Therapeutic approaches to DMD 1 and 2

Question 1

What is another therapeutic approach to DMD?

Answer 1

Antisense oligonucleotide-mediated exon skipping

Question 2

How does Antisense oligonucleotide-mediated exon skipping work?

Answer 2

• Opposite of ASO therapy in SMA – exon exclusion (DMD) instead of exon inclusion (SMA)
• ASO causes “skipping” of exon that neighbours deleted exon, transforming out-of-frame
(nonsense) transcript into in-frame transcript capable of dystrophin production
• Thus, dystrophin lacks some amino acids but retains function, similar to BMD phenotype

Question 3

How many exons in open reading frame for healthy dystrophin?

Answer 3

• 79 exons fit precisely together
• Start of each exon coincides with position 1, 2, or 3 of codon, which encodes specific AA
• ASO promotes skipping of exon adjacent to mutation [i.e., deleted exon(s)] to restore ORF

Question 4

What does the ASO specifically target on the exon?

Answer 4

The ESE (part of splicing process)

Question 5

What is used for the ASO mediated exon 51 skipping?

Answer 5

Exondys 51

Question 6

What did the study of DMD patients given exondys 51 intravenous for 48 weeks daily show?

Answer 6

• 23% and 52% dystrophin-positive fibers after 24 and 48 weeks of treatment, respectively
• Presence of nNOS, β- and γ-sarcoglycan restored at sarcolemma, indicating DAPC reassembly

Question 7

What does nNOS do?

Answer 7

Regulates blood flow

Question 8

What test did they use to test if the DMD patients retained function?

Answer 8

6 minute walk test

Question 9

What did the 6 minute walk test show?

Answer 9

• In 4 year, open-label study, Exondys-treated patients (n = 12) experienced slower disease
progression than matched historical controls amenable to exon 51 skipping (n = 11)
• Distance lost on 6MWT 160m less over 4 years, and treated boys (2/12) demonstrated lower
incidence of loss of ambulation compared to control group (6/13) during 4-year study

Question 10

What did they call Exondys 51?

Answer 10

Eteplirsen

Question 11

what was the 1st treatment approved for DMD

Answer 11

aso exon skipping

Question 12

What is the ASO that skips exon 53?

Answer 12

Vyondys 53

Question 13

With both groups; low does and high does what was seen with the study using vyondys 53?

Answer 13

Significant muscle dystrophin production in both cohorts (+5.8% of normal) observed by WB

Question 14

What tests were used to see improvements of function using vyondys 53?

Answer 14

Timed function tests

Question 15

What were the results of the tests?

Answer 15

Walked at faster velocity and less than historical controls, quicker to get up from supine.

Question 16

What are the 4 ASO treatments? And which exon do the skip?

Answer 16

Exondys 51, Vyondys 53, Viltepso (exon 53), Amondys 45

Question 17

What percentage of DMD patients benefit from exon 51 skipping?

Answer 17

13%

Question 18

What percentage of DMD patients benefit from exon 53 skipping?

Answer 18

7.7

Question 19

What percentage of DMD patients benefit from exon 45 skipping?

Answer 19

8.1%

Question 20

How common are premature termination codon (PTC) mutations (nonsense) in DMD patients?

Answer 20

15%

Question 21

What does a premature stop codon do to the mRNA?

Answer 21

Prevents translation of mRNA and results in a truncated version of the polypeptide and is degraded

Question 22

What happens in the presence of a premature stop codon?

Answer 22

There is still an assembly of ribosome complex and early stages of translation occurs but when they reach the stop codon everything stops and disperses.

Question 23

How many stop codons? Can there be more how so?

Answer 23

3, can be created by nonsense mutations

Question 24

What does Translarna do?

Answer 24

promotes production of functional dystrophin by enabling
read through of premature stop codon associated with nonsense mutation

• Does not bypass normal stop signal, does not interfere with transcription or mRNA stability

Question 25

Is translarna a big or small molecule and how is it injected?

Answer 25

a small molecule that can be taken orally

Question 26

What do tRNA do?

Answer 26

Bring amino acids to ribosome for translation

Question 27

In the absensce of translarna, what happens when there is a premature stop codon?

Answer 27

Eukaryotic release factors are recruited, and go to ribosmes which cause disassociation and then a trunctaed polypeptide (most of the time dysfunctional and degraded)

Question 28

With a molecule that promotes read-through like translarna, what happens?

Answer 28

near-cognate tRNA decodes PTC & new AA incorporated in place of stop codon New AA forms sense or missense mutation instead of nonsense

Question 29

what did 28 day oral, 3X/day Translarna in DMD | patients with PTC mutations do in terms of dystrophin and serum ck levels?

Answer 29

* 60% of patients increased dystrophin levels (mean change 11%) * Reduction in serum CK in 85% of patients

Question 30

What did 48 week oral, 3X/day Translarna (ataluren) treatment in DMD patients with PTC mutations show functionally and what test did they use?

Answer 30

• Primary endpoint of 6MWD demonstrated significant difference versus placebo (31.3 m) • Timed function tests of 10 m walk/run, 4-stair ascend and 4-stair descend, also demonstrated modest preservation of ambulation with low dose Translarna used the 6 min walk test

Question 31

Who has approved translarna?

Answer 31

European union

Question 32

What is the point of micro dystrophin gene that was created?

Answer 32

Dystrophin is so large to give through a virus so they found the minimal size that will result in a functional protein. That fits in the virus

Question 33

What goes into the virus?

Answer 33

Micro dystrophin, muscle specific promoter

Question 34

Describe the Systemic viral delivery of micro-dystrophin to DMD patients study

Answer 34

1-year open-label non-RCT, 4 DMD patients aged ~5 years, single IV dose • Recombinant adeno-associated virus serotype rh74 with micro-dystrophin driven by skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7) • Skeletal muscle biopsy (gastroc) after 12 weeks for muscle histology, dystrophin IF and WB

Question 35

What did the study show?

Answer 35

* Treatment was well tolerated with minimal adverse events | * Improvement in function scores and reduction in serum CK for 1 year after dose

Question 36

What did the staining of muscle biopsy show?

Answer 36

The central myo-nucleus, fibrous and adipose tissue pre and post

Question 37

What did red staining do?

Answer 37

Red stain binds to collagen fibers so shows fibrosis (shows it less in post treatment), less creatine kinase

Question 38

What does IF and WB show?

Answer 38

Robust expression & correct localization of micro-dystrophin

Question 39

Disadvantages to study?

Answer 39

only 4 kids, and one dose