EFFICIENCY OF AAV9 NEURAL TRANSDUCTION AFTER INTRAMUSCULAR ADMINISTRATION

By

EMMANUELLE LAVASSANI

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

© 2016 Emmanuelle Lavassani

To my Mom

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ACKNOWLEDGMENTS

I would like to express my utmost gratitude for the guidance and support that my

mentors, Dr. Barry Byrne and Dr. Manuela Corti, have provided me over the past three

years. I am deeply appreciative for the opportunities they have given me to further my

knowledge of Pompe Disease through clinical and laboratory work. I would like to thank

Marda Jorgensen for the countless hours she has spent cultivating my histology skills.

Her patience and dedication to training and instructing me have been invaluable. My

sincere appreciation is extended to Dr. David Fuller for the advice and assistance he

generously provided to help me navigate through this process. I would like to express

appreciation for all of my committee members, Dr. Barry Byrne, Dr. Manuela Corti, Dr.

David Fuller, and Dr. Brian Harfe, for their valued guidance and insight.

Additionally, I would like to gratefully acknowledge Dr. Todd Golde and David

Fromholt for their assistance with the imaging of the spinal cord tissues. Special thanks

to Penn Vector Core in the School of Medicine Gene Therapy Program at the University

of Pennsylvania for supplying us with the AAV2/9 vector. I would to extend my gratitude

to Dr. Lin Yang, Fuyong Xing, and Yuanpu Xie for their analysis of the spinal cord

tissues. My deepest gratitude is extended to members of the Byrne Lab and Falk Lab

for their incredible support, training, and assistance. Lastly, I am eternally grateful to my

parents and grandparents for all of their love and encouragement.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 INTRODUCTION .................................................................................................... 12

Overview of Pompe Disease ................................................................................... 12

Pompe Disease CNS Pathology ............................................................................. 13 Enzyme Replacement Therapy ............................................................................... 15 AAV-Mediated Gene Therapy ................................................................................. 16

Properties of Recombinant AAV ............................................................................. 16

2 MANUSCRIPT ........................................................................................................ 19

Introduction to Pompe Disease ............................................................................... 19

Gene Therapy Targeting the CNS .......................................................................... 20

Materials and Methods............................................................................................ 22 Experimental Animals ....................................................................................... 22 AAV Vector ....................................................................................................... 24

In Vivo Vector Administration ........................................................................... 24 Histological Processing .................................................................................... 24

Immunohistochemistry...................................................................................... 25 Microscopy and Motor Neuron Quantitative Analyses ...................................... 27

Findings .................................................................................................................. 28

AAV9 Transduction Diminishes Gaa Expression .............................................. 28 Motor Neuron Morphology Associated with Pompe Disease ............................ 29 AAV9-Mediated eYFP Expression .................................................................... 31 Retrograde Transport of AAV9 ......................................................................... 32

Discussion .............................................................................................................. 33

3 CONCLUSIONS ..................................................................................................... 41

Significance ............................................................................................................ 41 Future Studies ........................................................................................................ 41

LIST OF REFERENCES ............................................................................................... 43

6

BIOGRAPHICAL SKETCH ............................................................................................ 46

7

LIST OF TABLES

Table page 2-1 Experimental animals.. ....................................................................................... 36

8

LIST OF FIGURES

Figure page 2-1 Experimental design. .......................................................................................... 35

2-2 Immunohistochemical acid α-glucosidase (GAA) staining of Gaa Flox/Flox one month post-injection and R26-stop-lacZ; Chat-Cre spinal cord sections. ........... 37

2-3 Immunohistochemical acid α-glucosidase (GAA) and NeuN staining of R26-stop-lacZ; Chat-Cre and post-injection Gaa Flox/Flox spinal cord sections. ........... 38

2-4 Immunohistochemical Green Fluorescent Protein (GFP) and NeuN staining of spinal cord section in R26-stop-eYFP one month post-injection with AAV. .... 39

2-5 Immunohistochemical Green Fluorescent Protein (GFP) and CD31 staining of spinal cord section in R26-stop-eYFP one month post-injection with AAV. .... 40

