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CHARACTERIZATION OF THE EFFECTS OF REDUCED MUTANT HUNTINGTIN
IN THE R6/1 MODEL OF HIUNTINGTON' S DISEASE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
I dedicate this work to my loving and ever-supportive wife Erica and my daughter
Arianna; and to my parents, the inspiration of my life.
I would like to acknowledge the support and enduring patience that my mentor, Dr.
Ronald J. Mandel, had towards me from the beginning of this proj ect. I also would like to
make special mention of Dr. Alfred S. Lewin, whose wisdom and guidance made me the
confident scientist that I am today. I would like to thank all of my committee members,
Dr. Lucia Notterperk, Dr. Dennis Steindler and Dr. Nick Muzyczka for their
insightfulness. I gratefully acknowledge the help and support from Dr. Eileen Denovan-
Wright and her laboratory; she was that second pair of hands we all wish we had.
I thank everyone at the Mandel laboratory, especially Izzie Williams, for her
technical and moral support. Special thanks go to my beloved friend Carmen Peden. I
gratefully acknowledge all of the members of the Lewin laboratory, past and present, for
their unconditional friendship and support. I also would like to thank Dr. Corinna Burger
and Dr. Kevin Nash for lending me their time and for their ideas.
No words convey the gratitude that I have towards my parents. It is with much love
that I acknowledge and deeply appreciate the sacrifices and difficulties that my loving
wife, Erica, patiently endured. Lastly, but most importantly, I thank God for keeping us
safe and healthy through it all.
TABLE OF CONTENTS
ACKNOWLEDGMENT S ................. ................. iv.............
LIST OF FIGURES .............. ....................vii
AB S TRAC T ......_ ................. ..........._..._ viii..
1 INTRODUCTION ................. ...............1.......... ......
Huntington' s Disease ..........__._ .... ..._. ...............1....
Genetics of HD .............. ...............3.....
Huntingtin .........._.... ... ....__ ...............3.....
CAG Triplet Repeat Disorders .............. ...............5.....
Neuronal Inclusion Bodies ............... ...............5....
Transcriptional Dysregulation in HD .............. ...............6.....
Transgenic Models of HD .............. ...............8.....
Gain of Function vs. Loss of Function ............ ......__..... ..........1
Nucleic Acid-Based Gene Therapy .............. ...............12....
Ribozym es .............. ...............12....
RNA Interference............... ..............1
Gene Delivery in the CNS .............. ...............17....
2 RIBOZYME-MEDIATED REDUCTION OF STRIATAL MUTANT
HUNTINGTIN INT VIVO .............. ...............20....
Introducti on ................. ...............20.................
Materials and Methods .............. ...............22....
Defining Target Sites................ .... ...... .............2
Preparation of Short Ribonucleic Acid Target ................. ............... ...._...22
Time Course Analysis .......................... ..............2
In Vitro Transcription of m-Htt mRNA ................. ............. ........ .......24
Cloning of Ribozymes into rAAV Vectors ................. ................. ..........24
Human Embryonic Kidney 293 Cells................. ....... .............2
Transfections using Lipofectamine on HEK 293 Cells............... ..................2
RNA Isolation and Northern Analysis .............. ...............26....
rAAV Vector Production ........._....._ ...._.._......_._ ...........2
R6/1 Transgenic Colony ............._ ......__ ........._ ... .........2
Surgical Procedures ...._.. ................ ......._.. ..........2
Tissue Processing .............. ...............28...
In Situ Hybridization Analysis .............. ...............29....
R e sults............... ......... .. .... .............3
Time Course of Ribozyme Cleavage ................. .. ....... .... .........__ .......3
HD6, HD7 and dsCAG1 Activity Against hr Y7tro Transcribed m-Htt mRNA .31
HD6 and HD7 Ribozyme Activity Against m-Htt mRNA hIn Yvo ................... ..36
hr vivo Activity of rAAV-HD6 and rAAV-HD7 Ribozymes ................... ...........36
Biological Effects of rAAV5-HD6 and HD7 Ribozyme Expression in the
R6/1 Mouse Striatum .............. ...............3 8....
D discussion ............... .... ...... .... ..... ... ....... ... ........... .......4
Ribozymes Can Modulate the Expression of Cellular Genes .............................42
bI Vivo use of Ribozymes Directed Against the R6/1 Trans gene ................... ....42
Conclusion ................. ...............44..__.........
3 RNA INTERFERENCE OF MUTANT HUNTINGTIN INT VIVO ................... ........46
Introducti on ................. ...............46.................
Materials and Methods .............. ... ...............47.
rAAV-shRNA Plasmid Construction .............. ...............47....
Testing of shRNA Efficacy in Cultured Cells............... ...............48.
Western Blot Analysis using Hum-1 Antibody ................. ................. ......49
rAAV Vector Production ..........._..__.. ....._.._... ........_._ ............5
Intrastriatal Inj section of shRNA-Expressing AAV Vectors. .............. ..............50
In Situ Hybridization Analysis .............. ...............55....
Effect of Anti-mHtt shRNA on Phenotype ................. ............................56
Stati sti cs ................. ...............56........... ....
Re sults................. .............. ....... .. .... ..... .............5
RNA Interference of Mutant Huntingtin In Vitro ........._._......... .... ..............57
Long-Term Striatal Expression of rAAV5-shRNAs in the R6/1 Mouse ............60
Reduction in the Size and Amount of NIIs is Observed in R6/1 Mice Treated
with rAAV5-siHUNT-1 or rAAV5-siHUNT-2. ............. .... ..................6
Reduced Levels of Striatal mHtt Affect Levels of Striatal-Specific
Tran scri pts .................. ....... ....... ............... ..... ..... ..........7
Long-Term Bilateral Striatal Expression of rAAV5-siHUNT-1 in the R6/1
Mouse is Associated with a Delay in the Clasping Phenotype. ................... ....75
Discussion ................. ...............78........... ....
4 CONCLUSIONS .............. ...............83....
Unresolved Issues ................. ...............8.. 4..............
Future Studies ................. ...............86.......... .....
LIST OF REFERENCES ................. ...............88........... ....
BIOGRAPHICAL SKETCH ................ ............. ............ ..97..
LIST OF FIGURES
1-1. Neuropathology in HD. ............. ...............2.....
1-2. Structure domains of huntingtin. ........._._._ ....___........ ........___.........4
1-3. Molecular cascades in HD. ..........._...... ._ ...............10.
1-4. The hammerhead ribozyme. ............. ...............14.....
1-5. The RNAi pathway. ............. ...............16.....
2-1. Ribozyme design. ............. ...............32.....
2-2. Time course cleavage analysis. ............. ...............33.....
2-3. Target accessibility ................ ...................... ............................34
2-4. In vivo expression of rAAV ribozyme vectors. ....._.__._ ... .....___ ........._......37
2-5. rAAV5-HD7 expression results in a loss of striatal-specific transcripts. .................. .40
3-1. rAAV-shRNA constructs mediate the silencing of m-Htt in vitro. ................... .........58
3-2. Experimental design. ............. ...............61.....
3-3. Long-term in vivo striatal expression of rAAV5-siHIUNT 1 and rAAV-siHIUNT2
decrease mHtt transgene mRNA expression. .............. ...............63....
3-4 Analysis of mHtt protein aggregates after expression of rAAV5-shRNAs. ...............67
3-5 In situ hybridization (ISH) analysis of ppEnk and DARPP-32 transcripts. ...............73
3-6 Bilateral long-term striatal expression of rAAV5-siHIUNT-1 delays the clasping
phenotype of R6/1 mice. ............. ...............76.....
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF THE EFFECTS OF REDUCED MUTANT HUNTINGTIN
IN THE R6/1 MODEL OF HIUNTINGTON' S DISEASE
Chair: Ronald J. Mandel
Major Department: Neuroscience
Huntington's disease (HD) is a fatal, inherited, autosomal dominant neurological
disorder that currently affects over 30,000 individuals in the United States alone. HD is
characterized by motor impairment and psychological manifestations. Expression of an
abnormally expanded poly-glutamine domain in the N-terminus of the protein called
huntingtin is responsible for the genesis and progression of the disease. The aim of this
thesis was to develop molecular tools that could mediate the reduction in the levels of
mutant huntingtin in the striatum of the R6/1 mouse model of HD. We hypothesized that
reduced levels of striatal mutant huntingtin would ameliorate the progressive HD-like
phenotype that is observed in the R6/1 mouse.
Our initial studies focused on the development of RNA enzymes, called ribozymes,
and their ability to specifically target and efficiently knockdown the levels of mutant
huntingtin in vivo. We found that, although anti-mutant huntingtin ribozymes were highly
efficient in vitro, expression in the R6/1 striatum led to neuronal dysfunction in both
wild-type and R6/1 mice. Attempts at re-designing new ribozymes were hindered by
limitations in target site accessibility and availability. Thus, we focused our efforts
towards developing a new gene knockdown strategy based on the RNA interference
In vivo expression of anti-mutant huntingtin short-hairpin RNA molecules resulted
in a significant reduction in the levels of striatal mutant huntingtin protein in the R6/1
mouse brain. This reduction was associated with a partial clearance of neuronal
intranuclear inclusions. In addition, reduced levels of mutant huntingtin led to an increase
in the levels of two neuronal-specific transcripts known to be downregulated in HD. Last,
there was a delay in the onset of the rear paw clasping progressive phenotype observed in
the R6/1 mouse.
These results demonstrate that reduced levels of striatal mutant huntingtin are
beneficial in a genetic model ofHD. Furthermore, the studies presented here address the
therapeutic potential of RNA interference in the treatment of dominant disease. Finally,
our ribozyme studies suggest the existence of an unidentified gene, or genes, whose
expression is critical for the proper function of striatal neurons in the mouse brain.
In 1872 a young American physician named George Huntington Jr. accurately
described the symptoms of chorea and dementia as well as the adult-onset and genetic
inheritance associated with the disease that would ultimately come to bear his name (40,
62). HIUNTINGTON' S DISEASE (HD) is an inherited, autosomal dominant neurological
disorder characterized by choreiform abnormal movements, cognitive deficits and
psychiatric manifestations associated with progressive striatal atrophy (3, 11). Formerly
known as Huntington's chorea, HD affects approximately 1 in every 10,000 individuals
of Caucasian origin with close to 30,000 HD patients currently diagnosed in the United
States alone. There are at least 150,000 individuals who currently live with a 50 percent
chance of developing HD, as well as thousands of family members and friends that must
take on the challenge of caring for those with such a chronic devastating disease.
Although HD was first described over a century ago, there is currently no effective
therapy and, in the case of adult onset HD, the disease always leads to death within 10 to
15 years after the onset of symptoms (11, 97).
Huntington's disease affects different brain regions to various degrees; however,
the neuropathological hallmark of HD is the enlargement of the lateral ventricles (3).
Expression of the HD mutant gene leads to the selective degeneration of the gamma-
amino-butyric-acid (GABA) medium sized spiny neurons that reside in the caudate-
Figure 1-1. Neuropathology in HD. Panel (A) is a coronal section obtained from the brain
of an HD affected individual. Notice the striatal degeneration that results in
enlarged ventricles. Panel (B) is shown to demonstrate the size of the striatum
in a normal brain.
putamen nucleus (striatum) (Fig. 1-1). This marked striatal susceptibility is not fully
understood and although it results in gross neuroanatomical damage, it is only evident
during the later stages of HD and it is not solely responsible for the etiology of the
disease (45, 78). In fact, symptoms associated with HD are evident prior to any detectable
striatal atrophy (45, 60). These symptoms, which include involuntary twitching, lack of
coordination, depression, mood swings and forgetfulness, typically arise during mid-life
(35-40 years of age) although close to 10 percent of affected individuals develop a more
severe and aggressive form of the disease termed Juvenile HD, in which symptoms are
evident before the age of 20 (94). The onset of symptoms prior to any significant cell
loss, a phenomenon observed also in several animal models of the disease (7, 74, 84),
suggests that neuronal dysfunction, and not cell death, is responsible for the initial stages
Genetics of HD
The advancements in modern molecular techniques during the 1980s and the
unprecedented collaborative effort between groups of scientists from different institutions
led to the discovery of the HD gene in 1993 (81). The study found that HD is caused by
the inheritance of an unstable and excessively repeated cytosine-adenine-guanine (CAG)
codon within the coding sequence in exon 1 of the IT-15 gene (HD gene). Unaffected
individuals were found to have repeats with lengths of 34 CAGs or less, while HD
affected individuals had anywhere between 40 to 121 repeats. In HD, the severity of
symptoms seems to be correlated with the number of repeats while the age of onset
appears to be inversely proportional to the repeat amount (69), however, recent data
obtained from the largest known HD kindred (50,000 individuals in Lake Maracaibo,
Venezuela) suggest that genetic modifiers influence both severity and age of onset (90).
HD is developed when the CAG expansion is translated into a poly-glutamine (pQ)
repeat domain in the N-terminus of the protein encoded by the HD gene named
huntingtin (Htt) (81). Expression of expanded mutant Htt (m-Htt) results in the initiation
of a cascade of events that progressively disrupt neuronal homeostasis. Importantly,
expression of the expanded HD gene leads to activation of caspases, which in turn cleave
m-Htt into small N-terminal pQ-containing fragments (46, 61). These fragments can
readily cross through the nuclear pore and become aggregated inside the nucleus.
Abnormal nuclear translocation of expanded m-Htt N-terminal fragments is required for
the pQ-induced cell death observed in cellular models of HD (73).
Huntingtin is a large, 348 kDa, cytoplasmic protein of unknown function. It is
localized to many subcellular compartments and expressed in all tissues of the body with
P 0 HEA Ts
Figure 1-2. Structure domains of huntingtin. Shown above is a depiction of the structure
domains present in huntingtin. The poly-glutamine domain (pQ) lies just
upstream of a poly-proline rich domain (pp). A stretch of 36 HEAT-like
domains is thought to serve as docking sites for cellular proteins.
higher concentrations found in the brain and testis (24, 50). Htt has three maj or domains:
a polymorphic poly-glutamine domain, a proline-rich domain and 36 HEAT-like repeat
structures (Fig, 1-2); however, its amino-acid sequence has no major homology to any
known protein (75). Even though Htt's function has been difficult to elucidate, new
insights have emerged from studies in culture and animal models of HD.
Initially, approaches to the study of Htt' s function focused on the disruption of the
murine HD homolog gene (Hdh). Ablation of the Hdh gene in the mouse results in
embryonic lethality associated with aberrant brain development and increased apoptotic
cell death while post-natal conditional deletion in the mouse forebrain leads to abnormal
brain development and neurodegeneration reminiscent of HD (19, 95). These
observations suggest a critical role for Htt during neurogenesis and development. Recent
evidence suggests that Htt may function as a scaffolding protein (50, 66) and that it is
involved in many cellular processes such as neuronal vesicle transport in both the
endocytic and secretary pathways, protein trafficking and transcriptional regulation (13).
Studies have demonstrated that Htt colocalizes with vesicles and that expression of m-Htt
can interfere with normal neuronal vesicular transport (24). Additionally, Htt was found
to be a crucial member of the dynactin complex and this interaction was required for the
proper cortico-striatal anterograde transport of brain-derived neurotrophic factor (BDNF)
(24, 38). Also, wild-type Htt is proposed to have anti-apoptotic properties and may play a
role in neuronal survival (87). The abnormal level of apoptotic cells found in the Hdh
knockout mouse supports this hypothesis. Finally, it was recently showed that Htt
interacts with the REST-NRSF complex and that this interaction promotes the expression
of neuron specific genes such as BDNF (97). Taken together, these data suggest a
complex cellular role for Htt; however, what that specific role is, remains inadequately
CAG Triplet Repeat Disorders
The presence of a pQ domain in m-Htt makes HD a member of a family of at least
nine neurological disorders known as the CAG triplet repeat or pQ repeat disorders which
include Dentato-rubral pallido-luysian atrophy (DRPLA), Spinobulbar muscular atrophy
(SBMA) and the Spino-cerebellar ataxias (SCA) 1, 2, 3, 6, 7 and 17 (20, 23). Although
all of these disorders differ in the context of the protein containing the pQ domain, they
share many similarities including threshold CAG expansion lengths, genetic inheritance,
adult-onset and progressive neurodegeneration (23, 28, 65). Additionally, all of these
disorders are well characterized by the aberrant nuclear translocation of the mutant
protein as well as by the abnormal aggregation of cellular proteins that results in the
formation of intracellular bodies both in and outside the nucleus known as inclusion
bodies (30, 71, 89).
Neuronal Inclusion Bodies
Many of the known neurodegenerative disorders such as HD, Parkinson' s disease,
Alzheimer' s disease and amyotrophic lateral sclerosis (ALS) are characterized by the
presence of proteinaceous bodies readily visible under the light microscope. In the case of
HD, insoluble protein aggregates are observed late in the progression of disease (7, 70,
85, 87). It has been suggested that this process is modulated by the activation of caspases
that mediate the cleavage of m-Htt into small, toxic and aggregate-prone N-terminal
fragments (88). m-Htt sequesters into these inclusions, proteins that are critical for
maintaining neuronal homeostasis, i.e., transcription factors such as Spl, effectively
interfering with their proper cellular localization and function (14). This sequestration is
proposed to take place via a variety of different mechanisms such as the formation of
polar-zipper structures, transglutamination and SH3-domain dependent binding, all
resulting in aberrant protein-protein interactions that take place in and are modulated by
the pQ domain region of m-Htt (7, 66). Also, it has been suggested that inclusion bodies
can lead to neurite and neuropil dystrophy and can physically interfere with cellular
processes critical for neuronal function such as protein transport and proteosomal
processing (11, 32, 49, 50).
In contrast, other researchers have proposed that the formation of inclusion bodies
in HD is a neuronal defense mechanism designed to neutralize the toxicity associated
with the expanded pQ domain in m-Htt. In fact, a strong correlation between the
formation of inclusion bodies and survival rate was recently observed in neurons
engineered to express m-Htt in culture (2, 63). Whether the presence of inclusion bodies
in HD forms an integral part of the disease process itself or is an attempt by neurons to
cope with an otherwise toxic protein remains controversial. Nevertheless, many agree
that inclusion bodies are a reliable marker of disease progression and suggest a common
pathological mechanism amongst many neurological disorders (6, 63).
