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RNAi Applications

RNA Interference Applications in medicine
Module

Mechanisms of Gene Expression

17 Documents
Students shared 17 documents in this course
Academic year: 2022/2023
Uploaded by:
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This document has been uploaded by a student, just like you, who decided to remain anonymous.
Imperial College London

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MGE 16- Applications of RNAi

Key Points

Any gene can be targeted to

find function of gene- reverse

genetic screens

A RNAi library can be

constructed which contains all

genes of C. Elegans:

1. PCR products on

genomic template

2. Production of dsRNA

using T7 system

3. Feed genes

individually

RRF-3 mutants useful to study

genes that in WT, do not show

a phenotype

Can result in protein synthesis

being shut down

HIGS: Host Induced Gene

Silencing

This could help with food

security

Goal is to reduce expression of

disease-causing genes

Relies on endosomal pathway-

uncoating of nanoparticle or

vector

Patisiran is an siRNA packaged

into lipid nanoparticle which

associates with apolipoprotein

to mediate endocytosis

TTR- Transthyretin (blood

protein)

Notes

RNAi Screening n C. Elegans (Lab is example):

- dsRNA synthesised in E. Coli which is complementary

to a target gene- fed directly to C. Elegans

- Uses bi-directional T7 RNAP constructs

- RNAi can be used to interfere with 86% of all C.

Elegans genes- mutant phenotypes identified for

10% of genes

- Many protein coding genes are Pseudogenes- non-

functional protein

- C. Elegans can be made hypersensitive to RNAi using

RRF-3 mutants- loss of function of RdRp

- Phenotypes include embryonic lethal, sterile and growth defective

5,690 genes screened 16,757 genes screened

Found genes important for

mitochondria

Found genes important for fat storage

and mobilisation

Mutation in the mitochondrial leucyl-

tRNA synthetase gene (lrs-2) impaired

mitochondrial function and was

associated with longer-lifespan-

coupling of metabolism and longevity

Gene inactivation’s cause reduced body

fat and some caused increased fat

storage- fat regulatory genes &

pathway-specific fat regulators

(signalling between brain and sites of

fat storage)

Whole genome RNAi screens in Mammals:

- Introduction of long dsRNA (>500bp) can produce an interferon

response- dsRNA specific protein kinase is activated as cell thinks it’s

under attack by virus

- Short RNAi can trigger RNAi without inducing interferon response

- siRNA transfected directly into

mammalian cells to induce RNAi and

mRNA degradation

- Example: Can protect plants against

pathogens by spraying dsRNA solution

or by transforming plants with a HIGS

construct

Therapeutics:

- Drugs can be toxic due to immunogenic reactions to dsRNA,

nanoparticle toxicity and off target effects

- Targeting RNAi and microRNA pathway can have adverse effects for

gene regulation (microRNA important in gene regulation)

- Delivery of dsRNA can be through nanoparticles. siRNA is labelled

with sugars such as GalNAc to

facilitate endocytosis

- First approved drug: Patisiran-

targets TTR mRNA which

contributes to Amyloidosis

- Amyloid deposits formed from

abnormal versions of TTR which

misfold when mutated

MGE 16- Applications of RNAi

Summary

RNAi screening in C. Elegans involves feeding on dsRNA producing RNA (T7 bidirectional systems) to identify gene function

(fat storage/mobilisation & longevity)

Screening in mammals involves direct transfection of siRNA- an example is spraying plants with dsRNA to protect against

pathogen invasion

Therapeutic approaches are usually unsuccessful due to a number of challenges including off target effects and toxicity

Examples include using nanoparticles for dsRNA delivery and targeting TTR mRNA, responsible for Amyloidosis

Was this document helpful?

RNAi Applications

Module: Mechanisms of Gene Expression

17 Documents
Students shared 17 documents in this course

University: Imperial College London

Was this document helpful?
MGE 16- Applications of RNAi
Key Points
Any gene can be targeted to
find function of gene- reverse
genetic screens
A RNAi library can be
constructed which contains all
genes of C. Elegans:
1. PCR products on
genomic template
2. Production of dsRNA
using T7 system
3. Feed genes
individually
RRF-3 mutants useful to study
genes that in WT, do not show
a phenotype
Can result in protein synthesis
being shut down
HIGS: Host Induced Gene
Silencing
This could help with food
security
Goal is to reduce expression of
disease-causing genes
Relies on endosomal pathway-
uncoating of nanoparticle or
vector
Patisiran is an siRNA packaged
into lipid nanoparticle which
associates with apolipoprotein
to mediate endocytosis
TTR- Transthyretin (blood
protein)
Notes
RNAi Screening n C. Elegans (Lab is example):
- dsRNA synthesised in E. Coli which is complementary
to a target gene- fed directly to C. Elegans
- Uses bi-directional T7 RNAP constructs
-RNAi can be used to interfere with 86% of all C.
Elegans genes- mutant phenotypes identified for
10% of genes
- Many protein coding genes are Pseudogenes- non-
functional protein
-C. Elegans can be made hypersensitive to RNAi using
RRF-3 mutants- loss of function of RdRp
-Phenotypes include embryonic lethal, sterile and growth defective
5,690 genes screened 16,757 genes screened
Found genes important for
mitochondria
Found genes important for fat storage
and mobilisation
Mutation in the mitochondrial leucyl-
tRNA synthetase gene (lrs-2) impaired
mitochondrial function and was
associated with longer-lifespan-
coupling of metabolism and longevity
Gene inactivation’s cause reduced body
fat and some caused increased fat
storage- fat regulatory genes &
pathway-specific fat regulators
(signalling between brain and sites of
fat storage)
Whole genome RNAi screens in Mammals:
- Introduction of long dsRNA (>500bp) can produce an interferon
response- dsRNA specific protein kinase is activated as cell thinks it’s
under attack by virus
- Short RNAi can trigger RNAi without inducing interferon response
-siRNA transfected directly into
mammalian cells to induce RNAi and
mRNA degradation
- Example: Can protect plants against
pathogens by spraying dsRNA solution
or by transforming plants with a HIGS
construct
Therapeutics:
- Drugs can be toxic due to immunogenic reactions to dsRNA,
nanoparticle toxicity and off target effects
-Targeting RNAi and microRNA pathway can have adverse effects for
gene regulation (microRNA important in gene regulation)
- Delivery of dsRNA can be through nanoparticles. siRNA is labelled
with sugars such as GalNAc to
facilitate endocytosis
-First approved drug: Patisiran-
targets TTR mRNA which
contributes to Amyloidosis
-Amyloid deposits formed from
abnormal versions of TTR which
misfold when mutated

Students also viewed

Related documents