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LIST OF ABBREVIATIONS

AAV Adeno-associated virus

CMV Cytomegalovirus

CNS Central nervous system

CSF Cerebrospinal fluid

GAA Acid α-Glucosidase

Gaa-/- Pompe disease mouse model lacking Gaa activity

GFP Green Fluorescent Protein

ERT Enzyme replacement therapy

eYFP Enhanced Yellow Fluorescent Protein

FDA Food and Drug Administration

NeuN Neuronal Nuclei antibody

PFA Paraformaldehyde

PBS Phosphate buffered saline

TA Tibialis Anterior

TBS Tris-buffered saline

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EFFICIENCY OF AAV9 NEURAL TRANSDUCTION AFTER INTRAMUSCULAR

ADMINISTRATION

By

Emmanuelle Lavassani

December 2016

Chair: Barry Byrne Major: Medical Sciences

Pompe disease is a degenerative neuromuscular disease caused by the

deficiency or absence of acid α-glucosidase (GAA), an enzyme required for glycogen

degradation in the lysosome. Pompe disease is characterized by progressive cardiac

myopathy, skeletal muscle weakness, and respiratory difficulty. Recent reports

uncovered central nervous system (CNS) pathologies, which prompts the need to better

understand the implications of glycogen accumulation in the brainstem and motor

neurons. Enzyme replacement therapy (ERT) is currently the only FDA-approved

treatment for Pompe disease. This therapy, however, suffers from a serious limitation as

it does not penetrate the blood-brain barrier and therefore does not treat the CNS

pathology of the disease. Gene therapy can offer a promising alternative.

Adeno-associated viruses (AAV) are single-stranded, non-pathogenic

parvoviruses with the ability to transduce into both muscles and motor neurons and are

therefore being implemented as a vehicle of choice to deliver gene therapy. This study

explores the efficiency and potency of the AAV9 vector in integrating into the lumbar

region of the spinal cord and treating the symptoms of Pompe disease via bilateral

tibialis anterior (TA) muscle injections. Findings indicate that one-month post-bilateral

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TA injections the AAV9 vector utilizes retrograde transport to transduce -motor

neurons with 20% efficiency.

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CHAPTER 1 INTRODUCTION

Overview of Pompe Disease

Pompe disease is an autosomal recessive glycogen storage disease caused by

the deficiency or absence of acid α-glucosidase (GAA). GAA is responsible for

lysosomal degradation of glycogen, a glucose-containing complex polymer utilized for

energy storage in skeletal muscles and liver. When energy is needed, glycogen is

converted into glucose then distributed to muscles by muscle cells and to the central

nervous system (CNS) by liver cells (Raben et al., 2001). Pompe disease occurs in

approximately 1 in 40,000 births and over 300 mutations in the GAA gene have been

identified to cause the disease (Byrne et al., 2011).

Pompe disease is characterized by severe cardiac hypertrophy, progressive

skeletal muscle weakness, and respiratory difficulties. As glycogen degradation is

necessary in all organs to some extent, the disease manifests to various degrees in

each organ. Production of glycogen is directly related to GAA production, and therefore

tissues with higher rates of glycogen synthesis would require more GAA protein.

Skeletal muscles, cardiac muscles, and the liver have the highest rates of glycogen

synthesis in the human body and therefore experience the most significant Pompe

Disease pathology. Patients follow the same general disease progression; initial

proximal lower limb and paraspinal trunk muscle weakness, followed by diaphragm and

respiratory insufficiencies. Loss of diaphragm contractile strength is a hallmark feature

of Pompe disease and patients exhibit increased respiratory difficulty due to this

progressive weakness. Respiratory distress and infections coupled with progressive

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muscle weakness cause many patients to become ventilator-dependent within the first

few months of life (Byrne et al., 2011).

Severity of Pompe disease and age of onset are dependent upon the amount of

GAA protein produced in the patient. Although all patients with Pompe disease are born

with mutated GAA alleles, it is the site and type of gene mutation that determines the

amount of GAA protein produced, as well as quantitative manifestation of glycogen

accumulation. Sufficient glycogen must accumulate in the tissues before symptoms are

apparent. Deficiency or near-deficiency of GAA causes early-onset (infantile) Pompe

disease, the most severe form of the disease. Even limited production of the enzyme

will result in a less severe phenotype and later disease onset (Kishnani et al., 2006).

