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Gene Knock-Down Technologies
Element 3BThe Future of Molecular Medicine Seminar 14
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