Inhibition of gene expression by means of nucleic...

Prof. Józef Dulak, PhD, DScDepartment of Medical Biotechnology

Faculty of Biochemistry, Biophysics and BiotechnologyRoom 3.025/3.07 Phone 664-63-75

Email: jozef.dulak@uj.edu.pl

15th December 2014

Inhibition of gene expressionby means of nucleic acids

Lecture 9

Genetic therapy

Acquired diseases

Inherited diseases Acquired diseases

Gene addition Gene inhibition Gene repairSubstitutingdelivery of

proper version of mutated gene

Enhancingdelivery of gene which

expression isimpaired

Suppresivedelivery of gene killing

the cells

Silencing ofgene expressionwith non-coding

nucleic acids(antisenseDNA decoyssiRNA/shRNA

microRNA)

Correction ofthe mutated

gene

DNA RNA

Genes(delivered by DNA vectors)

Non-coding sequences

Antisense oligonucleotides

DNA decoys

Genes(delivered by RNA vectors)

Non-coding sequences

ribozymes siRNAmicroRNA

Therapeutic nucleic acids

3

Watson-Crick based-dependent hybridisation is crucial for the inhibitory activity of several classes of inhibitory nucleic acid

However, this is not the only way in which nucleic acids caninhibit gene expression

4

Hybridisation of nucleic acids as a way to inhibitgene expression

DNA hybridizeswith DNA DNA hybridizes

with RNA RNA hybridizes

with RNA

U

U

U

U

U

U

U

U

5

Inhibitory nucleic acids

1. Antisense oligonucleotides: short, 12-20 nts. 2. Triple helix-forming oligonucleotides (TFO)

pyrmidine oligodeoxynucleotides that specifically bind to a major groove of polypurine region of dsDNA via the formation of triple helices according to recognition rules established by Hoogsten

3. Ribozymes – short catalytically active RNSs4. Deoxyribozymes (DNAzymes) – short catalytic DNA that

cleave sequence-specifically targeted RNA. More stable than RNA, it is easier to synthesize and to modify them.

5. siRNA/microRNA

I. Nucleic acids that bind to other nucleic acid

II. Nucleic acids that bind to proteins

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Antisense oligonucleotides

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Antisense oligonucleotidesShort fragments of single strand, chemically modified

DNA nucleotides (oligonucleotides), complementary to a given mRNA.

Paul Zamecnik (b. 1912)

discoverer of tRNA died October 2009

Later in his career, Zamecnik and Stephenson developed antisense technology, in which short, synthetic nucleotide sequences can be used to silence the activity of individual genes. They published their results, in which they used a 13-nucleotide sequence to halt production of Rous sarcoma virus in chicken embryos, in 1978. That paper appeared in Proceedings of the National Academy of Sciences,

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Antisense oligonucleotides

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Antisense oligonucleotides

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The enzyme RNase H (EC 3.1.26.4) is a ribonuclease that cleaves the 3'-O-P-bond ofRNA in a DNA/RNA duplex to produce 3'-hydroxyl and 5'-phosphate terminated

products. RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism, aided by an enzyme-bound divalent metal ion.

Members of the RNase H family can be found in nearly all organisms, from archaeaand prokaryota to eukaryota . In eukaryotic DNA replication RNase H is

responsible for cutting out the RNA primer, allowing completion of the newly synthesized DNA

RNAse H

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Antisense oligonucleotides

- 13-25 nucleotides long - hybridize to corresponding RNA - act by: a) modulation of splicing

b) inhibition of protein translation by disruption of protein assembly

c) utilise RNAse H enzymes

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Oligonucleotides fate in vivo

1.Degradation by serum nucleases 2. Degradation by liver cells (unspecific uptake) 3. Degradation by kidney cells and excretion with urine

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Modification of antisense oligonucleotide

1st generation

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Clinical application of antisense oligonucleotides

Vitravene - CMV-induced retinitis

Bcl2 antisense - melanoma

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Application of antisense nucleotides for DNA repair strategies -

To be disucssed in January

DNA RNA

Genes(delivered by DNA vectors)

Non-coding sequences

Antisense oligonucleotides

DNA decoys

Genes(delivered by RNA vectors)

Non-coding sequences

ribozymes siRNAmicroRNA

Therapeutic nucleic acids

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DNA decoys

Pułapki oligonukleotydowe

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DNA decoys

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DNA decoys

DNA decoys – inhibit transcription

Antisense – inhibit translation

DNA decoys – double strandedDNA fragments binding the specific transcription factor