Patients completely deficient of GAA present symptoms of congestive heart

failure and severe hypotonia within the first few months after birth. Due to progressive

skeletal and cardiorespiratory weakness, early-onset Pompe patients become

ventilator-dependent and may die within one to two years without treatment. Patients

with late-onset Pompe disease have partial GAA activity and express symptoms later

than infancy due to the partially present glycogen degradation pathway. These patients

typically experience less severe, slower progressing cardiac myopathy and muscle

weakness (Kishnani et al., 2006).

Pompe Disease CNS Pathology

The range of pathologies associated with Pompe disease extends to the central

nervous system, specifically spinal motor neurons. Motor neurons are the sole source of

communication from the brain to activate skeletal muscle contraction, and therefore are

responsible for life-dependent muscle activity, such as breathing. During embryonic

development, more motor neurons are initially generated than will persist into

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adulthood. Feedback of neurotrophic and neurotropic factors to support neuronal growth

from muscle innervation determines which cells will survive. In the postnatal period,

motor neurons continue to rely upon the surrounding tissues for trophic factors. Motor

neuron population size stabilizes and remains approximately the same during postnatal

maturation and throughout adulthood (Kanning et al., 2010). It is hypothesized that

treating spinal motor neurons will help correct the neuromuscular deficits experienced

by Pompe patients.

Glycogen is too large to be transported between axons and must be degraded

within the cell in order to be metabolized by axons. Typically neurons are devoid of

glycogen, as GAA efficiently degrades glycogen to prevent glycogen accumulation-

induced apoptosis of neural cells (Vilchez et al., 2007). Absent or decreased GAA

expression prevents the degradation of glycogen and causes the cells to enlarge

(Brown et al., 2004). Neurons are especially vulnerable to the effects of glycogen

accumulation, as there is limited cell regeneration potential with few metabolic pathways

to overcome the physiological impact of GAA deficiency.

There have been reported cases of glycogen accumulation in the cervical spinal

cord motor neurons of Pompe patients and Pompe mouse models lacking Gaa activity

(Gaa-/-), specifically in the phrenic motor neurons (Lee et al., 2011). Deposits of

glycogen in phrenic motor neurons cause swelling of the associated nerve and may be

the cause of diaphragm dysfunction and compromised ventilation. Motor neurons

experiencing glycogen accumulation can be identified by swollen perikaryon with

enlarged vacuoles and off-center nuclei (ElMallah et al., 2013). Reduced breathing

frequencies have been reported in Pompe patients, which may be indicative of a

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difference in respiratory neuron activity that decreases respiration when glycogen

accumulation occurs.

Swollen motor neurons are not as metabolically active and cannot transduce

signal as efficiently. Larger motor neurons need greater excitatory input to fire than

smaller motor neurons (Henneman et al., 1965). Enlarged motor neurons in Pompe

patients and Gaa-/- mice would require a higher action potentials, which reduces the

likelihood for motor neuron depolarization and could result in fewer signals to innervated

muscles. The entire motor unit should therefore be targeted in order to correct

downstream pathologies. Further studies need to be done to better understand

neuromuscular interactions and the downstream effects on innervated muscles.

Enzyme Replacement Therapy

Currently, there is no cure for Pompe disease, however, therapies are being

developed to correct for the lack of GAA and treat the symptoms of the disease.

Enzyme replacement therapy (ERT) is currently the only Food and Drug Administration

(FDA) approved treatment for Pompe disease. ERT involves bi-weekly intravenous

infusion of recombinant GAA enzyme to substitute functional GAA in patients. ERT

targets the cardiac and skeletal muscle manifestation of the disease while leaving the

central nervous system untreated. This is due to the inability of the protein to penetrate

the blood-brain barrier. Patients develop respiratory insufficiency and may require

mechanical ventilation even while receiving ERT, most likely as result of the lack of

CNS correction from the therapy. ERT delays the progression of muscle weakness but

is not a long-term solution for treating lysosomal glycogen accumulation (Fuller et al.,

2013).

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AAV-Mediated Gene Therapy

Gene therapy as a treatment for Pompe disease would provide long-term

correction for the deficient GAA gene. The most effective delivery of gene therapy with

the lowest immune response is via administration of adeno-associated virus (AAV).