Transcriptional Dysregulation in HD
Transcriptional dysregulation has recently been suggested as an important
pathogenic mechanism in HD (12, 14, 51, 79). Both HD models and patients display a
progressive loss in steady-state mRNA levels of a subset of striatal neuronal transcripts
(14, 79). Recent observations implicate the formation of intranuclear inclusions and the
sequestration of transcription factors into aggregates as possible explanations for the loss
in gene transcription (14). There is mounting evidence suggesting that soluble forms of
mutant huntingtin alter the expression of specific genes by interacting with their
transcriptional activators (55). X-ray diffraction studies have shown that the poly-
glutamine domain in m-Htt can mediate the aberrant interaction between m-Htt and poly-
glutamine-containing transcription factors via the formation of polar-zipper structures in
vitro (66). In addition, yeast-two hybrid screens have illustrated m-Htt's interaction with
several transcription factors such as p53, SP1 and the CREB-binding protein (CBP) in
vivo (74). This evidence suggests that the specific interaction of m-Htt with factors that
control transcriptional activation results in the early and progressive loss of striatal-
specific mRNAs in HD.
The cyclicAMP-responsive element (CRE) -mediated transcription pathway is
impaired in HD (11, 24, 75). CRE-mediated transcription is activated by the interaction
between phosphorylated CREB and CBP. This results in the recruitment of the
transcriptional machinery and initiates transcription. In HD, the transcriptional co-
activator CBP is recruited into m-Htt aggregates and leads to inhibition of CBP-mediated
transcription as well as cellular toxicity (41, 55). Overexpression of CBP can alleviate the
toxicity associated with the expression of m-Htt in a neuroblastoma cell line. In addition,
m-Htt can also sequester TAFIIl30, a critical regulator of CBP, CRE-mediated
transcription, into aggregates (14). These observations suggest that m-Htt expression can
block the proper targeting of the transcriptional machinery to CRE-dependent promoters
in affected neurons, resulting in the loss of transcriptional activity.
The interaction between m-Htt and regulators of transcription may provide
evidence of the "gain of toxic" function associated with the abnormally expanded pQ
domain in Htt. However, although the interruption on neuronal transcription happens
early in the evolution of the disease, the role that this plays in the molecular cascade
initiated by expression of m-Htt remains unresolved.
Transgenic Models of HD
The characterization of the mutation responsible for the neurological phenotype
observed in HD in 1993 led to the report of the first successful HD transgenic mouse line,
the R6 line, three years later (54). Since then, several transgenic and knockin lines have
been developed that accurately portray the progressive phenotype associated with HD (7,
72). All of these have in common the expression of an expanded CAG repeat although
they differ in the genetic context in which the abnormal expansion is expressed.
Importantly, the onset, rate of progression and severity of motor symptoms displayed by
these models are dependent on the number of repeats and the levels of m-Htt protein
expression. Of all the lines generated to date, the R6/1 and R6/2 transgenic lines remain
the best characterized.
In both patients and transgenic HD mice, the age of onset, disease progression and
severity of symptoms are correlated with the expression of m-Htt and the length of the
trinucleotide CAG repeat. The R6 HD transgenic lines, R6/1 and R6/2, were created by
inserting a transgene containing the 5'-untranslated region, exon1 with an expanded CAG
repeat, and part of the first intron of the human HD gene. This transgene is under the
control of the human HD promoter and generates a short N-terminal fragment with an
expanded pQ domain. Differences between the R6/1 and the R6/2 transgenic lines
include the length of the CAG repeat (R6/2 > R6/1) and the site of integration of the
transgene, thought to result in differences in the amount of protein produced (R6/2>R6/1)
(54, 72). R6/1 mice have a slower disease progression and a later onset of motor
abnormalities than the R6/2 mice. Previous work demonstrated that levels of m-Htt
protein are lower in the R6/1 mice compared to R6/2 and that neuronal intranuclear
inclusions (NIIs) form more slowly throughout the brain tissue in R6/1 compared to R6/2
mice (7, 54, 72). Therefore, the length of the polyglutamine tract encoded by the human
HD transgene and the relative levels of expression of the transgene affect the rate of HD
progression in these mice. The relatively slower rate of disease progression and longer
lifespan of the R6/1 mice make them more amenable for the study of slow acting
interventions. In this study, we focus on the use of R6/1 mice.
The R6/1 transgenic line is characterized by an early onset of pQ-associated
changes in neuronal function and a slow progression of motor phenotype. These mice
exhibit decreased striatal and total brain size; however reports of neuronal death have
been contradictory (7, 38). R6/1 mice also display ubiquitinated nuclear and cytoplasmic
neuronal inclusion bodies containing both mutant and wild-type Htt. Also, there is
increasing evidence of transcriptional dysregulation, which is modulated by the CAG
expansion and levels of transgene expression (36, 56). Finally, the expression of
expanded m-Htt results in the development of a neurological phenotype in 15 week-old
R6/1 mice, a much earlier time point than was initially reported. R6/1 mice exhibit
several behavioral abnormalities including gait disturbances, progressive clasping of the
hindlimbs and seizure activity, all associated with neuronal dysfunction (10, 58). Finally,
a failure to maintain body weight, as well as muscle wasting, has been reported.
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individuals the absence of a wild-type HD allele may progressively lead to neuronal
dysfunction and eventual susceptibility to insults, while in heterozygote patients, mutant
Htt may act in a dominant negative fashion against the wild type allele, effectively
interfering with the normal cellular function of wild-type Htt. This idea is further
supported by the observation that wild-type Htt is also sequestered away into inclusion
bodies by mutant Htt and appears to be depleted in both humans as well as animal models
of HD (95).
In contrast, others favor the idea that the mechanism underlying the pathogenesis of
HD is the gain of a new toxic function associated with the pQ expansion in mutant Htt
(53). In support of this theory is the fact that individuals with a deletion in one HD allele
do not develop HD (Wolf-Hirschorn syndrome) (53) and the observation that there is no
significant difference in the severity of symptoms between homozygote and heterozygote
HD patients (3). In fact, there is a stronger correlation between the severity of symptoms
and the number of CAGs in the HD gene (29, 53, 90). Furthermore, as described earlier,
mutant Htt is involved in aberrant protein-protein interactions that result in a disruption
of normal neuronal processes in a time dependent manner. The fact that Htt' s normal
function remains elusive is a giant hurdle in the quest for the understanding of the
molecular underpinnings of HD. It is likely that both models described above act in
concert and are both responsible for the damage observed in HD. However, it is of vital
importance to fully dissect the contributions of each during the disease process as this
would lead to the development of more effective therapies such as those aimed at
reducing the levels of Htt expression in the brain.
Nucleic Acid-Based Gene Therapy
The genesis and progression of disease in dominant genetic disorders require the
expression of the mutant protein. In the case of HD, this concept was first demonstrated
using a mouse model that carried an expanded HD allele engineered to shut down its
expression under specific pharmacological conditions (91). This conditional model
showed that the expression of m-Htt was necessary to maintain the progression of disease
and that interruption of mutant Htt expression, while maintaining wild type levels of
normal Htt, led to a reversal of HD-like symptoms including a clearance of inclusions
and behavioral improvements. This observation led to the hypothesis that suppressing
mutant Htt activity would ameliorate the HD phenotype in affected individuals. Although
several pharmacology-based mechanisms may be employed to achieve suppression of
gene expression, nucleic acid-based methods (NABM) are the most potent, specific and
cost-effective way of achieving post-transcriptional suppression of m-Htt gene expression
(18, 81, 82). Recent advances in molecular design and intracellular delivery of nucleic
acids further enhance the application of NABM for the treatment of dominant
neurological disorders. There are three different NABM that result in post-transcriptional
gene silencing: anti-sense oligodeoxyribonucleotides (AODN), ribozymes and small-
interfering RNAs (siRNA). This study focused on the use of small self-cleaving
ribozymes and siRNAs in order to suppress m-Htt gene expression in vivo.
Ribozymes are catalytic RNA molecules that mediate the sequence-specific
cleavage of other RNA molecules (34, 35, 48). Ribonucleotide enzymes (ribozymes)
catalyze the hydrolysis and phosphoryl exchange at the phosphodiester linkages between
RNA bases resulting in cleavage of the substrate. There are three main groups of
ribozymes which are classified as follows based on function and size: self-splicing
introns, Rnase P and small self-cleaving ribozymes (96). This study focused on the use of
small self-cleaving hammerhead ribozymes, naturally occurring enzymatic RNAs that
can catalyze the cleavage of RNA molecules in reactions that are devoid of proteins. The
most commonly studied small ribozymes include the hammerhead, the hairpin and the
hepatitis delta virus ribozymes (22, 48, 96). Of these three the hammerhead ribozymes
display great versatility as a tool for the study of gene function and disease.
Hammerhead ribozymes are approximately 34 base-pairs in length and can
mediate the cleavage of an RNA target in trans. The hammerhead ribozymes bind
substrate to form a structure that consists of a stem and three loops and a catalytic core
with a conserved nine- nucleotide sequence (96) (Fig. 1-4). Point mutations within the
conserved region prevent the cleavage of RNA. The hammerhead ribozyme cleaves the
substrate by a trans-esterifieation reaction, which is dependent on the presence of
magnesium and water. The enzymatic reaction results in the formation of two products
with distinct 3' (2'-3' cyclic phosphate group) and 5'ends (5'-hydroxyl group). Following
the cleavage of the RNA backbone, the reaction products diffuse away from the active
site leaving the ribozyme free to complete another reaction cycle. The hammerhead
ribozyme recognizes substrate sequences on either side of a NUX triplet cleavage site,
where N is any nucleotide and X is any nucleotide except G. The ribozyme anneals to the
mRNA substrate by means of two flanking arms, which hybridize to form helices III and
I (Fig 1-4). Cleavage occurs at the 3' end of the NUX site. There are varying degrees of
cleavage efficiency associated with the sequence of the NUX triplet. Cleavage occurs at
the 3' end of the NUX site.
A GCGC lN N N NN-5'
G AjG L
Figure 1-4. The hammerhead ribozyme. The hammerhead ribozyme binds substrate to
form a structure, which consists of a stem and three loops and a catalytic core
(shaded box) with a conserved nine-nucleotide sequence. The NUX cleavage
site is indicated. The arrow denotes the site of bond cleavage.
There are varying degrees of cleavage efficiency associated with the sequence of
the NUX triplet. In general, the triplet GUC is the most efficient cleavage site, followed
by CUC, UUC and AUC (78). Other cleavage sites are cleaved at least 10 times less
efficiently than the GUC site (77).
The turnover characteristic of ribozymes provides them with an advantage over
standard antisense technology, which only inactivates the target RNA without degrading
it. Due to their size, catalytic properties and lack of cellular toxicity, hammerhead
ribozymes show great promise as tools in molecular medicine. In fact, the successful
application of hammerhead and/or hairpin ribozymes in the treatment of dominant
genetic disorders has been reported (48). Specifically, the use of hammerhead ribozymes
against mutant rhodopsin was shown to effectively protect against photoreceptor
degeneration in a mouse model of Retinitis Pigmentosa (47). Additionally, new gene
regulation systems are being developed which incorporate the use of regulatory
sequences called aptamers into existing hammerhead ribozymes (17, 68). This fusion of
these technologies allows for the design of drug-responsive ribozymes, which only
catalyze the cleavage of target RNA in the absence of a regulatory drug while becoming
inactive in the presence of the drug.
To date, ribozymes are being used in a variety of gene transfer strategies including
the suppression of viral infection as well as in anti-oncogene strategies aimed at
correcting cancer associated with genetic defect (22, 48, 96). The ease with which
hammerhead ribozymes can be designed and regulated, coupled with the recent advances
in viral gene delivery systems, give these small versatile molecules great potential as
During recent years a new technology, called RNA interference (RNAi), has
completely revolutionized the study of gene function and the design and application of
nucleic acid-based therapies aimed at the silencing of disease-associated gene expression.
RNA interference is an innate cellular process associated with immune surveillance as
well as the regulation of gene expression during development in mammalian cells (82).
This mechanism specifically responds to the presence of double-stranded RNA (dsRNA)
molecules and directs the activity of post-transcriptional processes that lead to the
sequence-specific inhibition of genes with complementary sequence to the dsRNA
molecule (18). During RNAi, dsRNA molecules are rapidly processed by an enzyme
called DICER into small duplex RNA molecules of 20 to 21-nucleotides in length called
small-interfering RNAs (siRNA). Unidentified components of the RNAi machinery
recognize and incorporate a single strand of the siRNA molecule into a ribonucleo-
protein complex called the RNA-induced silencing complex (RISC) (82, 83). RISC can
then survey the mRNA population in order to mediate the sequence-specific cleavage of a
target mRNA (Fig. 1-5).
Fiue15 h N iptwa.Itaellrsothiri N saepoesdb
DICR 1)int sml-itrfrin RN oeue sRA.sR 7r
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inote el wihae hninoprae no ICan eiaetesiecngo h
exeiena ee fitret(8. motnty nmamlncels the sizeo h iN
death (83). Adtio nA allmthods. fo intracellular exrsino short-hairpin RNAs aepoesdb
(shRNA) E deivre as t plsmaid vetrs ae beng rN ecnly eveoe (16,A) 18).~ shRN
art te exprsse asic fl-ack sthem-lnoropsructure nomprisadedit of a senseandano atisens
strand and separated by a non-complimentary loop. The transcription of shRNAs is
normally placed under the control of RNA polymerase III, which is capable of generating
a transcript with a specified 3'-end terminal shown to be required for efficient silencing
activity (18). Once transcribed by the nuclear machinery, shRNAs are exported into the
cytoplasm, processed by DICER into functional siRNAs and incorporated into RISC.
Unlike anti-sense oligodeoxyribonucleotides (AODN) and ribozymes, siRNAs
achieve the silencing of genes through an endogenous host-cell, anti-viral defense
mechanism that has had the opportunity to evolve and become highly efficient over time.
Additionally, siRNAs act in concert with the RISC protein complex, which results in
increased stability and a more efficient capacity to turn over (18). Also, the stability of
RNA duplexes allows for siRNAs to be readily delivered into cells and makes it possible
to achieve biologically relevant concentrations inside the cell. Furthermore, low
concentrations of highly active siRNAs have been shown to induce long-term gene
silencing (82), making these molecules more pharmacologically attractive than either
AODNs or ribozymes. However, although this is a potent molecular tool, RNAi is a
relatively new and developing technology and unintended effects already known to be
associated with it, such as changes in expression of non-targeted genes, must be carefully
Gene Delivery in the CNS
Viral as well as non-viral vectors can be used to deliver therapeutic genes directly
into the central nervous system (CNS) in a process termed in vivo gene transfer. Although
non-viral vectors circumvent some of the potential toxic problems associated with viral
vectors, it has been difficult to achieve and maintain high levels of transgene expression
(43, 65). Viral vectors were designed based on the natural ability of viruses to infect,
transfer and express their genetic material in host cells. When compared to non-viral
delivery methods, viral vectors are capable of mediating longer, more widespread and
intense transgene expression in the CNS (16, 42, 64). The successful application of viral
vectors in the CNS depends on the vector' s capacity to mediate long-term transgene
expression in non-dividing cells. In addition, the immunogenecity associated with the
natural life cycle of the chosen vector most be assessed. Although different viral vectors
have been successfully used in the CNS, our study focused on the use of recombinant
Adeno-Associated viruses (rAAV).
The first rAAV vectors were described approximately 20 years ago (37). Since
then, their use has revolutionized the study of genetic disease in the CNS by allowing the
temporal and somatic regulation of gene expression (6, 8). AAV recombinant vectors are
small, single-stranded DNA viruses capable of infecting both dividing and non-dividing
cells (8, 37). Wild-type AAV is a non-pathological member of the parvoviridae family
able to integrate its genome into the host' s DNA. In contrast to its wild-type counterpart,
rAAV integration events seem to occur less frequently and more randomly (1, 21, 43).
rAAV vectors promote stable, long-term transgene expression in the CNS (4, 8).
Moreover, CNS exposure to rAAV capsid proteins does not lead to cellular toxicity.
However, the presence of circulating antibodies can block rAAV transduction and can
mediate a cellular immune response upon re-administration of the vector (86). Recently,
three different pseudo-typed rAAV vectors (rAAV-1, rAAV-2 and rAAV-5) where found
to efficiently transduce various regions of the rat CNS (13). The use of different pseudo-
typed rAAV vectors allows for the development of protocols that can circumvent the
immunological problems associated with circulating antibodies and vector re-
admini strati on.
The remarkable ability of rAAV vectors to efficiently transduce and sustain the
long-term expression of genes in neuronal tissue, with negligible induction of the
immune response, have led to the development of a variety of clinical protocols aimed at
the treatment of CNS disorders. Some of these gene transfer strategies include gene
replacement in Canavan's disease, Parkinson's disease and lysosomal storage disorders
(15). Also, rAAV-based strategies for the delivery of neuroprotective factors in
Alzheimer' s, Huntington's and amyotrophic lateral sclerosis (ALS) have been reported
(80). In conclusion, rAAV vectors can efficiently deliver genes into the CNS. rAAV
holds the promise as the future vector of choice for the delivery of therapeutic genes in
RIBOZYME-MEDIATED REDUCTION OF STRIATAL MUTANT HIUNTINGTIN INT
The progressive striatal pathology observed in Huntington's disease patients is
caused by the expression of an abnormally expanded poly-glutamine (pQ) domain in N-
terminus of the ubiquitously expressed protein termed huntingtin (Htt) (29, 53). The
uninterrupted expression of m-Htt is necessary for the genesis and evolution of HD as
shown by a conditional transgenic mouse model expressing a CAG expanded HD
transgene under the control of the tetracycline promoter (92). In this model,
transcriptional suppression of m-Htt expression, in both cortical and striatal regions of the
brain, led to a reversal of cellular and behavioral HD-like phenotype. However, the effect
that reduced striatal levels of m-Htt would have on the phenotype of transgenic HD mice
has not been assessed. We hypothesized that post-transcriptional knockdown of m-Htt
should lead to an amelioration of the HD-like phenotype in the R6/1 transgenic mouse
line. In vitro studies have demonstrated that anti-sense oligodeoxyribonucleotides
directed against an expanded human HD allele can mediate the suppression of m-Htt
protein expression in cultured cells (9, 33, 59). Similarly, DNA-enzymes, which are
composed of a ribonucleotide catalytic frame placed in between deoxy-ribonucleotide
hybridizing arms, were successfully used against m-Htt mRNA and resulted in specific
suppression of m-Htt protein expression (93). These data suggest that, unlike other
proteins such as those involved in cell signaling, reduced intracellular levels of m-Htt do
not induce HD promoter activity. More importantly, they demonstrate that the secondary
structure of m-Htt mRNA contains biologically accessible regions for Watson-Crick base
pairing. However, the application of these two technologies in in vivo models of HD is
limited. First, high intracellular concentrations of anti-sense oligo-deoxy-ribonucleotides
are required in order to achieve significant reduction of protein expression in vivo (9, 96).
