How mutated genes can be deleted or silenced using different techniques icluding SiRNA etc

1. Gene inactivation- in
vivo & in vitro
BY AMNA FAZAL

2. • Silence or deregulate expression of a gene
• Different approaches
o Reverse genetics (limitations)
o Cre/Lox recombinase target systems
o RNA interference technique (without altering DNA
seq)

3. RNA Interference Based Therapeutics
• use of RNA to inhibit gene expression
• Most common approach
• May involve either of these two
o Small interfering RNA(siRNA)
o microRNA
Incorporated into RNA-induced silencing complex (RISC)

5. Limitations
 Small RNA degraded by nucleases
 Cell membranes are impermeable to sRNA
 Could induce inflammatory response

6. Solutions to overcome the limitations of siRNA
• Synthetic siRNA mimics the structure of Dicer cleavage
products
• involves covalent modification of the nucleotide structure
for promoting siRNA silencing effectiveness, enhancing
resistance to nucleases, reducing nonspecific ‘off-target’
effects, and blocking innate immune responses

7. Increase resistance in
serum
Prevent immune
stimulation
Increase efficiency
and stability
• Increase gene slicing &
serum stability
• Lowers immune
response

8. siRNA conjugates and delivery vehicles
systemic delivery of the RNA in the micro or nanoparticles
attached to cholesterol groups or conjugated with antibodies
recognizing particular cell receptors.

9. Gene Inactivation via Genome Editing
• Deletion cassette (in vitro)
 e.g yeast

10. Knockout gene

11. THANK YOU