Janine Nicole Martin Gives Talk at American Society of Gene Therapy
Janine gave an excellent talk about Dystonia in San Diego at the 2009 convention of the
American Society of Gene Therapy. http://www.asgt.org/
419. Lethal Toxicity Caused by Striatal Delivery of shRNAs in Mouse Models of DYTI
Dystonia; Implications for Therapeutic Design
Janine N. Martin, Nicolle Wolken, Timothy Brown, William T. Dauer, Michelle E. Ehrlich,
Pedro Gonzalez-Alegre.
Genetics, U of Iowa, Iowa City, IA; Neurologv, Mt. Sinai School of MedIcine, New York City,
NY; Neurology and Pharmacology Columbia U, New York City NY,- Biochemistry and
Molecular Biology Thomas Jefferson U, Philadelphia, PA; Neuroiogy, U of Iowa, Iowa City, IA;
Neurology U of Iowa, Iowa City IA.
DYTI is the most common inherited dystonia, a disabling neurologically based movement
disorder. This incurable disease is caused by the deletion of a glutamic acid residue in the
protein torsinA (torA(DeltaE)). A common, dominantly inherited mutation likely resulting
from a dominant negative effect of torA(DeltaE) over torA(WT) indicates allele-specific
silencing of torA(DeltaE) could be a potential therapeutic strategy for DYT1. We have
previously tested shRNAs that achieve this goal in cultured neuronal cells without
triggering inflammatory responses and we completed the following studies to determine the
efficacy of those hairpins in vivo. Two different mouse models of the disease, DYT1 knockin
(KI) mice (129/SvJ strain) and transgenic (TG) mice overexpressing human torA(DeltaE)
under the DARPP32 promoter (DARPP32-T6rA(DeltaE)) (C57BL/6 background), along with
control littermates were used. The study design included bilateral striatal injections of
rAAV2,1.CMV.GFP vectors encoding either U6shTorA(DeltaE) (therapeutic vector), U6shMis
(control mismatched shRNA) or no shRNA at 2-6 months of age and behavioral testing (open
field, rotarod and staircase reaching tests) at baseline and post injection before sacrificing
the mice for biochemical and histological analysis. Unexpectedly, a cohort of DYTI KI and
control mice (n: 10 per genotype/vector) displayed mortality rates of 70% and 53% for those
receiving the therapeutic and control hairpins respectively, occurring 3-4 weeks post
injection, while the GFP only control vector did not induce toxicity. In parallel experiments
completed in DARPP32-TorA(DeltaE) TG mice and control littermates, the mortality rate
was 41% and 28% in those receiving the therapeutic vector and control shMis respectively,
with no toxicity in the GFP only group. Mortality in the TG model was reduced when
compared to the KI model and occurred 6-8 weeks post injection, perhaps reflecting the
different genetic background. This delay allowed us to complete behavioral analysis. While
baseline behavioral analysis did not display any difference, TG and control mice receiving
the therapeutic vector and control shRNAs exhibited significant hyperactivity in open field
behavior and did significantly worse on rotarod testing 6 weeks post injection when
compared to those injected with GFP only vectors, indicating striatal neuronal dysfunction.
In conclusion, our studies demonstrate that expression of U6-shRNA in the mammalian
brain can lead to fatal toxicity, even when cell culture studies did not predict toxicity.
Furthermore, the genetic background of rodents modifies their sensitivity to this form of
toxicity, a factor that should be taken into consideration in the design of therapeutic RNAi
trials. Future studies will explore the mechanism of toxicity and modifying the therapeutic
vector to abolish toxicity.
...... .
S164 Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright c The
American Society of Gene Therapy
Janine gave an excellent talk about Dystonia in San Diego at the 2009 convention of the
American Society of Gene Therapy. http://www.asgt.org/
419. Lethal Toxicity Caused by Striatal Delivery of shRNAs in Mouse Models of DYTI
Dystonia; Implications for Therapeutic Design
Janine N. Martin, Nicolle Wolken, Timothy Brown, William T. Dauer, Michelle E. Ehrlich,
Pedro Gonzalez-Alegre.