This leads to cellular toxicity and the induction of the immune response. Addionally,
anti-sense molecules are fairly unstable and the mechanism by which they mediate gene
silencing is currently unknown. Also, the efficient in vivo delivery and long-term
expression of anti-sense molecules and DNAenzymes, which cannot be encoded for,
represents a challenge that is not easily surmounted.
Hammerhead ribozymes are RNA enzymes that can be targeted to catalyze the
cleavage of a specific RNA molecule (5, 48, 96). High intracellular concentrations of
hammerhead ribozymes are not toxic and can be achieved with vector-based expression
systems (34, 47, 48). Ribozymes against the untranslated 5'end region of the HIV-1
genome have been shown to inhibit virus replication (5). Moreover, rAAV-mediated
delivery of a hammerhead ribozyme against mutant rhodopsin mRNA resulted in the
rescue of photoreceptors in a mouse model of retinitis pigmentosa (47). These results
suggest that hammerhead ribozymes can efficiently mediate the knockdown of target
RNAs in vivo.
In this study, we examined the effects of ribozyme-mediated post-transcriptional
suppression of striatal m-Htt in the R6/1 mouse. We specifically assessed whether rAAV-
ribozyme expression could affect the transcriptional dysregulation phenotype that is
associated with the expression of m-Htt in the R6/1 mouse striatum. hz vitro screening of
ribozymes against the HD transgene expressed in the R6/1 mouse resulted in the
identification of two highly active ribozymes. Surprisingly, rAAV-mediated expression
of these two ribozymes in the striatum of either R6/1 or age-matched wild-type mice
resulted in a marked decrease in the steady-state levels of pre-pro-enkephalin (ppEnk),
dompamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and the
dompamine receptor type-2 (D2R) mRNAs. We report that ribozymes targeting a region
just upstream of the CAG repeat domain in the human Htt mRNA mediate the cleavage
of an unidentified RNA molecule critical for neuronal function and survival.
Materials and Methods
Defining Target Sites
Target sites containing a GUC (HD6 and HD7), CUC (dsCAGl) or UUC
(dsCAG2) cleavage site were chosen from the human exon 1 Htt mRNA sequence
(Genbank accession number L273 50). Target regions with six nucleotides on either side
of the arms consisting of a 50% GC content were selected since an ideal length for the
target region is between 6-7 nucleotides. The presence of a U/A at the 3' cleavage site
also enhances the kcat ten fold. Selected target regions were examined using the RNA
folding algorithum designed by Micheal Zucker (REF) The folding program was used to
fold 100-200 nucleotides on either side of the NUX cleavage site to determine whether
the ribozyme binding location was acceptable. A BLAST search was also conducted to
ensure the absence of target sites on any other known human or mouse mRNA sequence.
Preparation of Short Ribonucleic Acid Target
The target oligonucleotide to be used in the cleavage reactions was radioactively
labeled at the 5'end using T4 polynucleotide kinase (New England Biochmeicals;
Beverly, MA). The reactions were set up as follows: 2C1l of the RNA target oligo
(10pmol/CIl, 20pmol total)(Dharmacon, Boulder, CO) was added to a mixture containing
lul of 10X polynucleotide kinase buffer (Promega, Madison, WI), lul RNASin
(Promega, Madison, WI), lul 0.1M DTT (Sigma, St. Louis, MO), 3 Cl water, 1Cl1 (gamma
32P) dATP (10uci) (ICN, Santa Clara CA) and 1Cl1 of polynucleotide kinase (5 units)
(Sigma, St. Louis, MO). The reaction was incubated at 37 OC for 30 minutes. 90C1l of TE
(Fisher, Swanee, GA) was added to the reaction prior to extraction of the unincorporated
nucleotides. A spin column (1ml syringe) was prepared with sterile glass wool and loaded
with sephadex (Sigma, St. Louis, MO) saturated in water. The column was centrifuged at
1000 RPM for 5 minutes to remove any excess water and to further pack the sephadex.
The 32P labeled target (100pl1) was loaded on to the column. The column was sealed with
parafilm and centrifuged again at 1000 RPM for 5 minutes. The labeled elute was
collected in a 1.5ml Eppendorf tube (Fisher, Swanee, GA) and was stored at -20 OC.
Time Course Analysis
Time course analysis was done by setting up a cleavage reaction as follows: 13C1l of
400mM Tris-HCL (Fisher, Swanee, GA), pH 7.4-7.5 were added to lul ribozyme
(2pmol) (Dharmacon, Boulder, CO) and 88ul of water. In order to properly fold the
ribozyme synthetic RNA, the above mixture was incubated at 65 OC for 2 minutes and
then left at room temperature for 10 minutes. 13 Cl of a 1:10 ratio of RNasin: 0. 1M DTT
was added to the reaction mixture along with 13 Cl of 200mM MgCl2 (20mM final)
(Sigma, St. Louis, MO). The reaction was incubated at 37 OC for 10 minutes. 1Cl1 of the
32P labeled (0.2 pmol) and 1Cl1 of unlabeled target (20pmol total) were premixed and
added to the reaction mixture at 37oC. For each time point, 10 Cll of the reaction mixture
was removed from 37 OC and added to a tube containing 10ul of formamide dye mix
(90% formamide (Sigma, St. Louis, MO), 50mM ehtylenediamine tetra acetic acid
(EDTA) pH 8 (Fisher,Swanee, GA), 0.05% bromophenol blue (Sigma, St. Louis, MO),
and 0.05% xylene cyanol (Sigma, St. Louis, MO). The samples were initially placed on
ice and then heat denatured at 90 OC for 3 minutes. The denatured samples were cooled
on ice before loading 6 Cll onto a 10% PAGE-8M urea gel to separate the products.
Bromophenol blue band was run about 2/3 down the gel. The gels were analyzed on a
molecular dynamics phosphoimager.
In Vitro Transcription of m-Htt mRNA
The human expanded HD transgene expressed in R6/1 mice was amplified from
genomic DNA using the sense primer HDPCR1 (5' -agggctgtcaatcatgctggc-3 ') and
antisense primer HDPCRla (5' -tctgggttgctgggtcactctg-3 '). This PCR fragment was
cloned into the TOPO TA vector following manufacturer's protocol (Invitrogen, San
Diego, CA). A HindIII / NotI fragment from the TOPO-R6/1 vector was cloned into the
pRC/CMV (Invitrogen, San Diego, CA) plasmid in the forward orientation with respect
to the cytomegalorivus immediate early promoter and a putative bacterial T7 promoter
sequence. Radio-labeled transcripts were generated using Ambion's MAXIscript In Vitro
Transcription Kit following manufacturer's instructions (Ambion, Austin, Tx).
Transcripts were kept frozen at -20 oC. Time course analysis was done with a ratio of
ribozyme to transcript of 5:1. Reaction conditions were similar to those described above.
Cloning of Ribozymes into rAAV Vectors
Two complimentary DNA oligonucleotides (Invitrogen, San Diego, CA) were
annealed in order to produce a double stranded DNA fragment coding for each
hammerhead ribozyme. All DNA oligonucleotides were synthesized with 5'phosphate
groups. The DNA oligonucleotides were designed to generate a cut HindIII site at the
5'end and a cut Nsil site at the 3' end after annealing. The DNA oligonucleotides were
incubated at 90 OC for 2 minutes and annealed by slow cooling to room temperature for
30 minutes. The resulting double stranded DNA fragment was ligated into the HindIII
and Nsil sites of the rAAV vector pTRUF-12 (UF vector Core,
http://www.gtc.ufl .edu/gtc-home.htm). A self cleaving hairpin ribozyme has been cloned
downstream of the inserted hammerhead ribozymes into a downstream Clal site.
Expression of the ribozyme cassette was placed under the control of the CMV, chicken-
beta-actin chimeric enhancer-promoter. The ligated plasmids were transformed into
SURE electroporation competent cells (Stratagene, La Jolla, CA) in order to maintain the
integrity of the rAAV inverted terminal repeats. Ribozyme clones were sequenced in the
University of Florida' s DNA sequencing core (ICBR).
Human Embryonic Kidney 293 Cells
HEK 293 cells were obtained from American Type Culture Collection (ATCC)
(Manassas, VA) and plated onto a 150 mm tissue culture dish. The cells were allowed to
attach and grow to about 80% confluency. To ensure a homogenous population of HEK
cells, the morphology of the HEK cells was recorded using a Zeiss microscope (Zeiss,
Transfections using Lipofectamine on HEK 293 Cells
HEK 293 cells were obtained from the American Type Culture Collection (ATCC)
(Manassas, VA) and plated onto a 150 mm tissue culture dish. HEK 293 cells were fed
with lX high glucose Dubellco's modified Eagle medium (Invitrogen, San Diego, CA)
containing 5 % fetal bovine serum (Invitrogen, San Diego, CA) twice weekly. The cells
were cultured at about 80% confluency at which time they would be split on a 1:10 ratio.
For transfection experiments, HEK 293 cells (ATCC, Manassas, VA) were seeded onto a
10-cm CorningTM tissue culture dish and allowed to attach and grow to 70-90%
confluency. LipofectamineTM and PlusTM reagents (Invitrogen, San Diego, CA) were used
for the transient co-transfection of pCMV-R6/1 and rAAV-ribozyme expression vectors
at a 1:5 or 1:10 pCMV-R6/1 to rAAV-ribozyme ratio. In order to control for transfection
efficiency, 293 cells were also co-transfected with pCMV-R6/1 and a pTR-UF 11
(rAAV-GFP) GFP vector at the same ratios. GFP expression was determined using a
fluorescence microscope at 24hr and 48hr. Cells were harvested 48hr post-transfection
RNA Isolation and Northern Analysis
Total RNA was isolated from HEK 293 cells by using the TRlzol reagent
(Invitrogen, San Diego, CA). Northern blot analysis was performed using standard
techniques. Briefly, 25 Cg of total RNA were separated on a formaldehyde-containing 1.2
% agarose gel. Fractionated RNA was then transfer unto Hybond N+ membranes using
alkalize transfer and probed with a 32P product of a Hindlll/Agel digest of the pCMV-
R6/1 vector (~ 150-bp of sequence spanning the 5'UTR and ATG translational start
sites). Sample loading was normalized by stripping and re-probing the same membranes
with a probe that recognizes nucleotides 150-270 of the human p-actin mRNA. Blots
were exposed to phosphoimager-ready intensifying screens and intensity of bands was
measured using a Phosphoimager scanner (Molecular Dynamics)
rAAV Vector Production
All rAAV vector preparations used in this study were made by the University of
Florida Vector Core facility (Powell Gene Therapy Center). Briefly, rAAV vector is
produced in human HEK 293 cells by transient CaPO4 precipitation-mediated co-
transfection of the rAAV expression vector plasmid and a helper-plasmid that encodes
the AAV rep and cap genes along with certain adenovirus genes (26). After 72 hours,
cells are harvested and stored at -80 oC. Cell are resuspended in 0.5% sodium
deoxycholate in 20 mM Tris, pH 8.0 and 150mM NaC1, treated with benzonase, and
cellular membranes are disrupted by three cycles of repeated freeze-thaws. Crude lysates
are purified using affinity chromatography, followed by cation exchange
chromatography. The Einal product is concentrated to a Einal titer of 1-5 X 1013 genOme
copies per ml.
R6/1 Transgenic Colony
Animal experiments were performed in the laboratory of Eileen Denovan-Wright,
PhD (Dept of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada).
Transgenic R6/1 HD mice were originally obtained from Jackson Laboratories and
breeding colonies were maintained at Dalhousie University. R6/1 males were crossed
with unrelated CBAxC57BL/6 females. After weaning, the mice were group housed
under 12 h light-dark cycle with ad libitum access to food and water. All mice were
genotyped at 3 weeks of age and at the time of death by amplifying a region of the human
huntingtin transgene using primers 5' AGG GCGT GTC AAT CAT GCT GG 3' and 5'
GGA CTT GAG GGA CTC GAA 3'. These primers correspond to a region upstream of
the CAG repeat at nts 77-96 and 347-364, respectively, of human huntingtin (Genbank
Accession number XM 003405). DNA was extracted from ear punches that were
digested with Proteinase K and used as the substrate for PCR using the REDExtract-N-
Amp Ready Mix (Sigma). Animal care and handling protocols were in accordance with
the guidelines detailed by the Canadian Council on Animal Care and were approved by
the Carleton Animal Care Committee at Dalhousie University.
Isofluorane was delivered by inhalation to anesthetize the mice. After induction of
anesthesia, the mice were placed into a sterotaxic frame (Kopf Instruments, Tujunga,
CA). All injections were performed using a continuous infusion system (Carnegie
Medicin, Sweden), attached to a 10 Cll Hamilton microsyringe fitted with a glass
micropipette with an outer diameter of 60-80 Clm (Mandel et al., 1999). The anterior-
posterior (AP) and medial-lateral (ML) stereotaxic coordinates were calculated from
bregma and the dorso-ventral (DV) coordinates were calculated from the dural surface.
A burr hole was drilled in the skull at the calculated AP and ML coordinates. Mice
received intrastriatal inj sections of rAAV2 or rAAV5 expressing HD6 or HD7 or GFP
suspended in phosphate-buffered saline (PB S) at a dose of 2 Cll/site and an infusion rate
of 0.5 Cll/min. During the infusion of rAAV, the glass pipette was slowly retracted 1 mm
every min. One minute after the cessation of the infusion, the micropipette was retracted
an additional 1 mm, allowed to remain at this position for 4 min and then slowly retracted
from the brain. The stereotaxic coordinates used for two injections within the same
striatum were: Site 1: AP = +1.0, ML = + 1.8, DV = -3.3; Site 2: AP = +0.4, ML = + 2.1i,
DV -3.4. The DV coordinate was calculated with the tooth bar set at 0.0 mm.
Ten-weeks after the intrastriatal infusion of rAAV, the mice were deeply
anesthetized with sodium pentobarbital (65 mg/kg i.p.) and decapitated. The brains were
removed and frozen at -70oC. Tissue sections (14 Clm) were cut using a Micron cryostat
through the rostral-caudal axis of the striatum, thaw-mounted onto Fischer SuperFrost
slides and stored at -70oC. For each slide, 5 sections, each separated by approximately
3 50 Clm, were placed on a single slide. This distribution of tissue was used to ensure that
each slide contained sections taken throughout the anterior-posterior regions of the mouse
In Situ Hybridization Analysis
In situ hybridization was performed on coronal sections (Bregma +1.70 to -0.50;
Franklin and Paxinos, 1997) of mouse brains using radiolabeled antisense gene-specific
oligonucleotide probes. Frozen sections were allowed to reach room temperature, fixed
with 4% paraformaldehyde in lX phosphate-buffered saline (PBS) for 5 min, rinsed
twice for 3 min in lX PB S, once for 20 min in 2X sodium chloride-sodium citrate (SSC),
and then air dried. Each slide was covered in 200 ul of hybridization buffer (50%
deionized formamide, 5X SSC, lX Denhardt' s reagent, 0.02 M sodium phosphate, ph
6.8, 0.2% SDS, 5 mM Na2EDTA, 10 ug/ml Poly(A)n, 10% dextran sulfate, 50 ug/ml
salmon sperm DNA, 50 ug/ml yeast tRNA) containing ~1 x 106 c.p.m./ml of
oligonucletide probe that had been 3' end-labeled with [ot-33P]dATP for 90 min at 37oC
using terminal deoxynucleotidyl transferase (Promega). Prior to use, unincorporated
nucleotides were removed from the labeled probes using a Sephadex G-25 spin column
(Pharmacia). The slides were coverslipped with parafilm and incubated overnight at 420C
in a humidified chamber. The coverslips were removed in 2X SSC and the slides were
washed for four times for 30 min at 550C in lX SSC, four times for 30 min at 550C in
0.5X SSC, two times for 30 min at 550C in 0.25X SSC, then rinsed briefly in H120 and
allowed to air dry overnight. Slides were exposed to Kodak Biomax MR film for up to 4
weeks at room temperature. The hybridization signals were analyzed using Kodak 1D
Image Analysis Software as described in Hebb et al. (2004). The sequences of the probes
used were: ribozyme probe (5'-cttacaccc cactcgtgcaggctgcccaggg-3 '), ppEnk
(5 'tctgcatccttcttcatgaaaccgccatacctcttggcaaca-'), DARPP-32 (5'-
tecacttggtcctcagagttgccatctctc-3 '), NGFi-A (5' -ccgttgctcagcagcatgatgtc
ctccagtttggggtagttgtcc-3 '), D2 receptor (5' -ggcagggttggcaatgatacactcattctggtcttgatt-',-
acti n (5 '-ggcgatccacacggagtacttgcgctcagg aggagcaatgatct-3 ') .
Time Course of Ribozyme Cleavage
We initially screened ribozymes that were directed against four different regions along
the mRNA sequence of the human HD transgene expressed in the R6/1 mouse. Figure 2-
1A is a schematic of all four ribozymes and their target sequences. HD6 and HD7
ribozymes (Rbz) were targeted to unique and overlapping sequences that lie between the
ATG translational start site and the polymorphic CAG repeat domain. dsCAG1 and
dsCAG2 were targeted to unique sequences immediately downstream of the expanded
CAG domain (Fig. 2-1B). Time course analysis of target cleavage was done for the HD6,
HD7, dsCAG1 and dsCAG2 hammerhead ribozymes as described in the methods section.
