Oligonucleotides

Question 1

antisense

Answer 1

RNA strand complementary to mRNA strand

Question 2

siRNA

Answer 2

[small interfering RNA]

short double-stranded RNA that plays role in RNA interference

Question 3

how do gapmers work?

Answer 3

1. bind to target mRNA = DNA-RNA complex
2. RNAse H recognises hybrid + cleaves RNA strand

= RNA degradation
= downregulation of corresponding protein

Question 4

how does steric blocking work?

Answer 4

1. modified ASO (with high affinity binding) binds to target RNA
2. blocks mRNA translation / exon skipping

• no RNA degradation

Question 5

RISC

Answer 5

= RNA-inducing silencing complex

1. removes 1 strand of siRNA and uses it as template for recognising comp. mRNA
2. when it finds comp. strand, it activates RNase and cleaves RNA

Question 6

advantages of ASOs

Answer 6

only require gene sequence

can potentially target all genes

following chemical modification, antisense/siRNA can be stable (> 6 months)

Question 7

disadvantages of ASOs

Answer 7

stability - rapidly degraded by RNAses (require chemical modification or packaging into vesicles)

immune response

delivery

Question 8

chemical modifications

Answer 8

cell itself modifies its own RNA to avoid immune response

can’t modify base pairs as it’s involved in mRNA binding (backbone modified instead)

Question 9

chemical modifications - phosphorothioate

Answer 9

O -> S

improves circulation time in body (up to 24hrs)

Question 10

chemical modifications - 2’-MOE

Answer 10

OH -> C2H4OCH3

increases binding affinity and potency

half-life = 2-4 weeks

broad reduction in toxicity

Question 11

chemical modifications - 5’-methylcytosine/uracil

Answer 11

additional CH3 group

improves binding affinity and potency

reduces immune stimulation

Question 12

chemical modifications - PMO

Answer 12

O -> NH2

high binding affinity

high stability + safety (not very effective)

Question 13

chemical modifications - 2’-O-methyl

Answer 13

OH -> OCH3

modest increase in binding affinity and potency

half-life = 2-4 weeks

modest reduction in immune stimulation

Question 14

chemical modifications - 2’ fluoro

Answer 14

OH -> F

increased binding affinity and potency

no effect on stability or pharmacokinetics

improves RISC action

Question 15

problems with ASO delivery

Answer 15

oligonucleotide therapies = large and -vely charged

plasma membrane also negatively charged

Question 16

methods of delivery

Answer 16

liver, kidney + muscle = IV and subcutaneous

eye, CNS, lungs and joints = topical delivery

Question 17

liver delivery using GalNac

Answer 17

GalNac = amino sugar derivative galactose

recognised by ASGPR = highly selective and expressed in hepatocytes

conjugation to siRNA increases liver uptake by 10-100 fold

also means lower dose can be used

Question 18

mRNA-based vaccines

Answer 18

mRNA encapsulated in lipid nanoparticles (LPNs)
-> protects from degradation
-> facilitates entry into cells

after injection, LNPs taken into cells (APCs, macrophages) via endocytosis

mRNA released from LNPs inside cells

protein encoded by cell’s ribosomes = identical to pathogen (non-infectious)

binds to MHC class I molecules = activation of immune pathway

Question 19

nucleoside modified mRNA vaccine

Answer 19

UTR on 3’ and 5’ end

from globulin genes = highly expressed

optimizes codon sequence

Question 20

lipid nanoparticles

Answer 20

ionizable lipids

neutral at physiological pH

+vely charged

cholesterol -> enhances stability

helper lipid -> stability and fusion

PEGylated lipid -> stability + size

easy to manufacture