Antisense Oligonucleotides:

Question 1

Chemically modifeid ASOs can achieve (5):

Answer 1

• Increase stability – resistance to endogenous nucleases.
• Increase half-life.
• Increase binding affinity.
• Increase specificity.
• Reduce toxicity.

Question 1

What are ASOs? (3)

Answer 1

• Short single-stranded DNA or RNA sequences
• Anneal to gene by Watson-Crick base pairing.
• Oligonucleotides are chemically modified (not naturally occurring)

Question 2

Two major mechanisms of ASO action:

Answer 2

1. gene silencing
2. steric blocking

Question 3

difference between siRNA and ASO gene silencing

Answer 3

siRNA: double-stranded and can only work in cytoplasm
Antisense: single-stranded, can target cytoplasm (mRNA) can also go to nuclease and target primersa nd RNA.
- can target exon or intron on pre-mRNA to achieve gene silencing

Question 4

ASO: gene silencing happens by:

Answer 4

a. RNase h mediated mRNA degradation

Question 5

ASO: Splice-switching happens by either:

Answer 5

i. Exon skipping
ii. Exon-inclusion

Question 6

ASO: RNase H cleavage, what is a gapmer ASO:

Answer 6

Gapmer AON.
- Mixture of RNA and DNA.
- At each end RNA.
- DNA gap (gapmer)

RNA to RNA has much stronger binding affinity then RNA to DNA.

causes RNA degradation

Question 7

ASO: occupancy only mechanisms (steric blocking mechanisms) (5)

Answer 7

• Just RNA
• Binds to target gene.
• Combine to start ATG code and open reading frame, and microRNA (which upregulates expression of target gene).
• Can work in cytoplasm and nucleus. – important to induce exon skipping or exon inclusion for splice switching antisense.
• Splicing happens in the nucleus.

Question 8

Splicing modulation -

Answer 8

ASO needs to work at pre-mRNA stage
either exon skipping
or exon inclusion

Question 9

ASO: exon skipping

Answer 9

target enhancer to reduce splicing efficiency induce exon skipping – to get rid of exon skipping and save the reading frame.

Question 10

ASO: Exon inclusion

Answer 10

You want to save exon that is normally won’t be transcribed in typical condition. This is done in spinal muscular atrophy.

Question 11

WHAT IS TANGO and what does it involve: (4 + name)

Answer 11

TANGO: targeted augmentation of nuclear gene output

• By promoting poison exon skipping
• By blocking the translation of non-canonical open reading frames (uORFs)
• By blocking translation inhibitory elements
• By blocking regulatory naturally occurring antisense transcripts (NAT).

Question 12

Angelman syndrom: ASO targeting

Answer 12

• Target gene is UBE3A.
• All up to maternal allele
• But if the maternal allele is a mutation – the gene loses function
• The paternal allele is there – but due to imprinting it is not translated.
• SO – you want to reactive the paternal allele – so design AON to block the binding of long non-coding RNA to the paternal allele (which normally inhibits its translation) – this releases

Question 13

SPINAL MUSCULAR ATROPHY

Answer 13

Involves the degeneration of alpha-motor neurons in the spinal cord and lower brainstem. And is the second most common autosomal recessive disease. Effects 1 in 6,000 – 10,000. (Recessive).

Associated with loss of SMN protein. Effects all the organs in the body.

Question 14

What are the three drugs available for SMA

Answer 14

1. Nusinersen
2. Small molecule
3. Gene therapy

Question 15

EXON inclusion ASO therapy for SMA:

Answer 15

In SMA, the function gene is called the SMN1 gene, which has a tuned gene called SMN2. There are only five nucleotide differences, none of which affect AA coding. The C to T change changes the functional side of exons’ splicing.

SMN1 = full product
SMN2 – exon 7 not translated. Most of the product is not translated – and the product is very unstable.

Can design an AON therapy to restore the splicing of exon seven in SMN2.

Question 16

How is nusinersen administered

Answer 16

intrathecal injections regualrly

Question 17

Issues with systemic delivery accumulation of ASO therapy?

Answer 17

40-50% will accumulated and digested in the liver, another 40% will be cleaned out by kidney – so for this reason delivery raises huge issues because most of the AON lost.

Question 18

what is bio-conjugation (ASO)

Answer 18

Conjugate antisense with AB/small peptide – which can be tissue specific. And therefore carry the AON to the targeted organ/cell – works well if target is liver.