Figure 2-2 shows autoradiographs from 10% polyacrylamide-8M urea gels used to
separate the products of cleavage of each of the four ribozymes and their targets
following reactions performed at 37 OC and at 20mM MgCl2. The autoradiographs show
an increase in the 5'cleaved products over time and a corresponding decrease in the levels
of target. Significant product accumulation was detected almost immediately after the
addition of target to the reaction. Figure 2-2B shows a graphical representation of the
percent of product cleaved as a function of time for all four ribozymes tested. This
analysis demonstrated that HD6, HD7 and dsCAG1 could mediate the efficient cleavage
(~90% at the 10 minute time point) of target RNA in vitro. In contrast, dsCAG2 Rbz was
unable to cleave more than 5% of its target after 60 minutes of reaction time.
HD6, HD7 and dsCAG1 Activity Against In Vitro Transcribed m-Htt mRNA
Success of ribozyme-mediated suppression of gene expression requires the identification
of RNA target sites that are not masked by intrinsic RNA secondary structures. Although
algorithms such as the M-fold program predict RNA secondary structure, determining the
bio-availability of target sites requires an experimental approach. To examine the
accessibility of target sites, we cloned the expanded human HD transgene that is
expressed in the R6/1 mouse into a pRC/CMV (Invitrogen) expression vector (pCMV-
R6/1). This vector can drive both in vitro and in vivo transcription of the cloned gene. We
generated in vitro transcripts of the R6/1 transgene and performed cleavage reactions
under identical conditions as described for the short-target time course cleavage reaction
(see methods section). Figure 2-3A shows autoradiographs from 5% polyacrylamide gels
used to separate the products of cleavage of both HD6 and HD7 Rbz following
incubation with the R6/1 derived m-Htt transcript. Significant product accumulation from
the HD7 Rbz reaction was initially detected 15 minutes after the addition of Rbz to target
(Fig 2-3A, HD7 Rbz lane 1). There was an increase in the accumulation of the expected
size product over time. In contrast, HD6 Rz mediated cleavage of m-Htt mRNA in vitro
was not as efficient as the one observed with HD7 Rz (Fig 2-3). Nevertheless, the
accumulation of product of the expected size suggests that this target site is available for
binding and cleavage. In fact, as shown in Eigure 3-1B, HD6 and HD7 Rz have
overlapping sequences. The small but significant
Figure 2-1. Ribozyme design. Four ribozymes were designed to target unique sequences
in the human HD exon 1. In (A) ribozymes are shown annealing to their target
sequences with the NUX triplet depicted in color. B. Illustration of the four
target sites in relation to the CAG triplet repeat domain in human HD exon 1.
difference in turnover activity between these two Rbz might be explained by factors
influencing the release of product. dsCAG1 Rbz was unable to mediate the cleavage of
m-Htt transcript in vitro. This lack of enzymatic activity was likely due to the inability of
dsCAG1 Rbz to properly bind to its target site within the context of m-Htt RNA' s
0' i 10' IS' 30' 60' 120'
Time (mins) O' 5' 10' 30' 60' 120' 180' O/N
Target L "
O' 5' 10' 30' 60' 120' 180 O/N
0' 15' 30' 60' 120' 180' O/N
Time (mins) O 2 5' 10 30' 60 120' 180' O/N
Target *r at
Figure 2-2. Time course cleavage analysis. A. Autoradiographs from 10% acrylamide
gels used to separate the product of the reaction from the target. Target signal
decreased while a corresponding product signal accumulated as a function of
time. B. Graphical representation of the reactions shown above. Data is
presented as percent target cleaved. Ribozymes HD6 (closed circles), HD7
(open triangles) and dsCAG1 (closed triangles) were capable of cleaving >
80% 10 mins into the reaction while dsCAG2 was much slower (closed
12 34 5
GFP HD6 HD7
b-actin mnRNA L ---
Figure 2-3. Target accessibility. A. Autoradiographs from 5 % acrylamide gels separating
the products of a time course analysis of two highly active ribozymes, HD6,
HD7. Both ribozymes were incubated with an in vitro transcribed human HD
exon1 transcript (top left arrow) to determine the accessibility of the target
sites within the context of a folded RNA molecule. Lanes 2-through-4 are the
15, 30, and 60 min time points for each reaction. Lane 1, in both
autoradriographs, is a 60 min time point of a reaction that was devoid of
ribozyme. Expected size products for both HD6 and HD7 accumulated over
time (top and bottom right arrows). HD6 Rbz was less active against the in
vitro transcribed human HD exon1 transcript (Lane 5 is an overnight
incubation time point). Panel (B) is a northern blot analysis (top
autoradiograph) of m-Htt mRNA. Total RNA was obtained from HEK 293
cells that were transiently co-transfected with pCMV-R6/1 (m-Htt) and either
a GFP, HD6 or HD7 expression plasmid. Increasing the concentration of HD6
Rbz vector from a 1:5, target to ribozyme ratio (lane 3), to a 1:10, target to
ribozyme ratio (lane 4), resulted in an increase in activity as determined by the
intensity of the m-Htt mRNA band. HD7 Rbz activity against m-Htt mRNA
Figure 2-3 continued
remained constant at either 1:5 (lane 5) or 1:10 (lane 6) target to ribozyme
ratio. The same blot was probed with a p-actin probe to normalize sample
loading. Both ribozymes reduced m-Htt mRNA levels by > 60% when
compared to controls.
HD6 and HD7 Ribozyme Activity Against m-Htt mRNA Inz Vivo
We next examined the activity of HD6 and HD7 Rbz against m-Htt mRNA in
cultured HEK293 cells. HD6 and HD7 Rbz were cloned into a rAAV vector specifically
engineered for the intracellular expression of HH Rbz (Figure 2-4A). This expression
cassette generates a transcript that is processed by a self-cleaving hairpin ribozyme
strategically placed downstream of the sequence encoding for either HD6 or HD7 Rbz.
The self-cleaving activity of the hairpin ribozyme results in the release of a small HH
Rbz-containing RNA and a transcript with an internal ribosomal entry site (IRES)
sequence to facilitate the translation of the downstream GFP sequence.
In order to test the activity of these Rbz against m-Htt mRNA in culture, HEK293
cells were transiently co-transfected with CMV-R6/1 and either rAAV-HD6 or rAAV-
HD7 Rbz vectors. Northern blot analysis of total RNA obtained forty-eight hours post-
transfection showed a significant decrease (>60% of control) in the levels of m-Htt
mRNA in samples obtained from both rAAV-HD6 and rAAV-HD7 transfected cells (Fig.
2-3B). This decrease was not evident in cells that were co-transfected with a rAAV-GFP
expressing vector. Interestingly, increasing the rAAV-HD6 Rbz to target ratio from 1:5 to
1:10 resulted in a significant increase in cleavage activity (Fig 2-3B lanes 3 and 4).
In2 vivo Activity of rAAV-HD6 and rAAV-HD7 Ribozymes
The focus of this study was to evaluate the effects that reduced striatal levels of
m-Htt would have in the striatal-specific transcriptional dysregulation associated with the
expression of m-Htt protein in the R6/1 mouse model of HD. Initially, we generated
rAAV type 2 (rAAV2) particles encoding for HD6, HD7 or GFP control (see Methods).
Intrastriatal inj sections of high-titer rAAV2 resulted in intense and localized transduction
of the mouse striatum (Fig. 2-4B). Although the levels of viral expression attained with
Figure 2-4. In vivo expression of rAAV ribozyme vectors. (A) Diagram depicting the
ribozyme expression cassette that was packaged into rAAV viral particles. A
hairpin self-cleaving ribozyme (self-cleaving Rz), placed immediately
downstream from the HD6 and HD7 cloning sites, was used to mediate the
processing of the primary Rbz-containing transcript. B. In situ hybridization
analysis of the viral RNA transcript was done on coronal sections obtained
from mice inj ected with either rAAV2 or rAAV5-HD7 vector. The ribozyme-
specific probe was allowed to hybridize with the sections under identical
rAAV2 were remarkably high, the feasibility of this study was dependent upon extensive
striatal transduction. We therefore generated rAAV viral vectors pseudotyped with the
capsid from rAAV serotype-5 (rAAV5) encoding for HD6 (rAAV5-HD6 Rbz), HD7
(rAAV5-HD7 Rbz) and GFP (rAAV5- GFP). Our rationale was based on a recent elegant
b-actin Exon1 Active Rbz
study showing that intrastriatal delivery of rAAV5 particles in the rat brain results in a
larger transduced area when compared to rAAV2 vectors (13). Intrastriatal delivery of
rAAV5-HD6 or HD7 Rz in R6/1 and age-matched wild-type mice resulted in a dramatic
increase in the brain transduced area when compared to rAAV2 particles as demonstrated
by in situ hybridization (ISH) analysis using a Rbz-specific probe (Fig. 2-4B).
To determine the in vivo activity of rAAV5-HD6 and rAAV5-HD7, R6/1 and
age-matched wild-type littermate controls were divided into the following experimental
groups: rAAV5-HD6 (R6/1 n=8, wild-type n=5), rAAV5-HD7 (R6/1 n=8, wild-type n=5)
and rAAV5-GFP (R6/1 n=5, wild-type n=5). Six week-old mice were injected
unilaterally at two different striatal sites in the right hemisphere while the left hemisphere
remained uninj ected and served as control (Fig 2-4B). Ten weeks post-surgery fresh
frozen brains were obtained, sectioned and maintained frozen throughout the remainder
of the experiment.
Biological Effects of rAAV5-HD6 and HD7 Ribozyme Expression in the R6/1 Mouse
Alterations in gene transcription occur prior to the display of motor abnormalities
in the R6/1 mouse ofHD. Striatal-specific genes known to be downregulated in HD
include the dopamine-, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), pre-
pro-enkephalin (ppEnk), nerve growth factor-inducible A (NGFi-A) and the dopamine
type-2 receptor (D2R).Reduction in the steady-state mRNA levels of these transcripts is
both progressive and modulated by the length of the expansion repeat and the expression
levels of m-Htt. Thus, ribozyme-mediated knockdown of m-Htt expression should lead to
an increase in the transcript levels of ppEnk, DARPP-32, D2R and NGFi-A.
To test this hypothesis, we performed in situ hybridization analysis with probes
specific to either NGFi-A, DARPP-32, ppEnk and D2R mRNAs (Fig 2-5). Analysis of
coronal sections obtained from R6/1 mice unilaterally injected with rAAV5-HD6 or -
HD7 ribozyme showed an unexpected sharp decrease in the mRNA levels of ppEnk,
DARPP-32 and D2R around the area of transduction. Unexpectedly, expression of either
rAAV5-HD6 or HD7 Rz exacerbated the rate of mRNA loss for most of the analyzed
transcripts in the R6/1 mouse striatum (Fig 2-5A bottom panel). In contrast, there was a
significant induction of NGFi-A mRNA in the right inj ected striatum of the R6/1 mouse.
To further characterize this loss in mRNA steady-state levels, we analyzed sections
obtained from inj ected age-matched wild-type mice. ISH analysis revealed a similar
pattern of loss in transcriptional activity, albeit to a lesser degree (Fig. 3-5A top panel).
This effect was specific to the intracellular expression of Rbz in striatal tissue since
cultured HEK293 cells did not exhibit any HD6 or HD7 Rbz-associated toxicity.
Furthermore, analysis of sections from rAAV5-GFP animals showed that long-term
expression of rAAV5-GFP does not lead to detectable changes in the steady-state levels
of either NGFi-A or D-32 mRNA (Fig. 2-5B).
We next investigated if this effect was associated with the expression of the self-
cleaving hairpin ribozyme or the IRES-GFP transcript, which are encoded by our rAAV
expression cassette (Fig. 2-4A). We inj ected wild-type mice with a rAAV2-HD7 Rz in
the right hemisphere and rAAV2-hAAT Rz in the left hemisphere. hAAT Rz targets the
human alpha-1 anti-trypsin (hAAT) liver enzyme which is not expressed in neuronal
tissue. As shown by ISH analysis, rAAV2-HD7 Rz increased the steady-state levels of
NGFi-A while causing a loss in the levels of D-32 mRNA (Fig. 2-5C).
151I Probe: Rz NGFiA D 32 ppEnk D2R b-actin
ISH Probe: GF;P NGFiA D -32
ISH Probe: hkAAT/ HD7 N17ri : D-32
Figure 2-5. rAAV5-HD7 expression results in a loss of striatal-specific transcripts. A. Dr
situ hybridization analysis of coronal sections obtained from a wild-type (WT)
and a R6/1 (HD) mouse. Comparison between genotypes revealed a marked
difference in the mRNA levels of NGFi-A, DARPP-32 (D-32), ppEnk and D2
receptor (left hemispheres). rAAV5-HD7 expression led to a remarkable loss
in DARPP-32, ppEnk and D2 receptor mRNAs in both inj ected R6/1 and
wild-type mice. In contrast, NGFi-A mRNA was increased to levels that were
~20% above wild-type in both wild-type and R6/1 inj ected mice. Panel (B)
are coronal sections obtained from wild-type mice inj ected with a rAAV5-
GFP vector. Dr situ hybridization analysis demonstrated that the loss in
hybridization signal intensity of DARPP-32 and the increase in the levels of
Figure 2-5 continued
NGFi-A were specific to striatal expression of the HD7 ribozyme. C. Coronal
sections obtained from wild-type animals expressing either rAAV2-HD7
ribozyme or a ribozyme targeting hAAT (rAAV2-hAAT). Analysis of
DARPP-32 and NGFi-A expression revealed that the loss and gain in mRNA
levels was specifically associated with the HD7 target sequence. Arrows on
(A) and (C) denote the inj ected hemisphere.
Ribozymes Can Modulate the Expression of Cellular Genes
There are a number of reports on the ability of the hammerhead ribozymes to
control expression of specific genes in cell culture. For example, a hammerhead ribozyme
designed to cleave mRNA encoding C-Ha Ras mutation inhibited formation of foci of
transformed cells by 50% (22). Hammerhead ribozymes have also been used to target
oncogenes. The use of hammerhead ribozymes targeted to survivin, which is expressed in
carcinoma cells, resulted in up to 74% reduction in the levels of surviving mRNA (76).
Finally, hammerhead ribozymes have also been shown to effectively reduce the levels of
a rhodopsin mutant protein in vivo (34, 47). Taken together these data demonstrate that
hammerhead ribozymes are capable of modulating the expression levels of targeted
genes. This study demonstrates that hammerhead ribozymes can also be designed to
suppress the expression of m-Htt in culture. However, upon detailed examination, our in
vivo results showed an off-target effect associated with the expression of both HD6 and
HD7 hammerhead ribozymes. This effect raises questions concerning the efficiency and
safety of ribozymes as tools in molecular medicine.
Inz Vivo use of Ribozymes Directed Against the R6/1 Transgene
HD is a progressively devastating neurological disorder that currently affects the
lives of approximately 30,000 individuals in the United States alone. Although
pharmacological interventions exist that are designed to alleviate some of the
excruciating symptoms associated with this disorder, HD remains largely without an
effective treatment (53). The overall goal of this project was to develop a gene transfer
strategy based on the rAAV-mediated delivery of anti-m-Htt hammerhead ribozymes in
the striatum of the R6/1 mouse model of HD. This strategy was aimed at reducing the
levels of striatal m-Htt in order to prevent, reverse or ameliorate the symptoms associated
Four hammerhead ribozymes directed against the HD transgene expressed in the
R6/1 mouse were designed and characterized in vitro. We showed that co-expression of
either rAAV5-HD6 or HD7 Rbz with a m-Htt expression vector (pCMV-R6/1) results in
significant reduction in the relative levels of m-Htt mRNA (> 60%) as determined by
densitometric analysis. However, in vivo expression of rAAV5-HD6 or HD7 Rbz in the
striatum of R6/1 mice resulted in a loss in the mRNA levels of striatal-specific genes
known to be critical for neuronal function. This reduction in striatal mRNA levels was
not due to a global suppression of transcriptional activity since the steady-state levels of
the NGFi-A transcript were induced to levels that were ~20% that of control.
This result was unexpected and contrary to our initial hypothesis. Furthermore, in
situ hybridization analysis of sections obtained from age-matched treated wild-type mice
showed a reduction in the steady-state mRNA levels of the same striatal-specific genes.
Since both of these ribozymes were directed against the human HD transgene in the R6/1
mouse and should not have cleaved any other known mouse RNA sequence, the loss of
mRNA levels in wild-type animals lead us to conclude that there is an off-targeting effect
associated with the striatal expression of HD6 or HD7 ribozymes which was not evident
in cultured HEK293 cells. This off-targeting effect results in the cleavage of an
unidentified RNA transcript that codes for a protein whose expression is necessary to
neuronal function as evidenced by the loss of a subset of neuronal and striatal specific
mRNAs in wild-type mice.
In an attempt to identify this transcript, we performed BLAST searches for short,
nearly exact matching sequences. We limited the search, to sequences exhibiting greater
than 50% sequence similarity to the HD6 and HD7 target site. Additionally, we only
analyzed sequences that contained the required GUC triplet cleavage site and are
expressed in neural tissue. Initial screens did not yield any significant matches to the
human HD sequence spanned by the HD6 and HD7 target sites. However, the inclusion
of expressed sequence tags (ESTs) during the search resulted in the identification of 6
sequences that have up to 60% similarity with the ribozymes target sites, contained the
GUC triplet and are expressed in cultured neurospheres. The identification of the gene, or
genes, responsible for the loss in striatal-specific mRNA levels might lead to the
elucidation of an unknown gene function.