High transduction of AAV-GAA in cardiac tissue has been reported to decrease

glycogen accumulation and restore cardiac structure and function. Previous studies with

AAV have demonstrated sustained GAA correction in Gaa-/- mice models via systemic

and intramuscular administrations (Mah et al., 2013). AAV vectors are being utilized in

clinical trials to treat a variety of diseases, such as alpha-1 antitrypsin deficiency, Limb-

girdle muscular dystrophy, and hemophilia B (Corti et al., 2014). AAV-mediated gene

therapy is being implemented in Phase I/II clinical trials for Pompe disease and has

been shown to restore GAA activity and improve cardiorespiratory and muscular

function (Byrne et al. 2011).

Properties of Recombinant AAV

Recombinant AAV are single-stranded, non-pathogenic human parvoviruses

containing DNA. The vector induces low inflammation and toxicity while maintaining

long-term expression in tissues. It has not been shown to cause any diseases in

humans to date. Wildtype AAV integrates into the AAVS1 site in human chromosome

19; however, recombinant AAV lacks the machinery to integrate into the genome.

Recombinant AAV therefore cannot be an insertional mutagen, although it is worth

noting that random integration of recombinant AAV occurs randomly with a 0.1%

frequency. Self-complementary vectors contain modifications that enable AAV

replication to occur independent of a replication helper virus, which results in higher

vector transduction and activity levels. Self-complementary recombinant AAV vectors

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are responsible for a stronger innate immune response and contain less safety control

mechanisms. Neither wild type nor recombinant AAV has been known to cause toxicity

in animal models or humans.

Recombinant AAV vectors are ideal for clinical application as they persist as a

double stranded circular episome in the nucleus of the cell and do not integrate into the

host genome. Recombinant AAV episomes can develop a chromatin-like structure and

persist for years in the nucleus without damaging the cell, which enables stable, long-

term expression. AAV can transduce dividing and non-dividing cells, which makes it an

ideal candidate to target the entire motor unit and correct for the lack of GAA in the

muscles and CNS of Pompe patients and animal models. At least nine different AAV

serotypes are being developed as candidates for gene therapy delivery.

The abilities of AAV9 to transduce muscles and penetrate the blood-brain barrier

make it an ideal candidate for delivery of gene therapy to the CNS. AAV9 specifically

targets the liver, muscles, heart, CNS, and alveoli in the lungs. AAV9 robustly infects

motor neurons via retrograde axonal viral delivery from muscles, which enables delivery

of gene therapy to specific motor neuron populations. AAV9 had been reported to

transduce hypoglossal motor neurons with 30% efficiency eight weeks post-injection

directly into the tongue. Additionally, a single intramuscular injection of AAV9 is

sufficient for sustained vector expression in the target muscle as well as the associated

motor neurons (ElMallah et al., 2012).

The high transduction efficiency of the AAV9 capsid yields high amounts of

transgenic expression in the injected tissues. This high efficiency enables low-

concentration delivery of gene therapy, which is critical for clinical application of the

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vector (Pacak et al, 2008). Effects of integration of the vector into the muscle have been

shown as early as two weeks post-intramuscular injection, which indicates that the

treatment could be delivered quickly to patients. The AAV9 retrograde transport

mechanism via intramuscular injection is still unclear; although it has been hypothesized

that vector integration occurs at the neuromuscular junction (Falk et al., 2014).

AAV2 was the first identified AAV serotype and has been shown to have safe,

long-term expression in vivo (Niemeyer et al., 2008). The low transduction efficiency of

AAV2, particularly to the CNS, limits expression of the target gene. Additionally, the

prevalence of pre-existing antibodies against AAV2 in the human population limits the

efficiency of the vector (Chirmule et al.,1999). However, it still remains one of the most

widely used vectors for human gene therapy.

The AAV capsid is comprised of proteins encoded by cap genes, which confer

serotype-binding specificity. Unique amino acids sequences in the variable regions of

external capsid proteins bind particular cell types and molecules with varying affinities.

Rep genes encode the proteins necessary for AAV replication within the cell nucleus.

Pseudo-typed vectors show capsid-associated tissue tropism and transduction

efficiency. An AAV2 genome plasmid pseudo-typed into an AAV9 capsid creates a

vector with higher neural transduction. A pseudo-typed AAV2/9 vector consists of the

AAV2 rep and AAV9 cap genes. AAV2/9 will elicit an immune response in response to

AAV9 as well as transduce cells targeted by that serotype, while expressing the target

gene encoded in AAV2. Pseudo-typing allows for vector transduction via AAV9 with rep-

mediated activity of the insert located in the AAV2 plasmid (Holehonnur et al., 2014).