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Narrowing of blood vessels after angioplastyor CABG

narrowing occurs also in vessels used for by-pass grafting

- STENOSIS

RESTENOSIS

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cdkCyclin A Rb

E2F

TTTCGCGC

c-mycc-mybcdc2 kinasePCNA

silent

cdkCyclin A Rb

E2FTTTCGCGC

c-mycc-mybcdc2 kinasePCNA

transactivate

P

Transcription factor E2F

Mann MJ, Mol Med. Today 2000

Quiescence

Proliferation

E2F induces proliferation of vascular smooth muscle cells what maycause vessel narrowing

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cdkCyclin A Rb

E2FTTTCGCGC

c-mycc-mybcdc2 kinasePCNA

transactivate

P

cdkCyclin A RbE2F

TTTCGCGC

c-mycc-mybcdc2 kinasePCNA

silent

P

DNA decoy

TTTCGCGC

TTTCGCGC

TTTCGCGC

TTTCGCGC

Mann MJ, Mol Med. Today 2000

Inhibition of cell proliferation by E2F decoys

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Testing an Edifoligide, an E2F-decoy in preventionof bypass graft stenosis

Mann et al., Lancet. 1999 Oct 30;354(9189):1493-8.

Failure of phase III trials with E2F decoys...

edifoligide failed to show any benefit for primary and secondary end points in two phase III trials...

In the PREVENT III trial of 1,404 patients with critical limb ischemia who needed peripheral artery bypass graft surgery, there was no difference between edifoligide and placebo on the primary end point of limb amputation. No differences were seen in secondary end points of critical graft stenosis, recurrent limb ischemia, or quality of life.

Similar results were reported in the PREVENT IV trial, which tested edifoligide against placebo in 3,014 patients for the prevention of vein graft failure after coronary artery bypass surgery.

Multiple isoforms of E2F exist, and the drug may not have inhibited them all. Edifoligide's pharmacokinetics may not have allowed it to inhibit E2F adequately

Reasons?

JAMA 2005, November, 294: 2495-2497 25

Macular degeneration

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Types of AMD AMD is the #1 cause of severe vision loss in people over age 60. Today, at least 15 million people in the United States have this health problem.

Dry AMD is the most common form of AMD, representing approximately 90% of all AMD cases. However, dry AMD accounts for only 10% of the severe vision loss associated with macular degeneration. Dry AMD is characterized by development of yellow-white deposits underneath your retina, known as drusen, and can also be determined by deterioration of your retina. There is no generally accepted treatment for dry AMD, although vitamins, antioxidants and zinc supplements may slow its progression. Over time, dry AMD cases often develop into wet macular degeneration

Wet AMD occurs when abnormal blood vessels start to grow under the center of your retina. These new blood vessels may be very fragile and often leak blood and fluid. The blood and fluid can damage your macula or create a scar on your retina, causing vision problems. Damage to the macula can occur rapidly, causing a noticeable blurring or even loss of central vision. The vision loss may be permanent, because abnormal blood vessels and scar tissue are actually destroying normal retina tissue. Once lost, these light-sensitive cells in your retina cannot be replaced.

It is estimated that about 1.6 million people in the United States currently have wet AMD, with 200,000 new cases per year.

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VEGF-A – a major mediator of blood vessels formation

Receptors on endothelial

cells

VEGF-A is a major angiogenic growth factor. It acts on endothelial cells, being produced by

numerous cell types, including vascular smooth muscle cells (VSMC), fibroblasts or tumor cells.

Various isoforms of VEGF

Exons 1-5 8

Exons 1-5 86A1 A2

Exons 1-5 87

VEGF-A121

VEGF-A145

VEGF-A165

VEGF-A189

VEGF-A206

Exons 1-5 876A1 A2

Exons 1-5 876A1 A2 6B

VEGF165 isoform appears to be the most important29

Treatment for AMD

1. Photodynamic therapy

1. Antibodies – anti-VEGF2.1. Bevacizumab (Avastin) 2.2. Rivacizumab (Lucentis)

3. Aptamers – pegaptanib sodium (Macugen)

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MACUGEN (PEGAPTANIB)

Pegaptinib sodium is a pegylated oligonucleotide aptamer that binds to and inactivates VEGF165

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Pegaptanib sodiumPegaptanib sodium is a covalent conjugate of an oligonucleotide of twenty-eight nucleotides in length that terminates in a pentylamino linker, to which two 20-kilodalton monomethoxy polyethylene glycol (PEG) units are covalently attached via the two amino groups on a lysine residue.