In this study we limited the designed of hammerhead ribozymes to sequences that
are contained within the human HD transgene expressed in the R6/1 mouse. This
transgene contains approximately 1 kb of 5'UTR sequence, an expanded exon 1 (117
CAG repeats) and 262 bases of intron 1. The design of ribozymes is limited by the NUX
triplet rule where N is any nucleotide, U is uracil and X is any nucleotide but guanosine
(Fig. 2-1). The small size of the transgene made the search for cleavage target sites
extremely challenging. Furthermore, the identification of potential target sites does not
necessarily correlate with the design of highly active hammerhead ribozymes as it was
demonstrated in this study (Fig. 2-2A). These two factors limit our ability to test our
underlying hypothesis using hammerhead ribozymes. Although the use of a full-length
HD mouse model would increase the success probability of applying the use of
hammerhead ribozymes to post-transcriptionally modulate the levels of m-Htt protein,
these models have slow, late-onset phenotypes that have not been fully described.
Recently, the use of RNAi in a neurological model of disease led to cellular and
behavioral improvements (91). This new technology is not dependent on NUX target
rules and results in a more stable and long-term silencing effect. The application of RNAi
in HD will be the focus in the next chapter.
RNA INTERFERENCE OF MUTANT HIUNTINGTIN IN VIVO
The genesis and progression of disease in dominant genetic disorders, such as HD,
require the presence and expression of a mutant allele. Nucleic acid-based molecules
have been designed to inhibit gene expression by sequence-specific targeting of mutant
mRNAs. It has been demonstrated that the expression of short, hairpin-like double-
stranded RNA (shRNA) molecules in neuronal cells, both in vivo and in vitro, results in
the sequence specific silencing of targeted genes (16). In fact, the successful in vitro
application of synthetic siRNAs against members of the polyglutamine family of
diseases, including HD, was recently reported (52, 57). This suppression was achieved by
targeting siRNAs to either the CAG repeat region or to adj acent gene sequences and
could even be designed to be allele-specific.
RNAi has also proven to be an efficient therapeutic tool in in vivo models of pQ
disease. A recent report demonstrated the full potential of this technology in a transgenic
mouse model of SCA-1 (91). In this experiment, cerebellar long-term expression of an
anti-ataxin-1 siRNA was achieved by delivering an shRNA expression cassette with a
recombinant adeno-associated viral vector. shRNA expression resulted in greater than
50% reduction of ataxin-1 protein in the cerebellum. Reduction in ataxin-1 transgene
expression was associated with a rescue of the Purkinke cells as well as significant
behavioral improvements in this model of SCA-1. This elegant study, addressing the
applicability of RNAi in neurological disease, suggests that the pathological mechanism
associated with HD could be effectively halted or reversed by introducing siRNAs that
can target the striatal expression of mutant Htt.
In this study, we examined the effects of RNAi mediated post-transcriptional
silencing of striatal m-Htt in the R6/1 mouse. We specifically assessed whether reduced
levels of striatal m-Htt could affect the transcriptional dysregulation phenotype that is
associated with the expression of m-Htt in the R6/1 mouse striatum. Long-term in vivo
expression of two rAAV5-shRNA vectors lead to the significant reduction in striatal m-
Htt mRNA and protein levels as determined by real-time quantitative RT-PCR, western
blot and immunohistochemistry analysis. This reduction was concomitant with a decrease
in both the size and number of neuronal intranuclear inclusions (NIIs). Finally, reduced
levels of striatal m-Htt resulted in an increase in the steady-state levels of ppEnk and
DARPP-32 transcripts as well as in mild-behavioral improvements.
Materials and Methods
rAAV-shRNA Plasmid Construction
Two complimentary DNA oligonucleotides (Invitrogen, San Diego, CA) were
allowed to anneal in order to produce a double-stranded DNA fragment coding for the
sense strand, 9 nucleotide loop and antisense strand of both anti-huntingtin shRNAs
tested. siHIUNT-1 targeted nucleotides 262-281 (5'-GCCGCGAGTCGGCCCGAGGC-
3', Fig. 3-1B) and siHIUNT-2 targeted nucleotides 342-363 (5'-
GGCCTTCGAGTCCCTCAAGTCC-3 ', Fig. 3-1B) of the human HD mRNA (Genbank
accession no. L12392). Double-stranded DNA fragments were ligated into the
Bgl2/HindlII sites of the rAAV vector pSOFF-Hlp-hrGFP. This vector contains the
human RNAse P H1 promoter downstream of the rAAV serotype-5 ITR and a cDNA
encoding the humanized Renilla reniformis GFP (Stratagene, La Jolla, CA) protein under
the control of the herpes simplex virus thymidine kinase (HSV-TK) promoter.
Testing of shRNA Efficacy in Cultured Cells
A portion of the mHtt transgene was PCR amplified from R6/1 mouse genomic
DNA using forward (5' -AGGGCTGTCAATCATGCTGG-3 ') and reverse (5'-
TCTGGGTTGCTGGGTCACTCTGTCTCTGCGGAGCCGGGGG-3') primers. The
resulting product was cloned into pCR 2. 1-TOPO TA cloning kit (Invitrogen, Carlsbad,
CA), sequenced and subcloned into the Hindlll/Nsil sites present in the pRC/CMV
(Invitrogen, Carlsbad, CA) eukaryotic expression plasmid. The resultant plasmid
(pCMV-R6/1) expressed part of the 5'UTR, the coding region within exon 1 with~-115
contiguous CAG repeats, and part of intron 1 of the mHtt transgene under the control of
the minimal cytomegalovirus (CMV) promoter.
Human embryonic kidney 293T (HEK293) cells (ATCC, Manassas, VA) were
seeded onto a 10-cm CorningTM tissue culture dish and allowed to attach and grow to 70-
90% confluency. LipofectamineTM and PlusTM reagents (Invitrogen, San Diego, CA) were
used for transient co-transfection of HEK293 cells with pCMV-R6/1 and rAAV-shRNA
vectors at a ratio of 1:4 and 1:8 respectively. In order to control for non-specific off-
targeting effects, we performed co-transfection of the pCMV-R6/1 plasmid with a rAAV-
shRNA vector targeting the dog rhodopsin mRNA (siRho-1). rAAV-siRho-1 has been
shown to be active against its target both in vitro and in vivo (M. Gorbatyuk and A.
Lewin manuscript submitted). Transfection efficiency was determined by analysis of
GFP-positive cells and ranged from 70-80%. All transfected cells were harvested 48 hr
post-transfection for northern and western analysis. Total RNA was isolated from
transfected HEK293 cells using TRlzolTM reagent (Invitrogen, San Diego, CA). Northern
blot analysis was performed using standard techniques ("Current Protocols in Mol Biol,
Wiley editing house). Briefly, 10 Clg of total RNA was fractionated on a 1.2 %
formaldehyde agarose gel and transferred to Hybond N+ (Amersham, Piscataway, NJ)
membrane by capillary action. pCMV-R6/1 was subj ected to restriction enzyme digestion
using HindlII and Agel, the 150 bp fragment spanning the 5' UTR and ATG initiation
codon of the mHtt transgene was gel-purified, radio-labeled using [ot-32P]dCTP (3000
mCi/ml; MP Biochemicals, Irvine, CA) and used as the hybridization probe. RNA
loading was normalized by removing the mHtt-specific probe and re-probing the
membrane with a radio-labeled fragment complementary to nucleotides 150-270 of the
human p-actin mRNA (Genbank accession no. BC002409). Blots were exposed to
phosphoimager-ready intensifying screens and the intensity of labeled bands was
determined using a Molecular Dynamics phosphoimager.
Western Blot Analysis using Hum-1 Antibody
A polyclonal antibody (Hum-1) was raised in New Zealand white rabbits against
the human Htt-synthetic peptide Ac-PQLPQPPPQAQPLLPQPQC-OH and affinity-
purified (New England Peptide, Gardner, Massachusetts ). Pre-immune serum was used
as a control. Western blot analysis was performed by blocking the membranes for 2 hr at
room temperature in 5% (w/v) skim milk powder in 20 mM Tris, 146 mM NaC1, 0.1%
Tween-20 (TBST), incubating the membranes in a 1:500 dilution of affinity-purified
polyclonal rabbit anti-human huntingtin transgene protein (Hum 1) antibody in 5% (w/v)
skim milk powder/TBST at 4o C overnight. Membranes were washed 2 x 20 min in
TBST. A peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:2000, Vector
Laboratories, Burlingame, CA) was incubated for 1 hr at room temperature in 5% skim
milk powder/TBST. Interaction between the primary and secondary antibodies was
detected using West Pico SuperSignal@ chemiluminescent substrate using the protocol
recommended by the manufacturer (Pierce Biotechnology, Rockford, IL) and Hyperfilm
ECLTM (Amersham Pharmacia Biotech, England)
rAAV Vector Production
All rAAV vector preparations used in this study were produced by the University
of Florida Powell Gene Therapy CenterVector Core facility using the method described
in . The rAAV5 vector used in this study consisted of rAAV5 capsids and AAV5
ITRs and is therefore not a pseudotyped vector (13). A standard triple transfection
method using the helper DNAs pDG  for required adenoviral proteins, our transgene
plasmid described above and pAAV5.2 which has rep and cap from AAV5. Crude
rAAV5 virus was then purified by iodixanol step gradients and Sepharose Q column
chromatography as previously described (13). The final product is concentrated to final
titers of 1 to 5 X 1013 genOme copies per ml.
Intrastriatal Injection of shRNA-Expressing AAV Vectors.
All animal care, handling and surgical protocols were in accordance with the
guidelines established by the Canadian Council on Animal Care and were approved by
the Carleton Animal Care Committee at Dalhousie University. Transgenic R6/1 HD
mice were obtained from Jackson Laboratories and used to establish a breeding colony at
Dalhousie University. R6/1 males were crossed with unrelated CBAxC57BL/6 females.
After weaning, the mice were group housed under a 12 h light-dark cycle with ad libitum
access to food and water. All mice were genotyped at 3 weeks of age and at the time of
death by amplifying a region of the human HD transgene as described previously (36).
Stereotaxic administration of AAV vectors were performed on 6 to 8 week-old
wild-type and R6/1 mice under isofluorance anesthesia. The anterior-posterior (AP) and
medial-lateral (ML) stereotaxic coordinates for inj section were calculated from bregma
and the dorso-ventral (DV) coordinates were calculated from the dural surface. Mice
received intrastriatal inj sections of rAAV5 expressing siHIUNT-1, siHIUNT-2 or TRUF 11
(rAAV5-hrGFP) suspended in phosphate-buffered saline (PBS) at a dose of 2 Cll/site and
an infusion rate of 0.5 Cll/min using a continuous infusion pump, attached to a 10 Cll
Hamilton microsyringe fitted with a glass micropipette with an outer diameter of 60-80
Clm. One minute after the cessation of the infusion, the micropipette was retracted an
additional 1 mm, allowed to remain at this position for 4 min and then slowly retracted
from the brain. The stereotaxic coordinates used for the two inj sections within the same
striatum were: Site 1: AP = +1.0, ML = + 1.8, DV = -3.3; Site 2: AP = +0.4, ML = + 2. 1,
DV -3.4 (All coordinates were measured with an empirically determined flat skull).
The effect of shRNA and control vectors on mHtt mRNA and protein levels was
assessed in a group of animals that received unilateral intrastriatal inj sections of rAAV
vectors. In these experiments, the contralateral hemisphere was used as an internal
control. Ten-weeks after the intrastriatal infusion of rAAV, mice were deeply
anesthetized with sodium pentobarbital (65 mg/kg i.p.) and decapitated. The brains were
removed and stored at -700C. Tissue sections (14 Clm) were cut using a Micron cryostat
through the rostral-caudal axis of the striatum, thaw-mounted onto Fisher SuperFrostTM
slides and stored at -700C. For each animal, 5 coronal brain sections, each separated by
approximately 3 50 Clm, were placed on a single slide. This distribution of tissue was used
to ensure that each slide contained sections taken throughout the transduced region of the
To determine the levels of mHtt RNA and protein, striatal tissue was isolated from
frozen sections that had been thaw-mounted on slides. Cortical tissue was removed using
a razor blade and discarded. To collect tissue mainly from the transduction area, sections
were first examined under epi-fluorescence to visualize the transduction area. The striatal
tissue from the right and left sides of 5 brain sections per animal were then manually
scraped from each slide taking only the approximate area of GFP positivity.
RNA was extracted using Trizol TM (Invitrogen, San Diego, CA), quantified
spectrophotometrically and used as the substrate for reverse-transcriptase (RT) reactions
to generate single-stranded cDNA. The reaction was optimized to reverse transcribe the
5' end of the human Htt transgene. Briefly, 1 Clg of total RNA and 0.75 Clg of random
hexamers were incubated with 1 Cll of 5X Q solution (Qiagen, Valencia, CA) in a total
volume of 5.75 Cll at 70oC for 3 min, mixed and placed on ice for 5 min. M-MLV RT
buffer and dNTPs were added to a final concentration of lX and 1.25 C1M, respectively,
in a final reaction volume of 10 Cll. 20 Units ofRNasin (Promega, Madison, WI) and 100
Units of M-MLV RT (Promega, Madison, WI) were added and the reaction was allowed
to proceed at 48oC for 60 min. The reaction was terminated by heating at 70oC for 10
min. -RT reactions differed from +RT reactions by the substitution of H20 for RT in the
reactions. Quantitative PCR (Lightcycler, Roche) was used to amplify a 115 bp region of
mHtt cDNA from striatal tissue. The PCR reactions contained lX QuantiTect SYBR
Green PCR master mix (Qiagen), 500 nM each of sense (5'-
AGAGCCCCATTCATTGCC- 3') and antisense (5' -GGACTTGAGGGACTCGAA-3 ')
primer. Cycling conditions included a denaturation step of 950C for 15 min, followed by
45 cycles of 940C for 15 s, 580C for 20 s, and 720C for 20 s, and ending with a melting
step from 650C to 990C over 30 s. Total SYBR green fluorescence was measured at the
end of each PCR cycle and continuously through the melting step. Known quantities of
mHtt cDNA were simultaneously amplified with experimental samples and used to create
a standard curve. The levels of cDNA in each sample were normalized to the levels of
hypoxanthine ribosyl transferase.
For western blot analysis, striatal tissue from 4-5 sections per animal were isolated
and homogenized in 0.32 M sucrose, quantified using the BCA protein determination
assay (Pierce), fractionated on SDS/PAGE gels using standard protocols and subj ected to
the immunoblotting conditions described for analysis of mHtt protein levels in transiently
transfected HEK293 cells.
For analysis of the distribution of immunoreactive inclusion bodies, fresh-frozen
coronal brain sections on SuperFrost slides were allowed to come to room temperature,
rinsed 3 x 10 min with phosphate-buffered saline (PBS: 100 mM phosphate, pH 7.4, and
0.9% NaC1) and the tissue was Eixed in 4% (v/v) parafomaldehyde for 15 min. The tissue
was incubated in 0.1% (v/v) H202, 10% methanol and PBS for 10 min and rinsed 3 x 10
min in PBS. Non-specifie binding sites were blocked in a solution of 5% normal goat
serum in 0.25% (v/v) Triton-X100/0.01 M PBS for 1 hr at room temperature.
Immunostaining was performed using a 1:500 dilution of affinity-purified Hum 1 or
1:4000 dilution of polyclonal rabbit anti-ubiquitin (IgG) antibody (DakoCytomation,
Carpinteria, CA) in 1% (v/v) normal goat serum, 0.25% (v/v) Triton-X100/ PBS. Tissues
were incubated overnight at 4o C. A 1:500 dilution of goat anti-rabbit biotin-labeled
secondary antibody (Vector, Burlingame, CA) was prepared in 1% (v/v) normal goat
serum, 0.25% (v/v) Triton-X100/0.01 M PBS. Slides were incubated in secondary
antibody solution for 2 hr at room temperature then rinsed 3 x 10 min in 0.01 M PBS.
Interaction between the primary and secondary antibodies was detected using the
Vectastain Elite ABC Kit (Vector, Burlingame, CA) and DAB using the company's
The size and distribution of Hum-1 immunoreactive NIIs were determined by using
the Analyze Particle function in the Image JNIH software (freeware available at
http://rsb .info.nih.gov/ij/). Briefly, the relative size (Clm2) and amount of NIIs were
determined by analyzing coded digital images of four different regions (left motor cortex,
right motor cortex, left medial striatum, right medial striatum) in coronal sections that
were subj ected to the stringent immunohistochemical procedure described above.
Fourteen Clm sections were too thin for unbiased stereological estimates of NII number
and the NIIs diameter was too small to use the rotator method of volume estimation .
The Image J software (NIH) was configured to detect particles of a specified size range
(0.03 Clm2-0.5 Clm2) and a constant upper and lower threshold value was used for all
grayscale images from all areas analyzed. The mean relative size area (Clm2) Of particles
(NIIs) as well as the mean number of particle counts within the specified size range was
calculated by a blinded operator for each of the four different regions in all of the
unilaterally inj ected R6/1 mice and reported as percent of the size and number of NIIs
present in the left motor cortex. Since absolute numbers or areas are not reported and
internal section controls were used to determine percent controls, this method of
quantification ofNIIs is valid.
In Situ Hybridization Analysis
In situ hybridization was performed on coronal mouse brain sections (Bregma
+1.70 to -0.50) using radio-labeled antisense gene-specific oligonucleotide probes (Table
1). Frozen sections were allowed to reach room temperature, Eixed with 4%
paraformaldehyde in lX PBS PBS for 5 min, rinsed twice for 3 min in lX PBS, once for
20 min in 2X sodium chloride-sodium citrate (SSC, 0. 15 M NaC1, 0.015M NaCitrate, pH
7.4), and then air dried. Each slide was covered in 200 Cll of hybridization buffer (50%
deionized formamide, 5X SSC, lX Denhardt' s reagent, 0.02 M sodium phosphate (pH
6.8), 0.2% SDS, 5 mM Na2EDTA, 10 Clg/ml Poly(A)n, 10% dextran sulfate, 50 Clg/ml
sheared salmon sperm DNA, 50 Clg/ml yeast tRNA) containing ~1 x 106 c.p.m./ml of
oligonucletide probe that had been 3' end-labeled with [ot-33P]dATP (2000 mCi/ml) for
90 min at 370C using terminal deoxynucleotidyl transferase (Promega, Madison, WI).