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CHAPTER 2 MANUSCRIPT

Introduction to Pompe Disease

Pompe disease is a rare metabolic, neuromuscular disorder caused by the

deficiency or absence of GAA. GAA is a critical enzyme in glycogen degradation

pathway, and deficiency of GAA results in glycogen accumulation throughout the body.

Progressive cardiac myopathy, respiratory insufficiencies, and muscle weakness are

hallmark features of the disease. Glycogen accumulation is most evident in the skeletal

muscles, cardiac muscles, and CNS (Byrne et al., 2011)

Recent studies have reported glycogen accumulation in the CNS, specifically in

in the spinal cord. Motor neurons experiencing glycogen accumulation contain enlarged

somas with off-center nuclei and distended vacuoles (ElMallah et al., 2013). It is

hypothesized that the CNS glycogen accumulation can contribute to the observed

progressive muscular weakness, as there is less signal transduction from the affected

neurons. Studies have shown that spinal motor neurons are particularly susceptible to

excessive glycogen accumulation, which could additionally compromise activity of the

associated muscles (DeRuisseau et al., 2009). The neurological pathology of the

disease remains unclear and further research is needed to understand how the disease

manifests in the CNS of Pompe patients.

Currently, ERT of recombinant GAA is the only FDA-approved therapy to treat

Pompe disease. Although ERT has been shown to prolong the lifespan of patients, it

cannot cross the blood-brain barrier and treat the neurological pathology of the disease

(Falk et al., 2013). AAV-mediated gene therapy can provide a safe, long-term correction

for GAA deficiency. AAV can penetrate the blood-brain barrier and effectively transduce

20

neurons and muscles, which makes it the ideal delivery system for Pompe disease

therapies. Intramuscular administration of AAV is preferred for treating the CNS as it is

non-invasive and the candidate target muscles are easily accessible.

Gene Therapy Targeting the CNS

AAV9 has been reported to utilize retrograde transport from the presynaptic

terminal at neuromuscular junctions through the length of the axon to reach the

projecting motor neuron cell nucleus and express target genes. In order for AAV to

travel to the neuronal cell body, the virus needs to bind viral receptors on the axon

terminal and use dynein-mediated microtubule transport to access the motor neuron

nucleus (Kaspar et al., 2003).

The goal of this study is to determine the efficiency of the AAV9 vector in

integrating into the spinal cord via bilateral intramuscular injections into the tibialis

anterior (TA) muscles. Targeting motor neurons with gene therapy can provide

sustained correction for GAA deficiency that prevents glycogen accumulation and the

resulting apoptosis. In order to treat the progressive muscle weakness of Pompe

disease, the underlying motor neurons innervating muscles need to be targeted and

corrected so atrophy of the motor unit does not occur. Congenital GAA deficiency could

cause early glycogen accumulation in motor neurons that prevents cell survival during

postnatal maturation, leaving muscles under-represented in spinal motor pools.

Previous reports have indicated that there is no loss of motor neurons during postnatal

maturation in healthy rats (Lowry et al., 2001). It can be assumed that the same occurs

in mice as well, and therefore intramuscular TA injections in adult mice will target motor

neurons without the issue of vector dilution from repeated cell division. This prevents

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the need for AAV re-administration, which is ideal as initial immune response to AAV

prevents the possibility of re-administration of the same AAV serotype.

Gaa Flox/Flox mice are conditional Pompe mouse models that replicate the

characteristics of the disease as it manifests in humans. R26-stop-eYFP mice are

functionally normal mice that conditionally express enhanced Yellow Fluorescent

Protein (eYFP). Gaa Flox/Flox and R26-stop-eYFP mice received injections of AAV9-CMV-

Cre to determine AAV9 neural transduction to the lumbar region of the spinal cord, as

shown in Figure 2-1. Injection of AAV9-CMV-Cre induces Gaa deficiency, and the

inferred Pompe disease pathology in Gaa Flox/Flox mice cells transduced with the vector.

