Premature Termination codon readthrough as a therapeutic strategy for NCL

1. Premature termination codon readthrough as a therapeutic strategy for
NCL
Michel Roberge and Alireza Baradaran-Heravi, Dept Biochemistry & Molecular Biology, University of British Columbia
Jill Weimer, David Pearce and Logan Langin, Sanford Health
The antibiotic gentamicin contains major compounds responsible for
antimicrobial activity, as well as minor components
Overview
In a large number of cases, NCL is caused by a nonsense mutation.
This type of mutation introduces a premature termination codon
(PTC) that causes cells to make a truncated inactive protein instead
of a full-length active protein. We have found that gentamicin B1, a
minor component of the antibiotic drug gentamicin, can force cells
to make full-length protein, a process termed PTC readthrough. As
a first step to investigate whether this approach has therapeutic
potential, we are testing whether gentamicin B1 can induce PTC
readthrough in cells derived from a patient with a nonsense
mutation in the CLN2 gene and in a mouse model with a nonsense
mutation in the CLN2 gene.
Induction of full-length protein by premature termination
codon (PTC) readthrough
The antibiotic gentamicin contains major compounds responsible for
antimicrobial activity, as well as minor components
The antibiotic gentamicin contains major compounds responsible for
antimicrobial activity, as well as minor components
Summary and outstanding questions
- Gentamicin B1 can induce CLN2 PTC readthrough in cultured
cells
- Gentamicin B1 administered systemically can induce CLN2 PTC
readthrough in mice in peripheral tissues
- Gentamicin B1 administered systemically does not appear to cross
the blood brain barrier
Outstanding questions
- Can gentamicin B1 induce PTC readthrough when administered
intracranially?
- Can gentamicin B1 ameliorate the Cln2R207X/R207X mouse disease
phenotype?
- Can gentamicin B1 be administered chronically at safe doses?
Gentamicin B1 was
administered intraperitoneally
once and TPP1 activity was
measured 48 h later.
Initial results indicate that gentamicin B1 administered systemically can induce PTC readthrough in
peripheral organs (liver) but not in the central nervous system (brain).
Preliminary testing in the Cln2R207X/R207X mouse model
W
T
C
ln2R
207X
(PB
S)
C
ln2R
207X
(100m
g/kg)
0
5
10
80
100
120
140
Liver
TPP-1EnzymeActivity
(%ofWT)
Liver
Brain
TPP-1EnzymeActivity
(%ofWT)
W
TC
ln2R
207X
(PB
S)
C
ln2R
207X
(50m
g/kg)
C
ln2R
207X
(100m
g/kg)
0
2
4
6
8
10
80
100
120
140 Brain
WT 0 100
mg/kg B1
WT 0 50 100
mg/kg B1
The antibiotic gentamicin contains major compounds responsible for antimicrobial activity, as well as minor
components
A B C
Results show that gentamicin B1 can induce PTC readthrough in fibroblast cells derived from a patient, as
measured by restoration of TPP1 activity (A) and formation of full-length TPP1 protein (B), and in
embryonic fibroblasts from the Cln2R207X/R207X mouse model (C).
PTC readthrough in cells with CLN2 nonsense mutations
The antibiotic gentamicin contains major compounds responsible for
antimicrobial activity, as well as minor components
This work is supported by a grant from the Batten Disease
Support and Research organization
The antibiotic gentamicin contains major compounds responsible for antimicrobial activity, as
well as minor components
These major components constitute >90% of
gentamicin components. They are potent
antimicrobials but show poor PTC readthrough
activity.
Gentamicin B1 is a minor component
(<3%) that shows potent PTC readthrough
activity.