Prior to use, unincorporated nucleotides were removed from the labeled probes using a
Sephadex G-25 spin column (Amersham Biosciences, Buckinghamshire, UK). The slides
were coverslipped with parafilm and incubated overnight at 420C in a humidified
chamber. The coverslips were removed in 2X SSC and the slides were washed for four
times for 30 min at 550C in lX SSC, four times for 30 min at 550C in 0.5X SSC, two
times for 30 min at 550C in 0.25X SSC, then rinsed briefly in H120 and allowed to air dry
overnight. Slides were exposed to Kodak Biomax MR fi1m for up to 4 weeks at room
temperature. The hybridization signals were analyzed using Kodak 1D Image Analysis
Software as described in (36).
For display purposes in order to make differences in hybridization signal easier to
visualize, hybridized sections were scanned and false-colored in Adobe Photoshop TM 7.0
by changing the image to RGB color and then a gradient map (as shown at the bottom of
Fig. 3-5) was applied. This was done to all coronal sections shown in figure 3-5 without
any further manipulation.
Effect of Anti-mHtt shRNA on Phenotype
The effect of anti-mHtt shRNA and control vectors on phenotype were assessed in
animals that received bilateral inj ectons of siHIUNT-1, siHIUNT-2 and control rAAV
vectors. Mice were weighed weekly following injection of rAAV. Average weight,
grouped by genotype and treatment, was determined for each week, and significant
differences determined using ANOVA Hind limb clasping behavior was assessed by
suspending the mice 30 cm above a flat surface by their tail for 60 seconds or until the
mice curled and clasped their hind limbs with their forelimbs and maintained the posture.
The percentage of mice per group at each time point that exhibited clasping was
recorded. Motor coordination was analyzed by placing the mice on rotarod that increased
from 0 to 40 rpm over 1 min. The time from placing the mouse on the rotarod until the
mouse fell off was recorded for each of 4 trials and the average time that each mouse
remained on the rotarod was recorded.
Analysis of variance (ANOVA) was used to evaluate the probability of differences
between experimental groups. Where appropriate, one-way ANOVA was performed and
individual post-hoc differences between groups was assessed using Fisher' s paired-least-
significant-difference test (Fisher's PLSD as available in Statview). Post-hoc differences
in two-way ANOVA designs were assessed in a hierarchical fashion as described by Kirk
et al (8) using simple main-effects analysis. The minimum probability accepted for
significance (a level) was 0.05.
RNA Interference of Mutant Huntingtin In Vitro
R6/1 trangenic HD mice express exon 1 of human Htt with -115 CAG repeats .
We designed short-hairpin RNAs (shRNA) (20 to 21 nucleotides in length) that could
target specific sequences of exon 1 of human Htt, but did not have significant sequence
similarity to the endogenous mouse Htt mRNA. We generated rAAV5-based DNA
constructs expressing two anti-mHtt shRNA molecules (Fig. 3-1A). The mHtt silencing
activity of these constructs was tested in vitro by transiently co-transfecting each
construct with a eukaryotic expression vector containing the R6/1 HD transgene (mHtt)
into HEK293 cells. Transfections were performed using a 1:4 and a 1:8 target vector to
shRNA vector ratio. Forty-eight hours post-transfection, we analyzed mHtt mRNA and
protein expression levels. Northern blot analysis of total RNA obtained from HEK293
cells co-transfected with a shRNA vector targeting nucleotides in the 5'-UTR of the mHtt
mRNA (siHIUNT-1) or a shRNA vector targeting a region immediately upstream of the
CAG repeat domain (siHIUNT-2) resulted in greater than 75% (p < 0.001) reduction of
mHtt mRNA compared to samples from cells that expressed mHtt and a GFP-only vector
(Fig. 3-1B). Co-transfection with a control shRNA vector targeted to the dog rhodopsin
mRNA (siRho-1) did not affect the levels of mHtt mRNA (Fig. 3-1B).
Immunoblot analysis of mHtt protein using a human amino-terminus specific Htt
antibody (Hum-1) showed that the reduction in mRNA levels was associated with a
significant decline in the levels of mHtt protein (Fig. 3-1C). Co-expression of the mHtt
vector with siHIUNT-1 shRNA reduced the levels of mHtt protein to 40% (p < 0.001) of
that observed in cells co-transfected with either a GFP-only vector or the siRho-1 shRNA
control vector. We also observed -55% (p < 0.001) reduction in the levels of mHtt
I 2 34 56 78 D
si-Rho-l aiHUNT I siHUNT-2
rAAV Silencing Construct
.j; 3 3
Figure 3-1. rAAV-shRNA constructs mediate the silencing of m-Htt in vitro. A. The
human H1 RNA promoter unit was used to drive the intracellular expression
of 20 to 21-nucleotide long shRNAs directed against the R6/1 HD transgene
(mHtt) from within the context of rAAV serotype-5 vectors. Expression of
hrGFP was used to positively identify transduced cells. B. Schematic
Figure 3-1 continued
representation of the Htt gene graphically showing the region of the gene that
is targeted by siHunt-1 and siHunt-2 shRNAs. C. Northern blot analysis of
mHtt mRNA obtained from transiently co-transfected HEK293 cells. Co-
expression of CMV-R6/1 (mHtt) with either siHUNT-1 or siHUNT-2 shRNA
constructs, at either a 1:4 (lane 5 and 7) or 1:8 (lane 6 and 8) target to shRNA
ratio, resulted in significantly reduced levels of mHtt mRNA when compared
to co-transfection with a control shRNA, siRho-1, (lanes 3 and 4) or CMV-
R6/1 alone (lanes 1 and 2). Comparisons of the average optical density signal
from three independent experiments performed with a 1:8 target to shRNA
ratio showed greater than 75% reduction in mHtt mRNA relative to levels of
p-actin mRNA (* = p<0.001, ** = p<0.001). D. Western blot analysis of total
protein from HEK293 cells treated as above. mHtt protein was detected using
a human-specific anti-Htt antibody (Hum-1). Analysis of the average (+ SEM)
optical density signal revealed that mHtt was silenced by intracellular
expression of both siHUNT-1 (>60% reduction, = p<0.0001) and siHUNT-2
(>55% reduction, **" = p<0.0001). Silencing of mHtt protein expression was
not observed in cells co-transfected with siRho-1. The same blot was probed
with an antibody to p-actin in order to normalize for total protein load. Error
bars represent + standard error of the mean (+SEM).
protein in samples from cells transfected with siHUNT-2 shRNA. mHtt protein levels
were unaffected by the expression of siRho-1 shRNA molecules (Fig. 3-1C). These in
vitro results demonstrate that intracellular expression of both siHUNT-1 and siHUNT-2
results in the specific and efficient knockdown of mHtt mRNA and protein levels in
cultured HEK293 cells.
Long-Term Striatal Expression of rAAV5-shRNAs in the R6/1 Mouse
We next investigated whether rAAV5-mediated long-term striatal expression of
siHUNT-1 or siHUNT-2 shRNAs could induce RNA interference of the mHtt transgene
in the R6/1 transgenic HD mouse. To test the in vivo activity of both shRNAs, 6-8 week
old R6/1 mice and wild-type littermates were divided into four groups and injected
unilaterally at two different sites in the right striatum. Each intrastriatal injection
delivered 2 Cll of high-titer rAAV5-siHUNT-1 (R6/1 n=12, wild-type n=6), rAAV5-
siHUNT-2 (R6/1 n=13, wild-type n=5), rAAV5-siRho-1 (R6/1 n=4) or rAAV5-GFP
(R6/1 n=5, wild-type n=5) viral vectors. Ten weeks post-surgery, fresh-frozen coronal
brain sections were obtained and viral transduction, mHtt mRNA levels, and protein
expression levels relative to the left untreated hemisphere were determined.
Inj section of rAAV5-GFP, rAAV5-siRho-1 and anti-mHtt rAAV5 shRNA vectors
resulted in widespread and intense neuronal transduction as was previously observed in
the rat striatum (13). In situ hybridization (ISH) analysis performed on coronal sections
against the rAAV5-encoded hrGFP mRNA revealed a non-uniform but widespread
rAAV5 transduction (Fig. 3-3A). hrGFP mRNA was efficiently expressed along the
dorsal-ventral and rostral-caudal axis of the striatum. A strong hybridization signal was
detected on either side of the corpus callosum but not in the white matter tracts (Fig. 3-
3A) suggesting that the maj ority of hrGFP signal was concentrated in neuronal cell
Onset of Motor Decficits
III I l l | I I I
Age 0 2 4 6 8' 10 12 14 .16
Bilatral Lct o como utio n ai
Figure 3-2. Experimental design. A. R6/1 and littermate control animals were injected
unilaterally with either rAAV5-siHUNT-1, -siHIUNT-2, -siRho-1 or -GFP
viral vectors (annotated below the line). B. Experimental design for bilateral
inj sections was identical to that shown above. Behavioral tests were performed
weekly between the time points indicated by the arrows above and below the
bodies. hrGFP protein expression was intense and paralleled the widespread pattern of
rAAV5 transduction detected by ISH analysis (Fig. 3-3B). Native hrGFP epiflourescene
was observed in all sections that contained rAAV5-transduced striatal tissue such as the
one shown in figure 3-3B. Long-term striatal expression of control rAAV5 vectors
(rAAV5-siRho-1, rAAV5-GFP) in the R6/1 mouse was not associated with any abnormal
changes in cellular morphology or astrocyte activation as determined by cresyl violet and
GFAP staining (data not shown). These observations demonstrated that the striatum of
the R6/1 mouse can tolerate strong, wide-spread and long-term rAAV5-mediated
expression of hrGFP and shRNA molecules.
To evaluate the silencing activity of rAAV5-siHIUNT-1 and rAAV5-siHIUNT-2 in
vivo, we extracted total cellular RNA from rAAV5-shRNA-transduced striatal regions
and subj ected the RNA to reverse-transcription reactions optimized to convert the GC-
rich mHtt transgene mRNA to cDNA. The levels of mHtt mRNA were determined using
real-time quantitative PCR. Baseline levels of striatal mHtt mRNA were established by
analyzing total RNA from striatal tissue contralateral to the rAAV5-shRNA inj section.
mHtt mRNA levels from control and rAAV5-shRNA inj ected striatum were normalized
to hypoxanthine phosphoribosyl transferase (HPRT) mRNA levels and subj ected to one-
way ANOVA. Long-term expression of rAAV5-siHIUNT-1 or rAAV5-siHIUNT-2 in the
striatum of the R6/1 mouse resulted in highly significant reductions in the levels of mHtt
mRNA (Fig. 3-3C) demonstrating that intrastriatal delivery of rAAV5 anti-mHtt shRNAs
in the R6/1mouse can induce efficient knockdown of mHtt mRNA levels. Moreover, this
reduction was specific to the expression of rAAV5-siHIUNT-1 or -siHU7NT-2 since mHtt
mRNA levels were unchanged following the expression of rAAV5-siRho-1 (Fig. 3-3C).
To determine the levels of total striatal mHtt protein in the R6/1 mouse, we
generated a rabbit polyclonal antibody against a specific peptide in the amino-terminus of
human Htt (Hum-1). The Hum-1 antibody recognized a high molecular weight band of
greater than 250 kDa that was enriched in nuclear fractions of protein extracts from R6/1
but not wild-type littermate controls (Fig. 3-4A). We measured the relative intensity of
the high molecular weight Hum-1 immunoreactive band to compare the levels of mHtt
protein in transduced and control R6/1 striatum. We normalized the levels of mHtt
120 e 120
100 a 100
E 80 80
1 0 60 o60
S 40 -a 40
20 I 20
ctl vector ctl vector ctl vector ctl vector ctl vector ctl vector
siHunt-1 siHunt-2 siRho siHunt-1 siHunt-2 siRho
Figure 3-3. Long-term in vivo striatal expression of rAAV5-siHUNT1 and rAAV-
siHU7NT2 decrease mHtt transgene mRNA expression. A. In situ
hybridization with a probe against the virally encoded hrGFP mRNA. Coronal
sections were obtained 10 weeks post-surgery. The panel was arranged in a
rostral-caudal axis to illustrate the extent of the transduced area as indicated
by the arrow. Two separate inj sections were placed into the same hemisphere
(arrows). These sections were obtained from one animal and are
representative of all other study animals. Scale bar = 2mm. B.
Photomicrograph of native hrGFP protein expression from the striatum of a
representative inj ected animal demonstrates that the vector derived mRNA
shown in (A) is transcribed into protein. Str = striatum, cc = corpus callosum,
and CTX = cortex. Scale bar = Imm. C. Real-time quantitative PCR analysis
of mHtt mRNA knockdown. Total RNA was extracted from the striata of
R6/1 mice. The levels of striatal mHtt mRNA on the uninj ected (black bars)
striata and injected striata with rAAV5 vectors (white bars). rAAV5-
siHU7NT-1, rAAV5-siHUNT-2, and rAAV5 siRho were measured,
normalized to the levels of HPRT mRNA and reported as percent of control.
Expression rAAV5-siHUNT-1 resulted in a significant 75% reduction of mHtt
mRNA expression (F[1,11] = 40.0 = p<0.001). Striatal rAAV5-siHUNT-2
treatment resulted in significant 78% reduction of striatal mHtt mRNA
expression (F[1.8] = 19.1, = p = 0.002). In contrast, striatal injection of the
control shRNA rAAV5-siRho did not reduce striatal mHtt mRNA levels
Figure 3-3 continued
(F[1,8] = 0.71, p > 0.40). D. Similarly to striatal mRNA analysis in (C), total
protein was obtained from the inj ected and untreated control striata of R6/1
mice. Similar to the mRNA levels, there was a significant rAAV5-sitHUNT-1
mediated 24.5% reduction in the levels of a Hum-1 immunoreactive high
molecular weight band (simple main effects, F[1,18] = 5.2, = p < 0.04). The
rAAV5-siHUNT-2 treated group displayed a significant 38% reduction of
striatal mHtt (simple main effects, F[1,18] = 8.4, = p = 0.01) while the
rAAV-siRho control groups did not have significant reductions of striatal
mHtt (simple main effects, F [1,18] = 0.50, p > 0.40) when compared to R6/1
control striata. Error bars represent +SEM.
protein to a cross-reactive band that was recognized by the Hum-1 antibody and was
present at constant levels in protein extracts isolated from R6/1 and wild-type mice.
rAAV5-siHUNT-1 and rAAV5-siHUNT-2 also reduced striatal mHtt protein levels but
not to the same degree observed with striatal mHtt mRNA levels. Thus, striatal rAAV5-
siHUNT-1 expression reduced striatal mHtt protein approximately 25% compared to
control striata or rAAV5-siRho treated mice (Fig 3-3D). In addition, rAAV5-siHUNT-2
reduced striatal mHtt protein 38% relative to untreated control striata or rAAV5-siRho
injected mice (Fig 3-3D.). Expression of the control shRNA, siRho had no effect on
striatal mHtt expression.
There is a clear discrepancy between the magnitude of si-HUNT-1 and si-HUNT-2
mediated mHtt mRNA reduction and the magnitude of reduction of mHtt protein from
the same striata. One potential reason for this discrepancy between mRNA and protein is
the differential kinetics of turnover of mRNA versus protein. Moreover, our tissue
sampling protocol included both transduced and non-transduced cells, which would lead
to an underestimate of the effectiveness of mHtt knock-down, although there is no reason
why this would differentially affect measured protein levels compared to mRNA levels.
In summary, long-term intrastriatal expression of rAAV5-siHUNT-1 and -siHUNT-2
shRNAs significantly reduced striatal mHtt mRNA levels and also resulted in diminished
striatal mHtt protein expression.
Reduction in the Size and Amount of NIIs is Observed in R6/1 Mice Treated With
rAAV5-siHUNT-1 or rAAV5-siHUNT-2.
One of the pathological markers of disease progression in mouse models of pQ-
disease such as HD is the presence of NIIs. Studies have shown that suppressing the
expression of the mutant pQ-containing protein in neurons results in the clearance of NIIs
[12; 23]. We examined whether the Hum-1 antibody could detect the presence of NIIs in
the R6/1 striatum. We reasoned that using an anti-Htt antibody instead of an anti-
ubiquitin antibody would allow us to directly correlate the levels of mHtt with the
presence or absence of NIIs. Immunohistochemical analysis of coronal brain sections
from untreated R6/1 mice subj ected to Hum-1 and anti-ubiquitin immunohistochemistry
revealed a similar pattern of immunoreactivity between both antibodies with respect to
the size and distribution of NIIs in the R6/1 mouse brain (Fig. 3-4A-D). Histological
analysis of sections obtained from R6/1 mice of various ages showed that Hum-1-positive
NIIs become visible in the light-microscope at about 8 weeks after birth (data not shown),
in agreement with the time that ubiquitin-positive NIIs become visible in the R6/1 mouse
. Hum 1- (data not shown) and anti-ubiquitin-  immunoreactive NIIs increase in
size and staining intensity during the progression of HD suggesting that protein
deposition in NIIs is cumulative.
We analyzed Hum-1 immunostained coronal sections from unilaterally inj ected
R6/1 mice and compared the relative area and intensity of NIIs present in four brain
regions including the left primary motor cortex, right primary motor cortex, left medial
striatum and right medial striatum (Fig. 3-4F). Three of these regions, left primary motor
cortex, right primary motor cortex and left medial striatum, corresponded to non-
transduced control tissue while the right medial striatum exhibited strong rAAV5-
mediated transduction (Fig. 3-3A,B & 3-4F). Comparison of Hum-1 staining in the left
and right primary motor cortex revealed no differences in the area or intensity of NIIs in
any of the R6/1 mice that were untreated or treated with either rAAV5-siHUNT-1 (Fig.
3-4G & H) or -siHUNT-2 (Fig. 3-4M & N). Areas shown in figure 3-4I,J,K,L,O,P were
3 4 5 6
aft right left right left riht left right.