R26-stop-eYFP mice express eYFP in the cytoplasm of cells positive for AAV9. Spinal

motor neurons innervating the TA muscles are located in lateral motor columns in the

lumbar region of the spinal cord, specifically L3-L5. It has been estimated that each TA

muscle is innervated by 188 motor neurons (McHanwell and Biscoe, 1981). It is

hypothesized that the vector will directly integrate into the injected TA muscles and

utilize retrograde transport to transduce the associated motor neurons located in L3-L5

of the spinal cord.

In order to determine the efficiency of AAV9 neural transduction, a baseline

lumbar motor neuron quantity needed to be established. Quantification of the total

number of motor neurons in the lumbar region of the spinal cord in control R26-stop-

lacZ; Chat-Cre mice yielded a value of 3,003 -motor neurons. Gaa Flox/Flox and R26-

stop-eYFP mice were administered the same vector and therefore histological

comparison of the respective spinal cords will help elucidate the transport efficiency of

the vector. The R26-stop-eYFP mice facilitate visualization of vector transduction in the

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spinal cord by fluorescence in vector-positive cells, thus providing an easy visual

demonstration of the target tissues of the vector and promoter. The R26-stop-eYFP

mice provide the same purpose as a control strain to compare the number of motor

neurons present after injection into the TA muscle versus the Gaa Flox/Flox mice, which

may undergo motor neuron loss from Gaa deficiency. The various experimental mice

strains outlined provide a comprehensive understanding of the transduction of the

vector through intramuscular injections, as well the viability of the TA as a candidate

tissue for gene therapy administration. The results of this study indicate that self-

complementary AAV9 can effectively transduce approximately the 20% of motor

neurons in the lumbar region of the spinal cord after bilateral intramuscular TA injection.

Materials and Methods

Experimental Animals

All experimental procedures were approved in accordance with the guidelines set

forth by the Institutional Animal Care and Use Committee at the University of Florida.

Mice were maintained on a 12 hour light/dark cycle with water and food provided ad

libitum.

R26-stop-lacZ; Chat-Cre mice were bred in house and obtained from Jackson

Laboratories, Bar Harbor, ME. R26-stop-lacZ; Chat-Cre mice are functionally normal

and serve as a control strain against Gaa Flox/Flox and R26-stop-eYFP mice. Histological

staining of the spinal cord sections of R26-stop-lacZ; Chat-Cre mice provided control

images of functionally normal motor neurons, and contributed to the quantification of

motor neurons located in the lumbar region of the spinal cord.

R26-stop-eYFP mice were developed at Jackson Laboratories and utilized as a

control strain against Gaa Flox/Flox mice. R26-stop-eYFP mice have a loxP-flanked STOP

23

sequence located upstream of the eYFP gene inserted into the Gt(ROSA)26Sor locus.

In the presence of Cre recombinase, the loxP sites remove the STOP sequences

blocking eYFP gene transcription. eYFP protein is expressed in the cytoplasm of cells

containing Cre. The mice are functionally normal until Cre recombinase is present. As

noted by Srinivas et al., 2001, there is no background expression of eYFP that could be

detected in R26-stop-eYFP mice.

Gaa Flox/Flox (line 11248) mice were obtained from Taconic, Albany, NY and are

conditional Pompe disease models. Gaa Flox/Flox mice have loxP sites exons 4-8 in the

Gaa gene located on chromosome 17. In the presence of Cre recombinase, the loxP

sites remove exons 4 - 8 in the Gaa gene and prevent Gaa expression. The mice are

functionally normal until Cre recombinase is introduced. Knocking down the Gaa gene

elicits a Pompe mouse disease model that replicates the disease characteristics as it

manifests in humans. Once the Gaa gene is inactivated, it is inferred that the mice will

experience glycogen accumulation and cardiac hypertrophy, progressive muscle

degeneration, and respiratory difficulties.

R26-stop-eYFP and Gaa Flox/Flox mice received bilateral tibialis anterior injections

of vector at 3 months of age. Administration of AAV9-CMV-Cre to Gaa Flox/Flox mice

eliminates expression of Gaa protein where Cre recombinase is expressed.