The chemical name for pegaptanib sodium is as follows: RNA, ((2'-deoxy-2'-fluoro)C-Gm-Gm-A-A-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2' -fluoro)C-Am-Gm-(2'-deoxy-2-fluoro)U-Gm-Am-Am-(2'-deoxy-2' -fluoro)U-Gm-(2'-deoxy-2'-fluoro)C-(2'-deoxy-

2'-fluoro)U-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2' -fluoro)U-Am-(2'-deoxy-2'-fluoro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2' -fluoro)C-Gm-(3'® 3')-dT), 5'-ester with a ,a '-[4,12-dioxo-6-[[[5-

(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[w -methoxypoly(oxy-1,2-ethanediyl)], sodium salt.

The molecular formula for pegaptanib sodium is : C294H342F13N107Na28O188P28 [C2H4O]n

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Macugen

-registered by FDA in December 2004

MACUGEN treatment given every 6 weeks for up to 2 years has been shown to significantly reduce the risk of moderate vision loss in patients with all types of wet AMD.

- Specifically binds VEGF-A165

administered by intravitreal injection every sixweeks for at least two years

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Aptamers

1. DNA decoys – bind transcription factors

Nucleic acids that bind proteins

Exist naturally – produced by viruses (HIV, adenoviruses)

2. Pegaptinib sodium – a pegylated oligonucleotide binding VEGF165

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DNA RNA

Genes(delivered by DNA vectors)

Non-coding sequences

Antisense oligonucleotides

DNA decoys

Genes(delivered by RNA vectors)

Non-coding sequences

ribozymes siRNAmicroRNA

Therapeutic nucleic acids

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RibozymesSmall RNA molecules which allow sequence-specific endoribonucleolytic

cleavage in a catalytic manner

Processing of rRNA in Tetrahymena

Self-cleaving RNAs from: - bacteriophages - plant satellite virusoids - satellite tobacco ringspot virus - human delta hepatitis virus

RNA components of a polysaccharide branching enzyme and RNAse P

Discovered in the begining of 80’s by Thomas Cech and Sydney Altman

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„Hammerhead” ribozymes

-They are present in genomes of viroids and virusoids, small single-stranded RNA pathogens of plants

- they are built from three helices connected by loops- modified ribozymes have only one loop

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H- A, C lub U

„Hammerhead” ribozymes

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Correction of mutation by means of ribozymes

/correct exon

mutated exon

complementary sequence allowing for hybridisation of ribozyme to mRNA

recovered, correct mRNA

Szala et al., Terapia genowa, 2003 39

Delivery of ribozymes to target cells

- exogenous - ribozymes must be chemically modified - endogenous - coding sequences for ribozymes are delivered

with viral/plasmid vectors

Degradation of ribozymes in the serum

Ribozymes are currently not tested widely for therapeutic purposes

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RNA interference

PSTG – post-transciptional gene silencing

Specific inhibition of gene expression by double-stranded RNA, which stimulates the degradation of a target mRNA

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Jorgensen i wsp, 1990

chalcone

THE PLANT CELL, Vol 2, Issue 4 279-289, Copyright © 1990 by American Society of Plant Biologists

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans

C. Napoli, C. Lemieux and R. Jorgensen DNA Plant Technology Corporation, 6701 San Pablo Avenue, Oakland, California 94608

We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNaseprotection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.

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dsRNA inhibits gene expression more effectively than ssRNA

Control Embryo

Embryo with Visualised

mRNA of mex-3

Embryo injected with single-stranded antisense RNA

Embryo injected with dsRNA

Fire at al., 1998

dsRNA several hundred nucleotides long

Nematode after injection of unrelated dsRNA

Nematode injected with specific dsRNA

- RNA interference is an inherited mechanism

- Only few dsRNA were required for inhibition of gene expression, indicating for an amplification process

DsRNA is more effective than single stranded

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Nobel prize 2007

Andrew Fire Craig Mello 46

Genetic inhibition by double-stranded RNAAbstract

A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA

and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods

Inventors: Fire; Andrew (Baltimore, MD), Kostas; Stephen (Chicago, IL), Montgomery; Mary (St. Paul, MN), Timmons; Lisa (Lawrence, KS), Xu; SiQun (Ballwin, MO),

Tabara; Hiroaki (Shizuoka, JP), Driver; Samuel E. (Providence, RI), Mello; Craig C. (Shrewsbury, MA)

Assignee: Carnegie Institute of Washington (Washington, DC)

Appl. No. 09/215,257

Filed: December 18, 1998

This application claims the benefit of U.S. Provisional Appln. No. 60/068,562, filed Dec. 23, 1997.