Cortex S3triatum Cortex Striatum '
~lri ~' ''~
1. *, I 'r
I r C
`r -'~~ t
Figure 3-4 Analysis of mHtt protein aggregates after expression of rAAV5-shRNAs. A-
D. Immunohistochemistry showed that the human Htt amino-terminus-
specific antibody Hum-1 antibody, like anti-Ubiquitin antibody, detects NIIs
in R6/1 but not wild-type mice. Panels (A) and (B) were striatal sections
immuno-stained using an anti-ubiquitin antibody. A. Striatal section from a 15
week-old wild-type mouse. B. Striatal section from a 15 week-old R6/1
mouse. Panels (C) and (D) were immuno-stained using the new Hum-1
"is r rs~
1 ii 'L
170- ggg (ll$ ,
Figure 3-4 continued
antibody. C. Striatal Hum-1 immuno-staining from the same 15 week-old
wild-type mouse as shown in (A). D. Striatal Hum-1 immuno-staining from
the same 15 week-old R6/1 mouse as shown in (B). Similar staining of NIIs
was found in both anti-ubiquitin and Hum-1 immuno-stained sections. Cross-
reactivity of Hum-1 antibody with cytoplasmic proteins in striatal neurons of
both wild-type and R6/1 mice is apparent in (C) and (D). E. Western blot
analysis of total (lanes 1 and 2), cytoplasmic (lanes 3 and 4) and nuclear
(lanes 5 and 6) protein derived from 15 week-old wild-type (lanes 1, 3 and 5)
and R6/1 mice (lanes 2, 4 and 6) using the Hum-1 demonstrated that the
antibody detects a high-molecular weight aggregate in R6/1 transgenic mice.
The lower panel shows a 170-kDa cross-reacting band that is present in all
samples and was used as loading control. F. Coronal section from rAAV5-
siHunt-1 inj ected R6/1 mouse processed for in situ hybridization with a probe
against vector specific sequences (as shown in Fig. 3A) to demonstrate the
transduction area. The boxes show the areas of Hum-1 staining used for
analysis of NII number and size (panels G, H, M, N, Q, R) and the
photomicrographs shown in panels (J-L), (O,P), (S,T). G-L. These panels
contain the analysis of NII count and size for rAAV-siHUNT-1 inj ected R6/1
mice. NII analysis from all vector-treated mice took place by examining the
transduction area using native hrGFP fluorescence in sections adj acent to the
Hum-1 immuno-stained section in order to correctly choose the boxed area
depicted in (F) for each section. G. Numbers ofNIIs were estimated in cortex
and striatum of long-term treated R6/1 mice, compared to identically
determined NIIs in control striata and expressed as percent of control
frequency. Cerebral cortex in either hemisphere was not transduced by rAAV
(F) and served as a region control. Cortical NII numbers were unaffected by
striatal injection of rAAV5-siHUNT 1 (Fisher' s PLSD post hoc test, p = 0.8).
The frequency of NIIs in cortex was generally greater than in the striatum of
the R6/1 mouse (F[1,20] = 22.0, *p < 0.001). In addition, treatment with
rAAV5-siHUNT 1 significantly reduced the frequency of striatal NIIs by 31%
compared to the control uninj ected striatum (Fisher' s PLSD post hoc test, Jf p
= 0.04). Error bars in (G-H), (M-N) H. Similarly to NII numbers, NIIs are
smaller on average in the striatum, regardless of treatment as compared to the
NII size in cortex (F[1,20] = 38.4,* p < 0.001). rAAV5 mediated siHUNT-1
expression reduced the size of the remaining NIIs by 39% compared to the
untreated control striatum (post-hoc, "f p = 0.0013). I. Photomicrograph of left
cortical Hum-1 stained coronal section from the area shown in the box in (F)
showing frequency of NIIs. J. Photomicrograph of the right cortical region
shown in the box in (F). Scale bar = 50 Clm and applies to (I,J,L). M-P. These
panels contain the data from rAAV-SiHUNT-2 treated R6/1 mice. M. The
frequency of NIIs in the R6/1 cortex was, in general, greater than the
frequency measured in the striatum (F[1,20] = 68.4, p < 0.0001). rAAV5
mediated siHUNT2 expression further significantly reduced the number of
striatal NIIs by 47% compared to the left striatum (Fisher's PLSD post hoc
test, Jf p < 0.0001). N. Similar to NII frequency, Hum-1 positive cortical NIIs
Figure 3-4 continued
are significantly larger than striatal NIIs (F [1,20] = 71.1, p < 0.0001).
Striatal siHUNT-2 expression further reduced striatal NII size by 35%
compared to the untreated striatum (Fisher' s PLSD post hoc test, Jf p < 0.03).
O. Photomicrograph from the control left striatal area depicted in (F) from a
representative rAAV5-siHUNT-2 treated mouse. P. Photomicrograph from
the right rAAV5-siHUNT-2 treated striatum from the same mouse depicted in
(0) clearly demonstrating reduced NII frequency and size. It should be noted
that untransduced striatal areas had Hum-1 positive NII frequency and size
that was indistinguishable from those seen in the opposite control striatum.
Q-T. These panels contain the NII analysis from the rAAV5-siRho treated
mice that serve as the control shRNA experimental group. Q. Quantitative
analysis of NIIs frequency of siRho treated mice. As with the other vector
treated groups cortical NIIs were more frequent than Hum-1 positive striatal
NIIs (F[1,12] = 20.5, p = 0.0007). However, there was no difference
between NII number between treated and untreated striata (Fisher' s post hoc
test, p > 0.2). R. Hum-1 positive NIIs are also smaller in striatum as
compared to the R6/1 cortex (F [1,12] = 16.8, *p < 0.002). However, control
rAAV5-siRho treated striatal Hum-1 positive NIIs were the same size as those
measured in the left, control striatum (Fisher' s PLSD post hoc test, p > 0.5).
S. Representative photomicrograph from the left, untreated cortex of a rAAV-
siRho treated R6/1 mouse. T. Representative photomicrograph taken from the
transduction area (F) from the right, inj ected striatum of the same mouse
shown in (S). There is no apparent difference in striatal NII frequency and
size when compared to the left striatum (S). Scale bar = 50 Clm and applies to
(O,P, and S). Error bars represent +SEM in (G-H), (M-N), and (Q-R).
taken from the same R6/1 mouse section and are representative of all other rAAV5-
siHUNT-1 and -siHUNT-2 treated R6 mice. Hum-1 immunoreactive NIIs in the left (Fig.
3-4K & O) and right (Fig. 3-40 & P) medial striatum were smaller and had less intense
staining than those present in the cortical regions of the same R6/1 mouse (Fig. 3-4I vs.
4K). This histological observation was also supported by the quantification ofNII count
(Fig. 3-4G) and area (Fig. 3-4H, see below).
A comparison of the non-transduced left and transduced right medial striatum
regions showed a marked reduction in the intensity of Hum-1 NII staining in the rAAV5-
siHUNT-1 (Fig. 3-4L) and rAAV5-siHUNT-2 (Fig. 3-4P) transduced right medial
striatum when compared to the left medial striatum (Fig. 3-4 K & P, respectively) of the
same animal. This observation was corroborated by the quantification ofNIIs (Fig. 3-
4G&M) count and area (Fig. 3-4H & N, see below). The reduction in Hum-1 NII
staining intensity was more pronounced in sections obtained from rAAV5-siHUNT-2-
treated compared to siHUNT-1-treated R6/1 mice. Sections from R6/1 mice treated with
either rAAV5-GFP (data not shown) or rAAV5-siRho-1 (Fig. 3-4Q-T) showed no
significant difference in the pattern of Hum-1 NII staining between the left and right
medial striatum regions (Fig. 3-4S versus 4T). The observation of rAAV5-siHUNT 1-
and rAAV5-siHUNT-2- mediated reduction of striatal Hum-1 NII staining further
corroborates the immuno-blot data indicating reduced striatal mHtt protein in vector-
In order to quantify the relative area and amount of NIIs present in shRNA treated
striata, we analyzed the same relative four regions shown in figure 3-4F in siHUNT-1
treated (n=11), siHUNT-2 treated (n=6) and siRho-1 (n=4) treated R6/1 mice. The mean
relative area (Clm2) and number of particle (NIIs) counts within a specified area range was
calculated for each of the four regions in all of the unilaterally inj ected R6/1 mice using
Image J software and reported as percent of the non-transduced corresponding area in the
opposite hemisphere. Using different anatomical regions from individual coronal sections
as internal controls revealed that medial striatal NIIs displayed reduced frequency as well
as reduced two-dimensional area compared to NIIs in motor cortex regardless of vector
treatment (Fig. 3-4G & H respectively).
Long-term expression of rAAV5-siHUNT-1 resulted in a significant reduction in
both the frequency (31%) and mean area (39%) of NIIs in the transduced right medial
striatum when compared to the contralateral non-transduced left medial striatum in the
same section (Fig. 3-4G&H respectively). Analysis of the right medial striatum in
rAAV5-siHIUNT-2 treated sections revealed a mean reduction of 47% in the estimated
frequency and of 3 5% in the mean area of NII particles when compared to the left medial
striatum (Fig. 3-4M). There was no significant difference in the number or area of NIIs
between the right and left medial striatum of rAAV5-siRho-1 treated R6/1 mice (Fig. 3-
4Q & R respectively). Our results show that long-term expression of rAAV5-siHIUNT-1
or rAAV5-siHIUNT-2 in the R6/1 striatum led to a significant reduction in the area and
frequency of striatal NIIs present within the region of viral transduction.
Reduced Levels of Striatal mHtt Affect Levels of Striatal-Specific Transcripts.
Expression of exon 1 of mHtt leads to a decrease in the level of a subset of striatal-
specific mRNAs of the R6/1 mouse   [29-31i]. These same transcripts are also
reduced in HD patients. At the very least, evaluation of these striatal-specific transcripts
can serve as an important metric of disease progression in R6/1 mice. To examine the
effects that silencing striatal mHtt expression might have on this molecular phenotype,
we performed in situ hybridization analysis of transcripts on coronal sections obtained
from unilaterally inj ected wild-type and R6/1 mice. Sections were hybridized with
oligonucleotide probes against preproenkephalin (ppEnk), dopamine- and cAMP-
responsive phosphoprotein, 32 kDa (DARPP-32), phosphodiesterase 10A (PDE10A),
phosphodiesterase 1B (PDE1B), nerve growth factor inducible-A (NGFi-A) and the
dopamine type 2 receptor (D2) receptor.
Densitometric analysis of rAAV5-siHUNT-1 inj ected R6/1 mice showed an
increase in the mean levels of striatal ppEnk (24%) and DARPP-32 (16%) mRNA in the
transduced striatum (Fig. 3-5A & B) as compared to the contralateral uninj ected side.
There was no significant side-to-side difference in the levels of PDE 10A, PDE-1B,
NGFi-A or D2 receptor mRNAs in R6/1 mice treated with rAAV5-siHUNT-1 (data not
In contrast, expression of rAAV5-siHUNT-2 in R6/1 mice resulted in a marked
decrease in the mean levels of both ppEnk (16%) and DARPP-32 (30%) mRNAs when
compared to the uninj ected left side (Fig. 3-5C & D). More importantly, this decrease
was also evident in unilaterally inj ected wild-type mice for both ppEnk (75%) &
DARPP-32 (56%) mRNAs (Fig. 3-5 panels C & D). This pronounced reduction in
mRNA levels was also observed in all other transcripts tested (not shown), except for
NGFi-A where the long-term expression of siHUNT-2 shRNA led to an increase in
transcript levels that exceeded that of wild-type uninj ected controls (not shown).
The P-actin-specific hybridization signal remained unchanged in all samples tested.
Based on the comparable reduction in transcripts in wild-type and R6/1 mice, we
attributed the reduction in the steady-state levels of these striatal-specific transcripts to a
ConRll siHunt-1 Control siHunt-1
left right left right
Control sinunt-a control Sinunt-
left _right leftriht
left _right left right
Figure 3-5 In situ hybridization (ISH) analysis of ppEnk and DARPP-32 transcripts.
Coronal sections shown to the left of (A), (C), and (E) were hybridized to a
ppEnk-specific oligonucleotide probe (Table 1) while sections in panels (B),
(D) and (F) were hybridized to a DARPP-32-specific oligonucleotide probe
(Table 1). The arrows above the coronal sections show the approximate vector
inj section site and the fill pattern of the arrows corresponds to the fill pattern of
the histograms in each panel to show which treatment was received in each
hemisphere. In order to better demonstrate the anatomical pattern of striatal
transcripts the coronal sections were false colored. The scale is shown at the
bottom of the figure. A. Evaluation of long-term intrastriatal rAAV-siHUNT-
1 expression on striatal ppEnk levels in R6/1 mice and wild-type littermate
controls. Quantitative analysis of normalized ppENK levels confirm that
ppENK levels are reduced in 22 week old R6/1 mice (F[1,66] = 99.5, p <
0.0001). rAAV-siHUNT-1 injection into wild-type control striata does not
lead to any alteration in striatal ppENK levels as compared to untreated
left _ri~ght R6/"
mHtt mRNA levels
Figure 3-5 continued
control left striata (Fisher' s PLSD post hoc test, p > 0.7). In contrast, in R6/1
mice, striatal rAAV5-siHUNT-1 treatment led to a mild 24% increase in
striatal ppEnk levels as compared to the ppEnk levels observed in the control
striatum (Fisher's PLSD post hoc test, Jf p = 0.01). B. Evaluation of long-
term intrastriatal rAAV-siHUNT-1 injection on steady state striatal DARPP-
32 mRNA levels. Similar to ppENK mRNA levels, DARPP-32 mRNA was
reduced in R6/1 animals as compared to littermate controls (F[1,66] = 114. 1, *
p < 0.0001). rAAV5-siHUNT-1 injection did not affect striatal DARPP-32
levels in wild-type controls (Fisher' s PLSD post hoc test, p > 0. 1). Striatal
rAAV5-siHUNT-1 treatment resulted in a significant 16% increase in
DARPP-32 mRNA levels (Fisher' s PLSD post hoc, "f p < 0.03) in R6/1 mice
as compared to DARPP-32 mRNA levels in the untreated striatum. C.
Analysis of the effect of intrastriatal inj section of rAAV-siHUNT-2 on striatal
ppEnk transcript levels. In stark contrast to the pattern seen with rAAV5-
siHUNT-1 inj section (A), rAAV5-siHUNT-2 long-term expression led to a
75% reduction of striatal ppENK levels in the inj ected striatum of wild-type
littermate controls (Fisher' s PLSD post hoc test, *p < 0.0001). Likewise,
rAAV5-siHUNT-2 treatment also resulted in a further 16% reduction in
striatal ppENK levels in R6/1 mice (Fisher' s PLSD post hoc test, Jf p < 0.05).
D. Analysis of the effect of intrastriatal inj section of rAAV-siHUNT-2 on
striatal DARPP-32 mRNA levels. Similar to the pattern seen in response to
rAAV5-siHUNT-2 expression striatal ppEnk levels (C), rAAV5-siHUNT-2
long-term expression led to a 44% reduction of striatal DARPP-32 mRNA
levels in the inj ected striatum of wild-type littermate controls (Fisher' s PLSD
post hoc test, *p = 0.0005). In addition, rAAV5-siHUNT-2 treatment resulted
in a further 70% reduction in striatal DARPP-32 mRNA levels in R6/1 mice
(Fisher's PLSD post hoc test, Jf p < 0.01). This further reduction in the treated
striatum of the R6/1 mice is particularly apparently in the right side of the
coronal section shown to the right of the histogram in this panel. E.
Evaluation of the effect of the long-term expression of the control shRNA,
siRho in the striatum of wild-type littermate controls and R6/1 mice on striatal
ppEnk levels. rAAV5-siRho injections had no effect on striatal ppEnk
transcript levels in either wild-type controls (Fisher's PLSD post hoc test, p >
0.9) or R6/1 mice (Fisher's PLSD post hoc test, p > 0.6). F. Evaluation of the
effect of the long-term expression of the control shRNA, siRho in the striatum
of wild-type littermate controls and R6/1 mice on striatal DARPP-32 mRNA
levels. rAAV5-siRho injections had no effect on striatal DARPP-32 mRNA
levels in either wild-type controls (Fisher's PLSD post hoc test, p > 0.7) or
R6/1 mice (Fisher' s PLSD post hoc test, p > 0.6). Error bars are + SEM.
siHIUNT-2 shRNA-mediated "off-targeting" effect. Further documentation of this
important off-targeting finding is the subj ect of a separate study (Rodriguez et al., in
Analysis of R6/1 mice inj ected unilaterally with the control rAAV5-siRho-1
shRNA revealed no significant differences in the levels of ppEnk, DARPP-32, PDE 10A,
PDE-1B, NGFi-A or D2 receptor mRNA between the left uninj ected and right inj ected
striatum (Fig. 3-5E & F). Additionally, there was no side-to-side difference in the steady-
state levels of any of the transcripts analyzed in the striatum of wild-type mice inj ected
unilaterally with rAAV5-siHIUNT-1 (Fig 3-5A&B). We conclude that rAAV5-siHIUNT-1
mediated reduction of mHtt levels in the striatum of R6/1 mice resulted in a small but
significant increase in the steady-state levels of striatal ppEnk and DARPP-32 mRNAs
relative to the levels observed in the untreated contralateral striatum.
Long-Term Bilateral Striatal Expression of rAAV5-siHUNT-1 in the R6/1 Mouse is
Associated With a Delay in the Clasping Phenotype.
R6/1 mice display a progressive neurological phenotype that includes clasping of
the hind limbs, and dyskinesias (10). Additionally, R6/1 fail to gain weight at a normal
rate and show a progressive decrease in retention times in the rotarod when compared to
age-matched wild-type littermate mice. In order to examine whether lowering the striatal
levels of mHtt would prevent the progression of these phenotypes, 6-8 week old R6/1 and
wild-type mice were inj ected bilaterally at two different striatal sites (4 injections total)
with rAAV5-siHIUNT-1 or rAAV5-GFP control. We initially assessed the progression of
the HD-like phenotype in the R6/1 mice by recording weights weekly beginning at 1
S35~ -r I I I I I ~ I C 35 -
2 2 0 -( -[ W T co t o (n 5 B 2 0 5- R / o t o n 5
-g- VVT GFP (n=5) -$- R6/1 GFP (n=5)
0 ~ ~~ ~ -V siHunt (n--5) 0 R6/1 siHunt(n=5)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Age (weeks) Age (weeks)
m -0- R6/1 control (n=5)
E -$ R6/1 GFP (n=5)
E R6/1 siHunt (n=5)
13 14 15 16 17 18 19 20 21 22
Figure 3-6 Bilateral long-term striatal expression of rAAV5-siHUNT-1 delays the
clasping phenotype of R6/1 mice. A. Animal weight was recorded weekly
starting at 1 week post-surgery (Fig. 2B). A. The mean (ASEM) weight for
wild-type littermate controls is shown. The symbol key is given in the lower
right corner of the graph. There was no effect of rAAV-siHUNT-1 treatment
on weight regardless of genetic background or vector treatment including
rAAV5-GFP (rAAVrepeated measures ANOVA, F [2,22] = 0.63, p > 0.5). B.
