presented by

What is Gene Silencing?• “Turning off" a gene• This process happens naturally• Occurs at a transcriptional or a post-

transcriptional level• An important laboratory technique• Epigenetic regulation of gene expression• Don’t confuse it “gene knockout”

Types

Transcriptional Post-transcriptional Meiotic

a) Genomic Imprinting

b) Paramutationc) Transposon

silencing d) Transgene

silencinge) Position effectf) RNA-directed

DNA methylation

a) RNA interferenceb) Nonsense

mediated decay

a) Transvectionb) Meiotic

silencing of unpaired DNA

Methods for PTGSAntisense Oligonucleotides

Ribozymes

RNA Interference (RNAi)

Antisense Oligonucleotides• Paul Zamecnik and Mary Stephenson (1978)• Short nucleic acid fragments• Bind to complementary target mRNA molecules• Mechanisms-

RNase H-dependent Steric blocking mechanism

• 80% to 95% decrease in the protein and mRNA expression.

Ribozymes• Sidney Altman and Thomas Cech (1989)• Cleave mRNA molecules• Types-

Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs group I and group II ribozymes RNase P ribozyme

• Similar mechanism as protein ribonucleases• Sequence-specific cleavage of target mRNA molecules.

RNA Interference (RNAi)• Andrew Fire and Craig Mello (1998)• Entry of double-stranded RNA (dsRNA)• Small double-stranded fragments by Dicer

Small interfering RNAs (siRNA) MicroRNA (miRNA)

• RNAi induced silencing complex (RISC)-> Argonaute proteins• "guide" strand, binds to RISC• “passenger” strand degraded• Cleavage or translational repression of the mRNA molecules

makes the genes inactive.

Dicer

Applications• Cancer• Infectious diseases

Virus Bacteria

• Respiratory diseases• Neurodegenerative disorders

Huntington’s disease (HD) Amyotrophic lateral sclerosis (ALS)

Cancer• Chronic myelogenous leukemia (CML) -> BCR-

ABL• Mutant tumor suppressor p53 molecules• Chemokine receptor 4 (CR4)• Antiapoptotic proteins (clusterin and survivin)• Colon adenocarcinoma (B-catenin)

Virus• Human immunodeficiency virus (HIV) ->

chemokine receptor 5 (CR5)• Hepatitis B and C• Human papilloma virus ->E6• West Nile Virus• Tulane virus• Norovirus

Bacteria• Host genes

Involved in immune response caused by infection Involved in mediating the entry of bacteria into

cells• Lipopolysaccharide (LPS) -> tumor necrosis

factor α (TNFα)• Psueomonas aeruginosa -> caveolin-2 (CAV2)

gene

Respiratory diseases• Asthma• Chronic obstructive pulmonary disease (COPD)• Cystic fibrosis

Cell hyperplasia Mucus hypersecretion Damaged lung tissue

growth factor (TGF)-αgrowth factor TGF-β

Huntington’s disease• Mutation in the huntingtin gene• Mutated huntingtin protein• Motor, cognitive, and behavioral deficits• Allele specific gene silencing

Antisense oligonucleotides-single nucleotide polymorphism (SNPs)

• Non-allele specific gene silencing Normal and mutated huntingtin proteins

Amyotrophic lateral sclerosis• Lou Gehrig’s disease• Brain and spinal cord; motor neurons

degenerate, death• Mutations in the Cu/Zn superoxide dismutase

(SOD1)• Allele-specific gene silencing

ConclusionDelivery and SpecificityFor neurodegenerative disorders, gene silencing

molecules must be injected directly or by pumpsViral vectors can be usedMore efficient methods to deliver and develop

specific gene silencing therapeuticsSafe and effective.

References http://en.wikipedia.org/wiki/Gene_silencing Kole, R; Krainer, AR; Altman, S (Jan 20, 2012). "RNA therapeutics:

beyond RNA interference and antisense oligonucleotides.". Nature reviews. Drug discovery 11 (2): 12540. doi:10.1038/nrd3625. PMID 22262036

https://www.google.co.in/search?q=gene+silencing+pictures&es_sm=122&source=lnms&tbm=nws&sa=X&ei=GaAtVa3-L4aOuATl7oHABw&ved=0CAgQ_AUoAQ

THANKYOU