Administration of AAV9-CMV-Cre to R26-stop-eYFP mice induces activation of eYFP

protein in cells where Cre recombinase is expressed. The AAV9-CMV-Cre vector

integrates fully into the tissue in about 4 weeks. Therefore one month post-injection, the

symptoms of Pompe disease manifest in Gaa Flox/Flox mice and cells express eYFP in

R26-stop-eYFP mice. The R26-stop-lacZ; Chat-Cre mice serve as control mice with

24

functional, wildtype Gaa expression and motor neuron physiology. All mice were age

and sex-matched and sacrificed at 4 months of age for histological assays.

AAV Vector

A self-complementary AAV2 genome plasmid containing a Cre recombinase

gene, under the control of the ubiquitous cytomegalovirus (CMV) promoter was pseudo-

typed into an AAV9 capsid. AAV9-CMV-Cre (AAV2/9sc.CMV.PI.Cre.SV40) was

obtained by the Penn Vector Core, Philadelphia, PA and injected into Gaa Flox/Flox and

R26-stop-eYFP mice. Self-complementary AAV vectors have been previously reported

to mediate transgene expression at substantially higher levels than single-stranded AAV

vectors, which require a helper virus (Nathwani, 2006). Each mouse was received a

dose of 1.5 × 1011 vector genomes per TA muscle, a dose determined sufficient for

vector transduction in prior studies (ElMallah et al., 2012). Final formulations of the

AAV2/9sc.CMV.PI.Cre.SV40 vector were diluted in Lactated Ringer’s solution.

In Vivo Vector Administration

Animals were three months of age at time of AAV9-CMV-Cre administration. Gaa

Flox/Flox (N=5) and R26-stop-eYFP (N=5) mice were anesthetized with 3% isofluorane in

oxygen in box induction, then 0.5%-2.5% maintenance using nose cone. Under sterile

conditions, the mice received a single subcutaneous injection of 25μl of vector directly

into each TA muscle. The insulin syringe (29.5 gauge) containing the vector was held at

a 45-degree angle and administered in the center of the muscle.

Histological Processing

One month post-injection the mice were anesthetized with 3% isofluorane in

oxygen in box induction, then 0.5%-2.5% maintenance using nose cone. R26-stop-lacZ;

Chat-Cre, Gaa Flox/Flox, and R26-stop-eYFP mice were euthanized via transcardial

25

perfusion, first with 0.1M phosphate buffered saline (PBS) then 4% paraformaldehyde

(PFA) in 0.1 M PBS. It should be noted that anesthetic depth just prior to euthanizing

any animal was confirmed by a lack of blood pressure and respiratory responses to toe

pinch. The spinal cords were extracted and post-fixed by immersion in 4%

paraformaldehyde (N= 5 animals per group) for 24 hours. The following day the spinal

cords were removed from the surrounding vertebrae and nerves with blunt end forceps

and scissors. The dura was then peeled off and the spinal cord was sectioned into

lumbar, thoracic, and cervical regions. TA muscles are innervated by motor neurons

located in the lumbar region, and therefore only the lumbar region of the spinal cord

underwent histological assays.

The spinal cords were washed three-times for 15 minutes in 0.01 M PBS to

remove 4% PFA residues then transferred into 30% sucrose solution for infiltration and

cryoprotection. The tissues remained in the sucrose solution for 2 to 3 days, at which

point the tissues sank to the bottom of the sucrose solution, signaling they were ready

for embedding. Upon removal from sucrose, the tissues were placed horizontally in

Tissue-Tek Intermediate Cryomolds (VWR) containing Tissue-Tek O.C.T. compound

(Sakura Finetek USA, Torrance, CA). Tissues were then rapidly frozen using

isopentane chilled with liquid nitrogen. Tissue blocks were stored at -80oC until

cryosectioning on a cryostat set at -22oC. Longitudinal spinal cord serial cryosections

(30 μm) were kept in 48-well plates containing cryoprotectant solution and stored at -20

oC for eventual staining.