The mean (ASEM) weight for R6/1 mice is shown. In general, R6/1 mice
weighed less than aged-matched littermate controls (n = 5) over the period of
the experiment (F [1,22] = 4.7, p = 0.04 A vs. B).C. Tail-suspension tests were
performed weekly beginning at 6 weeks post-surgery (corresponding to 13
weeks of age) on age-matched control R6/1 mice (n=5) and R6/1 mice
inj ected bilaterally with rAAV5-siHUNT-1 (n=5) or rAAV5-GFP (n=5). Data
are presented as percent of animals scoring positive for the test as a function
of time. Open circles denote age-matched control R6/1. Closed circles denote
R6/1 rAAV5-GFP. Open triangles denote R6/1 rAAV5-siHUNT-1.
As previously reported, we observed that R6/1 mice did not increase total body
weight at the same rate over time compared to age-matched wild-type littermate mice
(Fig. 3-6A versus B). Examination of the mean weekly weights of uninj ected wild-type
mice and wild-type mice inj ected with rAAV5-GFP or rAAV5-siHIUNT-1 revealed that
long-term expression of neither viral vector led to a significant change in the rate of
weight gain over the experimental period (Fig.3-6A). Similarly, long-term expression of
neither rAAV5-GFP nor rAAV5-siHIUNT-1 in R6/1 mice resulted in a significant change
in rate of weight gain when compared to untreated R6/1 (Fig. 3-6B).
We also analyzed the progression of the clasping phenotype in uninj ected, rAAV5-
GFP or rAAV5-siHIUNT-1 inj ected R6/1 mice by performing a weekly tail-suspension
test beginning at 13 weeks of age. Uninj ected and rAAV5-GFP inj ected R6/1 mice began
to display clasping of the hind limbs at 20 weeks of age. In contrast, R6/1 mice
expressing rAAV5-siHUNT-1 did not show evidence of clasping until 22 weeks of age,
when only 20% overtly displayed the phenotype in comparison with up to 80% of
uninj ected and rAAV5-GFP inj ected R6/1 mice, which overtly displayed the hind limb
clasping phenotype at this same time point.
Finally, we examined the locomotor activity, as determined by total distance
traveled, and rotarod retention times of both wild-type and R6/1 mice 16 weeks post-
surgery (22 weeks of age). Although there were significant differences between wild-type
and R6/1 mice in both tasks, we observed no significant differences among uninj ected,
rAAV5-GFP and rAAV5-siHIUNT-1 inj ected R6/1 mice in either total distance traveled
over time or rotarod retention times (data not shown).
We conclude that 6-8 week old mice expressing rAAV5-siHIUNT-1 bilaterally in
the striatum for up to 14 weeks did not display any overt toxicity associated with our
gene transfer approach. Furthermore, long-term expression of siHIUNT-1 anti-mHtt
shRNA resulted in a mild behavioral improvement of the R6/1 phenotype with respect to
clasping behavior but did not affect body weight abnormalities or muscle-dependent
activities such as rotorod retention and locomotion.
The expression of one expanded HD allele initiates a cascade of events that leads to
the neuronal dysfunction and pathology observed in HD (81). It has been hypothesized
that ablating the expression of mHtt might protect against or even reverse the effects
associated with poly-Q expanded Htt protein. In this study, we assessed the molecular
and behavioral effects of long-term intrastriatal rAAV5-mediated expression of shRNAs
designed to suppress the expression of the human mHtt transgene that is present in R6/1
mice. Our results demonstrate that post-transcriptional gene silencing of striatal mHtt in
adult R6/1 mice by one of two shRNAs tested, siHUNT-1, has a mild positive effect on
the rapid cellular and behavioral pathological changes that occur in this model. At the
cellular level, improvement occurred in only 2 striatal specific transcripts and this
amelioration was only found in the striatal subregion of rAAV5-mediated transduction.
These data therefore suggest that greater transduction efficiency could improve the
functional effects obtained here.
Intrastriatal inj sections of rAAV5 vectors resulted in widespread transduction of the
mouse striatum especially as compared to previously reported levels of transduction in
mice (35; 36). Long-term expression of rAAV5-siHUNT-1 in the R6/1 mouse striatum
resulted in mRNA levels that were reduced by 75% when compared to controls while
reduction in protein levels ranged from 25-38%. Aggregated, but not soluble, mHtt was
detected using our western blot protocol, which may have contributed to the discrepancy
between the magnitude of reduction of mRNA and mHtt protein. In fact, even though we
tested several published antibodies and a variety of extraction protocols, we were unable
to detect measurable levels of soluble mHtt N-terminal fragments in total striatal protein
samples from untreated adult R6/1 mice.
In agreement with the data obtained from Hum-1 immuno-blots indicating reduced
striatal mHtt in transduced regions, Hum-1 immunohistochemical analysis of sections
from rAAV5-siHUNT-1 treated R6/1 mice revealed a marked decrease in the size and
number of NIIs within the area of viral transduction. Thus, since aggregation of soluble
mHtt fragments leads to the formation of inclusions in vivo, our data suggest that
expression of rAAV5-siHUNT-1 significantly diminished the available cellular pool of
soluble mHtt N-terminal fragments in the striatum of the R6/1 mouse.
The functional significance of NIIs in HD mouse models is currently controversial.
NIIs have been hypothesized to be a protective mechanism in cells by functioning to
sequester detrimental toxic forms of Htt from damaging the cell (37). On the other hand,
NIIs may also be a pathological feature causing transcriptional dysregulation via binding
of vital transcription factors and sequestering these factors from regulatory sequences.
Along these lines, mHtt has been shown to affect the cellular localization and function of
several regulators of transcription known to be critical for proper neuronal function,
which results in the specific down-regulation of a subset of striatal mRNAs (3 8).
Expression of rAAV5-siHUNT-1 in the striatum of the R6/1 mouse resulted in a
significant increase in the steady-state mRNA levels of both ppEnk and DARPP-32
mRNAs as demonstrated by in situ hybridization analysis. No significant changes were
observed in either the dopamine type-2 receptor or NGFi-A mRNA levels. Additionally,
expression of rAAV5-GFP or the inactive control shRNA, rAAV5-siRho-1, did not result
in any detectable changes in the levels of any of the transcripts analyzed. The promoter
for ppEnk is regulated by the cAMP-responsive element (CRE-) transcriptional pathway
(39). The CRE-transcriptional pathway is extensively involved in the regulation of genes
needed for neuronal function and survival, and expression of mHtt has been shown to
interfere with this pathway (40-42). CRE-mediated transcription is modulated by
TAFIIl30, and the pQ domain of soluble mHtt N-terminal fragments can sequester
TAFIIl30, into aggregates. This aberrant protein-protein interaction is thought to
negatively affect CRE-regulated transcription by affecting the proper localization and
function of TAFIIl30 (41). Therefore, reduced levels of striatal mHtt might have a direct
effect on the activity of CRE-mediated transcription. In this study, while the frequency
and size of NIIs were reduced and disease-affected striatal transcripts were improved by
administration of siHUNT-1, the relationship between NIIs and disease progression and
the exact role of mHtt in transcriptional dysregulation remains poorly understood.
We have observed that the levels for both ppEnk and DARPP-32 mRNA begin to
decrease at ~ 4-5 weeks of age and reach a minimum steady-state level around 12 weeks
of age (Denovan-Wright, unpublished data). In this study, we initiated shRNA treatment
to reduce striatal mHtt expression in R6/1 mice that were between 6 and 8 weeks of age.
Since we did not fully restore ppENK and DARPP-32 mRNAs to wild-type levels, our
data suggests that lowering the levels of striatal mHtt affected the rate of loss in steady-
state mRNA levels. Alternatively, R6/1 striatal neurons expressing low levels of mHtt
protein may have maintained an increased level of transcriptional activity when
compared to striatal neurons expressing normal levels of mHtt.
Finally, we investigated whether long-term expression of rAAV5-siHUNT-1 would
affect the behavioral phenotype displayed in the R6/1 mouse. In this study, we evaluated
a battery of physiological and behavioral tests including effect on weight loss, clasping
phenotype, locomotor activity, and performance on the rotarod apparatus. There was no
difference in weight among the groups of R6/1 mice bilaterally treated with rAAV5-
siHUNT-1, rAAV5-GFP R6/1 and uninjected age-matched R6/1 controls. Additionally,
locomotor activity remained unchanged in rAAV5-siHUNT-1 treated R6/1 when
compared to controls. Analysis of retention times in the rotarod apparatus demonstrated a
clear difference between genotype; however, we observed no differences between all of
the three different R6/1 groups. In contrast, there was a delayed onset of the clasping-
phenotype on R6/1 mice that were bilaterally inj ected with rAAV5-siHUNT-1 when
compared to uninj ected or rAAV5-GFP inj ected R6/1 mice. This mild behavioral
improvement may suggest that suppression of mHtt expression in other regions of the
brain, i.e. cortex, may be required in order to achieve a more explicit positive effect on
behavior. Also, some of the behavioral phenotypes observed in the R6/1 mouse may be
affected by dysfunction of other systems outside the CNS. In fact, problems with
metabolism and muscle wasting have been observed in the R6 lines (43). These systemic
abnormalities would not be expected to improve after suppression of striatal mHtt
In addition to the observation that siHUNT-1 reduced mHtt levels and had a
positive effect on the cellular and behavioral phenotype of R6/1 mice, we observed that
another active anti-mHtt shRNA, siHUNT-2, reduced levels of striatal-specific transcripts
in wild-type and R6/1 mice indicating that this molecule had deleterious effects that were
independent of mHtt knock-down. As shown by these results, extreme caution should be
taken when interpreting data from shRNAs in vivo and that detailed analysis of cellular
gene expression can detect off-targeting effects associated with the intracellular
expression of shRNAs.
In conclusion, long-term rAAV5-mediated striatal expression of an anti-mutant Htt
shRNAs was well tolerated in both wild-type and R6/1 transgenic mice and leads to
changes that are consistent with reduced expression levels of mHtt protein. Moreover,
improved striatal transduction, use of a slower progressing HD mouse model,
transduction in additional critical brain regions such as cerebral cortex, intervention
earlier in the time-course of pathology, or a combination of these factors may lead to
better shRNA-mediated striatal mHtt knock-down effects. Nevertheless, we have
demonstrated that reduced levels of striatal mHtt can be achieved through the use of
RNAi and that this treatment results in a mildly improved cellular and behavioral
phenotype in the R6/1 line of HD. Since polymorphisms associated with the mHtt allele
have been described (44-46), these results suggest that mutant allele specific gene
silencing may be a clinically viable approach once remaining efficiency and safety issues
are resolved (19,34).
HUNTINGTON' S DISEASE (HD) is a severe and fatal autosomal dominant
neurological disorder characterized by a late onset of motor and cognitive deficits
including involuntary movements and psychiatric manifestations. Inheritance of an
abnormally expanded CAG-triplet repeat region is the genetic factor responsible for HD.
This expansion is translated into a poly-glutamine (pQ) domain in the N-terminus of a
protein termed huntingtin. Expression of this protein initiates the pathological molecular
cascade of events that leads, without remission, to death of the affected individual within
20 years after the presentation of symtpoms. At the time that these studies were
completed, there existed no clinical treatment to successfully slow down disease
Progression of HD is dependent on sustained mutant huntingtin expression.
Therefore, we hypothesized that reduced levels of striatal m-Htt would slow down the
rate of disease progression in a transgenic mouse model of HD. In order to test this
hypothesis, we tested the ability of anti-mutant huntingtin hammerhead ribozymes and
anti-mutant huntingtin shRNAs to mediate post-transcriptional silencing of m-Htt
expression in vivo. We specifically characterized the effect that the long-term expression
of anti-mutant huntingtin hammerhead ribozymes and anti-mutant huntingtin shRNAs
had on neuronal intranuclear inclusion (NIIs) bodies and the progressive transcriptional
dysregulation phenotype displayed in the R6/1 transgenic HD mouse line.
We have demonstrated that long-term expression of rAAV5-siHIUNT-1 short-
hairpin RNA against m-Htt RNA led to significant silencing of m-Htt expression in the
striatum of the R6/1 mouse (Chapter 4). Furthermore, RNAi-mediated silencing of m-Htt
expression led to a significant decrease in the size and numbers of neuronal intranuclear
inclusions, a marker of disease progression in the R6/1 mouse. In addition, in situ
hybridization analysis demonstrated that reduced striatal levels of m-Htt led to an
increase in the steady-state mRNA levels of both ppEnk and DARPP-32 but not of the
dopamine type-2 and NGFi-A transcripts. Finally, we showed that long-term bilateral
striatal expression of rAAV5-siHIUNT-1 led to a delayed in the onset of the clasping
phenotype displayed by R6/1 mice.
We are the first to demonstrate that long-term striatal expression of anti-mutant Htt
shRNAs result in reduced levels of m-Htt protein in vivo in the R6/1 model of HD. We
also showed that this reduction resulted in a decrease in the size and amount of NIIs,
concomitant with an increase in the mRNA steady-state levels of ppEnk and DARPP-32
and a delayed in the clasping phenotype in the R6/1 mouse.
The function of wild-type Htt remains unknown. Although there is data
supporting a role for Htt in neuronal survival, these studies have focused on the ablation
of Htt expression prior to neuronal survival. Our experimental approach tested the
hypothesis that the specific knockdown of striatal m-Htt protein would lead to a slower
progression of the HD phenotype in the R6/1 mouse. Allele-specific silencing of m-Htt,
while plausible in a transgenic mouse line, will be extremely difficult to achieve within
the context of the human genome. In HD, the inheritance and expression of one CAG-
expanded allele leads to disease. shRNAs directed against the CAG domain in huntingtin,
although efficient in silencing m-Htt expression, can not discriminate between the mutant
and wild-type alleles. Silencing of wild-type Htt in adult neurons could lead to
neurodegeneration. To circumvent these problems, synthetic siRNAs directed against
polymorphisms linked with the mutant allele have successfully reduced the levels of the
mutant allele while minimally affecting the levels of wild-type protein. However, this has
yet to be proven in vivo where the effects of off-targeting remain relatively unknown.
Long-term in vivo expression of both HD6 and HD7 hammerhead ribozymes as
well as the siHUNT-2 shRNA resulted in the remarkable loss of ppEnk, DARPP-32 and
dopamine type-2 receptor mRNA steady-state levels in both R6/1 and wild-type mice.
This reduction if striatal-specific mRNAs was not due to a global reduction in
transcriptional activity since NGFi-A mRNA was induced above wild-type levels
(Chapter 3). These three molecules targeted a 22-nucleotide region of the human Htt
mRNA. HD6 was designed to target nucleotides at positions 29-41 from the ATG start
site of the human Htt mRNA (accession # L34020). HD7 targeted nucleotides at positions
38-49 from the ATG start site while the shRNA siHUNT-2 targeted nucleotides at
positions 24-45 from ATG start site. BLAST searches have revealed no similarity to
known mouse mRNA sequences, however, there are up to six expressed-sequence tags
(ESTs) from adult mouse neurospheres that match the criteria required for the HD6, HD7
or siHUNT-2-mediated cleavage of the molecule. Because the phenotype associated with
the expression of HD6 HD7 and siHUNT-2 was reminiscent to an HD-like phenotype,
further studies into the gene or genes that are being directly affected by these molecules
are warranted. One initial approach would be to isolate the genes from neurospheres
using the EST sequences as probes. One could then design ribozymes or shRNAs against
different and unique sequences within the gene or genes in order to determine their
contribution to the HD-like phenotype that we observed.
We report a mild-behavioral improvement in the R6/1 mouse induced by the long-
term expression of rAAV5-siHIUNT-1. The R6/1 more accurately replicates Juvenile HD
were the progression of disease is much faster and severe. Therefore, the fact that we
only attained mild cellular and behavioral improvements in the R6/1 mice does not
diminish the potential therapeutic value of shRNAs in HD, especially on the more slow-
progressing, adult-onset type. In fact, others have evaluated the therapeutic potential of a
variety of molecules and environmental enrichment in the R6/1 mouse. These studies
have resulted in varying degrees of success that paralleled the findings in our study. For
example, the administration of fatty acids led to motor improvements but no recovery in
the levels of Dl or D2 mRNA levels. Also, environmentally enriched R6/1 mice have
increased rotarod rentention times when compared to controls, however, levels of Dl or
D2 mRNAs remained unchanged. Finally, cortical transplantation of wild-type donor
cortical tissue results in a delayed in the rear paw clasping phenotype. Thus, the mild
improvements attained in our study could translate into more profound effects in slower
progressing models of HD.
At the time of this study, RNAi was an exciting, albeit, relatively new and
unknown mechanism. New understandings of the pathway and the biochemical properties
of the RISC complex have paved the way for the development of more specific and more
efficient short-hairpin RNAs. This in turn has greatly enhanced the application of
shRNAs in animal models of neurological disease. However, issues concerning
intracellular expression and delivery into target sites are just now beginning to be
addressed. Developing drug-regulatable expression cassettes and enhancing the capacity
of viral vectors to spread into and be contained within target structures will expand the
application of RNAi from therapy into the study of gene function in vivo.
The search for a cure to Huntington's disease not only entails the development of
therapies but also the design and application of molecular techniques that will allow the
HD research community to unravel the molecular cascade of events that lead to death.
This study demonstrated the therapeutic potential of RNAi-meditated knockdown of
striatal m-Htt. In addition, we also demonstrate that shRNAs can be used to understand
gene function in a temporal and spatial manner.
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