Patent for RNA interference

47

Mechanisms of RNA interference

Dicer – an enzyme that cleaves long dsRNA

BL Davidson & PB McCray Jr, Nature Rev Genet, 201148

49

Methods for delivery of RNAi triggers

BL Davidson & PB McCray Jr, Nature Rev Genet, 201150

Strategies for delivery of siRNA in vivo

51

Some clinical trials for RNAi therapy

Z. Li & T. Rana, Nature Reviews Drug Discovery, August 2014

microRNA

53

K. Goljanek –Whysall et al., Clinical Science 2012

microRNAs regulate mRNA expression

Some microRNAs are encoded in introns on mRNA genes

Marquez & McCaffrey, Hum Gene Therapy 2008

The microRNA/RNA interference pathway

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microRNA

1. Regulate the expression of about 50% (or more…) mRNA genes

2. Regulation is strong and dependent on endogenous RNA interference system

3. microRNAs target specific sequences in mRNA

4. microRNAs are often expressed in a cell-specific manner

5. microRNA targeted vectors may allow cell-specific regulation of gene expression

• it is well documented that an individual miRNA can influence hundreds of gene transcripts to coordinate complex programs of gene expression and thereby, affect global changes in the physiologyof a cell

• miRNAs have been implicated as key molecular players in virtually all the cellular processes,

• numerous studies have demonstrated that alterations in the spectrum of miRNAs are correlated with various pathologicalsituations

microRNAs: a few basic facts

Exploiting and antagonizing microRNA regulation for

therapeutic and experimental applications

58

Cell-specific regulation of gene expression

1. Cell specific promoters

-eg, Flk-1, Tie -2: endothelial-specific

-Keratin 14 – keratinocyte specific

2. Vectors inhibited by microRNA

In 3’ region of the transgene the sequence recognized by microRNA expressed in the cells, in which we do not want to express the transgene is incorporated

eg. if we want the expression in hepatocyte but not in Kuppfer cell, we haveto add to the 3’ mRNA of the transgene the sequence recognized specifically by microRNA expressed in Kuppfer cells

Naldini i wsp. 2009

microRNA regulated vectors to drive expressionin a specific cell

Inborn errors of metabolism – Krabbe’s disease

Krabbe’s disease

From: Wikipedia

Krabbe’s disease

Psychosine is responible for demyelination in Krabbe’s disease

Krabbe’s disease

Treatment of globoid-cell leukodystrophy

microRNA-regulated gene therapy of globoid cell lecukodystrophy

microRNA-regulated gene therapy of globoid cell lecukodystrophy

Gene therapy for Krabbe’s disease

Orchard & Wagner, NEJM 2011

Gertner/Naldini, Science Transl Med. 2010

1. Krabbe’s disease- lack of galactocerebrosidase2. GALC can be delivered by HSC3. However, GALC is toxic for early HSC

Therapeutic efficacy of Krabbe’s disease gene therapy

microRNAs for genetic therapies

miR-34a as tumor suppressor microRNA

Hepatitis C

• An RNA virus discovered 25 years ago

• Major cause of liver cancer and liver transplant

• Globally 350,000 people die of HCV

• In USA more people die of HCV than AIDS

• Treatment: ribavirin and interferon – but with side effects: fever, headaches, fatigue,depression and anemia

• Therapy lasts up to 11 months and clears the infection in 5—70 %

• Current therapies: addition of protease inhibitors – improves cure rates and lessenstreatment time – but can be applied only against the most common type of HCV(so not equally effective around the world)

Role of miR-122 in HCV replication

HL Janssen et al., NEJM , 3 May 2013

miR-122, by binding to HCV RNA, promotes its propagation

Miravirsen – targeting HCV infection

HL Janssen et al., NEJM , 3 May 2013

Miravirsen – targeting HCV infection

N Engl J Med. 2013 May 2;368(18):1685-94. doi: 10.1056/NEJMoa1209026. Epub 2013 Mar 27

Miravirsen – targeting HCV infection

N Engl J Med. 2013 May 2;368(18):1685-94. doi: 10.1056/NEJMoa1209026. Epub 2013 Mar 27

Miravirsen – targeting HCV infection

N Engl J Med. 2013 May 2;368(18):1685-94. doi: 10.1056/NEJMoa1209026. Epub 2013 Mar 27

Selected anti-miR therapeutics curently in development

Z. Li & T. Rana, Nature Reviews Drug Discovery, August 2014

Oligonucleotide manipulation of microRNAfunction

Small & Olson, Nature 201180

Different ways of inhibition of gene expression

/miRNA

S. Richards., The Scientist, 03.2012

DNA decoys (inhibit transcription)

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Exam

2nd February 2015(Monday)

13 pm, room D107