emerging therapies for dmd

Sriharsha Gowtham

Connects actin to sacrolemmal plasma membrane

Likely a shock absorber as muscle contracts

(Finkel)

Mutations produce stop codons UAA, UAG or UGA in mRNA (Welch et al.)

Instability of transcript or truncation of protein can occur (Mendell and Dietz)

ptcbio.com

Can cause 5%-70% of cases of most inherited diseases (Welch et al.)

Cause 5-13% of DMD cases (Nelson et al.)

Degradation of truncated proteins

The nonsense-mediated mRNA decay pathway of the ribosome. This determines the decay rate of altered transcript during pre mRNA modification and extension of truncated protein

NMD pathway acts with the ribosome during “pioneering round of translation” as opposed to “productive translation”

Ribosome uses the exon-junction complex to determine the location of the nonsense mutation

(Schmitz and Famulok)

To suppress the ribosome’s ability to read nonsense codons (Welch et al.)

Results in only one change in amino acid Should help for other diseases with premature

stop codons such as cystic fibrosis (Finkel)

ptcbio.com

Two high through-put screens for a total of 800,000 low molecular weight compounds

1st used human embryonic kidney cells(HEK293) transfected with defective luciferase reporter gene

2nd used in vitro assay of luciferase mRNA and rabbit reticulocyte lysate

Non-toxic compounds that rescued the luciferase expression were isolated.

(Welch et. al Supp. info.)

.

PTC124 (C15H9FN2O3) isolated. no structural similarity to

aminoglycosides(potentially toxic compounds shown to have worked before)

Active at low concentrations- non-toxic

(Welch et al)

Welch et al.

PTC124 treated in cells of human DMD patients and mdx mice (contained premature UAA codon in Exon 23 of dystrophin gene) .

Antibody for dystrophin protein used Read through of nonsense codon

successful 40-60% of normal dystrophin: myosin in

humans, 35% in mdx samples

(Welch et al.)

PTC124 dosing in mdx mice: oral, intraperitoneal or both for 2-8 weeks

All three dosings improved strength Protection from contraction-induced

injury increased Serum creatine kinase decreased Full length dystrophin detected in

western blots Membrane localization determined with

immunohistological assays.

(Welch et al)

Microarray analysis of HEK293 cells treated with PTC124 or gentamicin showed few transcripts changed in expression vs control

Do normal stop codons differ from nonsense mutations?

Luciferase transcript with nonsense mutation and treated with PTC124 showed full length protein and no read through.

(Welch et al)

Reads through the mutation but not correct stop codon.

Possibly due to the secondary structure of mRNA near triplet codon (Finkel, Nelson et al.)

Or it prevents nonsense-mediated decay. (Nelson et al.)

Welch et al. claims that PTC124 induces read through of nonsense codons.

Auld et al claims that PTC124 stabilizes the protein with nonsense codon.

Many aspects of the experiment differed including the concentrations of PTC124 used by Auld et al.

Since PTC124 shows differing efficacy with differing nonsense codons, it is likely inducing read through.

The molecular target of PTC124 is unknown

Nelson et al.

After it was clear that no normal stop codon read through occurred in humans (Phase 1), a study regarding cystic fibrosis was conducted (Phase 2).

Nonsense codon was successfully suppressed

A study on DMD patients was conducted (Phase 3) showing dose dependent improvement.

Drug is being considered for Hemophilia A and B

Finkel

Finkel RS. Read-Through Strategies for Suppression of Nonsense Mutations in Duchenne/ Becker Muscular Dystrophy: Aminoglycosides and Ataluren (PTC124). Journal of Child Neurology. 25(9) 1158-1164

Mendell JT and Dietz HC. When the Message Goes Awry: Disease-Producing Mutations that Influence mRNA Content and Performance. Cell, Vol. 107, 411–414, November 16, 2001

Nelson SF, Crosbie RH, et al. Emerging genetic therapies to treat Duchenne muscular dystrophy. Curr Opin Neurol. 2009 October ; 22(5): 532–538.

Schmitz A and Famulok M. Chemical Biology: Ignore the Nonsense. Nature 447, 42-43 (3 May 2007) Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007;447:87–91 http://www.ptcbio.com/3.1.1_genetic_disorders.aspx