Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy.
OBJECTIVE
The objective of this study was to establish the feasibility of long-term gentamicin dosing to achieve stop codon readthrough and produce full-length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD).


METHODS
Two DMD cohorts received 14-day gentamicin (7.5mg/kg/day): Cohort 1 (n = 10) stop codon patients and Cohort 2 (n = 8) frameshift controls. Two additional stop codon DMD cohorts were gentamicin treated (7.5mg/kg) for 6 months: Cohort 3 (n = 12) dosed weekly and Cohort 4 (n = 4) dosed twice weekly. Pre- and post-treatment biopsies were assessed for dystrophin levels, as were clinical outcomes.


RESULTS
In the 14-day study, serum creatine kinase (CK) dropped by 50%, which was not seen in frameshift DMD controls. After 6 months of gentamicin, dystrophin levels significantly increased (p = 0.027); the highest levels reached 13 to 15% of normal (1 in Cohort 3, and 2 in Cohort 4), accompanied by reduced serum CK favoring drug-induced readthrough of stop codons. This was supported by stabilization of strength and a slight increase in forced vital capacity. Pretreatment stable transcripts predicted an increase of dystrophin after gentamicin. Readthrough efficiency was not affected by the stop codon or its surrounding fourth nucleotide. In 1 subject, antigen-specific interferon-gamma enzyme-linked immunospot assay detected an immunogenic dystrophin epitope.


CONCLUSIONS
The results support efforts to achieve drug-induced mutation suppression of stop codons. The immunogenic epitope resulting from readthrough emphasizes the importance of monitoring T-cell immunity during clinical studies that suppress stop codons. Similar principles apply to other molecular strategies, including exon skipping and gene therapy.
Malik
Vinod
V
Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Ohio State University, Columbus, OH 43205, USA.
Rodino-Klapac
Louise R
LR
Viollet
Laurence
L
Wall
Cheryl
C
King
Wendy
W
Al-Dahhak
Roula
R
Lewis
Sarah
S
Shilling
Christopher J
CJ
Kota
Janaiah
J
Serrano-Munuera
Carmen
C
Hayes
John
J
Mahan
John D
JD
Campbell
Katherine J
KJ
Banwell
Brenda
B
Dasouki
Majed
M
Watts
Victoria
V
Sivakumar
Kumaraswamy
K
Bien-Willner
Ricardo
R
Flanigan
Kevin M
KM
Sahenk
Zarife
Z
Barohn
Richard J
RJ
Walker
Christopher M
CM
Mendell
Jerry R
JR
D000293:Adolescent; D001299:Audiometry / Q000379:methods; D002648:Child; D002675:Child, Preschool; D018388:Codon, Terminator / Q000187:drug effects / Q000235:genetics*; D015331:Cohort Studies; D003402:Creatine Kinase / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005839:Gentamicins / Q000627:therapeutic use*; D006801:Humans; D032342:Muscle Cells / Q000473:pathology; D020388:Muscular Dystrophy, Duchenne / Q000097:blood / Q000235:genetics* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000187:drug effects / Q000473:pathology; D013997:Time Factors
D018388:Codon, Terminator; D005839:Gentamicins; D011500:Protein Synthesis Inhibitors; D003402:Creatine Kinase