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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 a) RNA interference a) Transvection
Imprinting b) Nonsense b) Meiotic
b) Paramutation mediated decay silencing of
c) Transposon unpaired
silencing DNA
d) Transgene
silencing
e) Position effect
f) RNA-directed
DNA
methylation
 Methods for PTGS
Antisense 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)-α
growth 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
 Conclusion
Delivery and Specificity
For neurodegenerative disorders, gene silencing
molecules must be injected directly or by pumps
Viral vectors can be used
More efficient methods to deliver and develop
specific gene silencing therapeutics
Safe 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
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