plants, fungi and animals

Currently it is estimated that vertebrate genomes may encode more than 1000 different miRNAs, which may regulate at least 20–30% of genes.

RNA interferenceWhat functions does it perform?

Regulates gene expression, e.g. loss of function associated with some cancersPart of antiviral defenceSame overall process with species-specific variation

RNA interference

RNAi• Transfect cell with dsRNA

• Dicer cuts into ~21bp duplexes with 3’ overhangs

• RiSC complex forms over duplexes and unwinds to give ssRNA template RNA

• Binding to target RNA induces message cleavage

• Translational repression also induced

Design of siRNAs basic rules:1. siRNA targeted sequence is usually 21 nt in length.2. Avoid regions within 50-100 bp of the start codon and the termination

codon3. Avoid intron regions4. Avoid stretches of 4 or more bases such as AAAA, CCCC5. Avoid regions with GC content <30% or > 60%.6. Avoid repeats and low complex sequence7. Avoid single nucleotide polymorphism (SNP) sites8. Perform BLAST homology search to avoid off-target effects on other genes

or sequences9. Always design negative controls by scrambling targeted siRNA sequence.

The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bases mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes.

Use computer based algorithms to design

Using RNA interference

A. Can introduce synthetic siRNA

B. Can transfect with vector expressing shRNA which is processed to siRNA

RNA interference

SpecificityUnexplained effects on unrelated proteinsMicroarrays

siRNAs can mediate transcriptional gene/ chromatin silencing

Heterochromatin inductionAffects chromosomal regions

Evidence of cell to cell movement

RNA interference

RNA interferenceApplications:

Research Tool

RNA interference

Huntington's disease (HD) is an inherited, autosomal dominant neurological disorder characterized by progressive development of motor abnormalities and cognitive impairments starting in midlife. HD is caused by the expression of an abnormally expanded polyglutamine domain (pQ) in the N-terminus of huntingtin (Htt), which is the product of the Htt gene. The presence of a pQ domain in mutant Htt (mHtt) results in dysfunction and progressive loss of the g-amino butyric acid-producing medium spiny neurons of the caudate and putamen.

Experimental mouse model of Huntington's disease is availableR6/1 transgenic HD mice express exon 1 of human HD with c115 CAG repeatsR6/1 mice display a progressive neurological phenotype that includes clasping of the hind limbs and dyskinesia

shRNAs were designed that could target specific sequences of exon 1 of human HD, but did not have significant sequence similarity to the endogenous mouse Htt mRNA.

neuronal intranuclear inclusions (NII)

In situ hybridization (ISH) analysis performed on coronal sections against the rAAV5-encoded hrGFP mRNA revealed a nonuniform but widespread rAAV5 transduction (A).

hrGFP mRNA was efficiently expressed along the dorsal–ventral and rostral–caudal axis of the striatum.

eGFP fluorescence was also detected (B)

Expression of siHunt-1 or siHunt-2 reduced the level of mRNA for Htt gene as measured by quantitative PCR

Protein level also reduced as measured by western blot

Lead to a mild improvement of disease progression

STILL SOME WAY TO GO…

Applications: Therapeutic Tool

Applications: Therapeutic Tool

Applications: Therapeutic Tool

Lox-1- Marker or active player in atherosclerosis?Involved in uptake of oxidised cholesterol

Can act as a ‘scavenger receptor’

Involved in inflammation

Experiment to determine involvement of Lox-1 in atherogenesis:

Create siRNA for Lox-1

Make shRNA expression vector, express from adenovirus

Instil vector in vivo into carotid artery: see if protects form atherosclerosis

RNA interferenceSummary

Conserved mechanism for specifically turning off gene expression, found in plants, fungi and animalsNaturally regulates gene expression, also part of antiviral defenceCan introduce synthetic siRNACan transfect with vector expressing shRNA which is processed to siRNAsiRNAs can be expressed from some gene transfer vectors e.g. lentiviral vectors, for sustained delivery

Intrabody

Summary of gene knock-down technology

Usually need to try multiple target sequences before achieving good gene silencing.

Secondary structure of RNA can protect sites being targeted.

Need to monitor non-specific effects

siRNA is becoming the most frequently used method.

Reading

NATURE. Vol 457, 22 January 2009, pages 426-433

References:1. Aagaard L, Rossi JJ. RNAi therapeutics: Principles, prospects and challenges. Adv

Drug Deliv Rev 2007; 59: 75-86.2. Bartel DP (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell;

116: 281-297.3. Scherer L, Rossi JJ, Weinberg MS (2007). Progress and prospects: RNA-based

therapies for treatment of HIV infection. Gene Ther; 14: 1057-1064.4. Weiss, B., Davidkova, G., and Zhou, L.W. (1999). Antisense RNA gene therapy for

studying and modulating biological processes. Cell. Mol. Life Sci. 55, 334-358. 5. DOHERTY, E.A., and DOUDNA, J.A. (2001). Ribozyme structures and mechanisms.

Annu. Rev. Biophys. Biomol. Struct. 30, 457-475. 6. Wood, M.J., Trulzsch, B., Abdelgany, A., and Beeson, D. (2003). Ribozymes and

siRNA for the treatment of diseases of the nervous system. Curr. Opin. Mol. Ther. 5, 383-388.

7. Ahn, J.D., Morishita, R., Kaneda, Y., Kim, H.S., Chang, Y.C., Lee, K.U., Park, J.Y., Lee, H.W., Kim, Y.H., and Lee, I.K. (2002). Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia. Gene Ther. 9, 1682-1692.

8. Dallas A, Vlassov AV. RNAi: A novel antisense technology and its therapeutic potential. Medical Science Monitor 2006; 12: RA67-RA74.

9. Saini HK, Griffiths-Jones S, Enright AJ (2007). Genomic analysis of human microRNAtranscripts. Proc Natl Acad Sci U S A; 104: 17719-17724.

RNA interference (Nature insight): http://www.nature.com/nature/insights/7006.html