Genetics, U of Iowa, Iowa City, IA; Neurologv, Mt. Sinai School of MedIcine, New York City,
NY; Neurology and Pharmacology Columbia U, New York City NY,- Biochemistry and
Molecular Biology Thomas Jefferson U, Philadelphia, PA; Neuroiogy, U of Iowa, Iowa City, IA;
Neurology U of Iowa, Iowa City IA.
DYTI is the most common inherited dystonia, a disabling neurologically based movement
disorder. This incurable disease is caused by the deletion of a glutamic acid residue in the
protein torsinA (torA(DeltaE)). A common, dominantly inherited mutation likely resulting
from a dominant negative effect of torA(DeltaE) over torA(WT) indicates allele-specific
silencing of torA(DeltaE) could be a potential therapeutic strategy for DYT1. We have
previously tested shRNAs that achieve this goal in cultured neuronal cells without
triggering inflammatory responses and we completed the following studies to determine the
efficacy of those hairpins in vivo. Two different mouse models of the disease, DYT1 knockin
(KI) mice (129/SvJ strain) and transgenic (TG) mice overexpressing human torA(DeltaE)
under the DARPP32 promoter (DARPP32-T6rA(DeltaE)) (C57BL/6 background), along with
control littermates were used. The study design included bilateral striatal injections of
rAAV2,1.CMV.GFP vectors encoding either U6shTorA(DeltaE) (therapeutic vector), U6shMis
(control mismatched shRNA) or no shRNA at 2-6 months of age and behavioral testing (open
field, rotarod and staircase reaching tests) at baseline and post injection before sacrificing
the mice for biochemical and histological analysis. Unexpectedly, a cohort of DYTI KI and
control mice (n: 10 per genotype/vector) displayed mortality rates of 70% and 53% for those
receiving the therapeutic and control hairpins respectively, occurring 3-4 weeks post
injection, while the GFP only control vector did not induce toxicity. In parallel experiments
completed in DARPP32-TorA(DeltaE) TG mice and control littermates, the mortality rate
was 41% and 28% in those receiving the therapeutic vector and control shMis respectively,
with no toxicity in the GFP only group. Mortality in the TG model was reduced when
compared to the KI model and occurred 6-8 weeks post injection, perhaps reflecting the
different genetic background. This delay allowed us to complete behavioral analysis. While
baseline behavioral analysis did not display any difference, TG and control mice receiving
the therapeutic vector and control shRNAs exhibited significant hyperactivity in open field
behavior and did significantly worse on rotarod testing 6 weeks post injection when
compared to those injected with GFP only vectors, indicating striatal neuronal dysfunction.
In conclusion, our studies demonstrate that expression of U6-shRNA in the mammalian
brain can lead to fatal toxicity, even when cell culture studies did not predict toxicity.
Furthermore, the genetic background of rodents modifies their sensitivity to this form of
toxicity, a factor that should be taken into consideration in the design of therapeutic RNAi
trials. Future studies will explore the mechanism of toxicity and modifying the therapeutic
vector to abolish toxicity.
...... .
S164 Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright c The
American Society of Gene Therapy
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Notes on the above:
GFP stands for Green Fluorescent Protein, or the gene used to create the GFP protein
which glows green.
C57BL/6 refers to a strain of Black Mice used for laboratory experimentation.
torA(WT) is Torsin A Wild Type.
KI or Knockin refers to the addition of a gene.
Compare with KO or Knockout or deletion or incapacitation of a gene.
GFP stands for Green Fluorescent Protein, or the gene used to create the GFP protein
which glows green.
C57BL/6 refers to a strain of Black Mice used for laboratory experimentation.
torA(WT) is Torsin A Wild Type.
KI or Knockin refers to the addition of a gene.
Compare with KO or Knockout or deletion or incapacitation of a gene.