Immunohistochemistry

Lumbar spinal cord sections were individually stained as floating sections in 48-

well plates to maintain the rostral to caudal sequence. Tissues were removed from

26

cryoprotectant and placed into new 48-well plates pre-loaded with 0.01M PBS. Sections

were washed two-additional times for 10 minutes each in 0.01M PBS, then equilibrated

for 10 minutes in 0.1M Tris-buffered saline (TBS)-0.2% Triton X-100. Next, the tissues

were blocked in a solution containing 0.1M TBS-0.2% Triton X-100 and 3% Normal

Horse Serum for one hour at 37oC. After blocking, tissues were incubated for 48 to 72

hours at 4oC in primary antibodies diluted in blocking buffer. The primary antibodies

used were as follows: rabbit anti-GAA at 1:2,000 (courtesy of Dr. Barry Byrne

Laboratory) for R26-stop-lacZ; Chat-Cre and Gaa Flox/Flox tissues, chicken anti-GFP at

1:1,500 (Aves Labs, Tigard, Oregon) for R26-stop-eYFP tissues, mouse anti-NeuN at

1:1,500 (EnCor Biotechology Inc, Gainesville, FL), 1:100 rat anti-CD31 (BD

Pharmingen, San Jose, CA) for R26-stop-lacZ; Chat-Cre, Gaa Flox/Flox, and R26-stop-

eYFP tissues. NeuN, meaning “neuronal nuclei,” is antibody that detects neuron-specific

nuclear proteins in post-mitotic neurons. NeuN is produced in mature neurons, however,

it is not present in neural progenitors, γ-motor neurons, Purkinje cells, oligodendrocytes,

glia, and astrocytes (Mullen et al., 1992). NeuN is used in this experiment specifically to

target α-motor neurons in the spinal cord. The Green Fluorescent Protein (GFP)

antibody binds eYFP with high affinity and is used to detect eYFP expression. CD31,

also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1), targets

endothelial cells and neutrophils and is utilized in only a few tissues to target vascular

endothelial cells in the spinal cord.

Following primary antibody incubation, the tissues were washed four-times for

20 minutes each in 0.01M PBS, then placed in the appropriate secondary antibodies at

room temperature for three hours. The secondary antibodies were used as follows: anti-

27

rabbit Alexa Fluor 594 at 1:2,000 (ThermoFisher Scientific, Waltham, MA) for R26-stop-

lacZ; Chat-Cre and Gaa Flox/Flox tissues, anti-chicken Alexa Fluor 594 at 1:1,500

(ThermoFisher Scientific) for R26-stop-eYFP tissues, anti-mouse Alexa Fluor 488 at

1:1,500 (ThermoFisher Scientific) for R26-stop-lacZ; Chat-Cre, Gaa Flox/Flox, and R26-

stop-eYFP tissues, and anti-rat Alexa Fluor 647 at 1:500 (Invitrogen, Carlsbad, CA).

After incubation, tissues were washed three-times for 30 minutes each in 0.01M PBS

then mounted onto Superfrost glass slides and coverslipped in Vectashield Antifade

Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA). A schematic

of the experimental animals and their respective histological assays is shown in Table

2-1.

Microscopy and Motor Neuron Quantitative Analyses

Fluorescent images were captured using an Aperio ScanScope Slide Scanner

(Leica Biosystems, Buffalo Grove, IL), a Slide Scanner Axio Scan.Z1 (Zeiss, Dublin,

California), an Olympus BX43 upright microscope, and an Olympus DP80 camera using

20x and 40x objectives. Measurements and analyses were performed using ImageJ,

Aperio Imagescope software (Leica Biosystems), ZEN lite Digital Imaging Software

(Zeiss, Oberkochen, Germany), Olympus CellSens software (Olympus, Pittsburgh, PA).

Multiple investigators qualitatively and quantitatively evaluated images and only

cells with visible processes or an identifiable nucleus were counted. In order to be

considered a motor neuron, cells were required to be NeuN positive, have a diameter of

18 μm – 60 μm, resemble typical motor neuron morphology, and fluoresce at sufficient

color intensities. Size parameters for cells were determined by literature values and

measurement of GFP-positive cell diameter size (Weber et al., 1997). Motor neuron

28

quantification was calculated using two channel (Alexa Fluor 488 and Alexa Fluor 594)

maximum intensity images of spinal cord sections.

For all mice, the NeuN-expressing cells with normal motor neuron morphology

were counted in the estimated lateral motor columns of the lumbar enlargement.

Quantification of R26-stop-lacZ; Chat-Cre NeuN-positive cells with the inclusion criteria

were counted as motor neurons for a baseline number of estimated motor neurons in

the lumbar region of the spinal cord. For Gaa Flox/Flox mice, the NeuN/GAA stained cells

with normal and Pompe disease motor neuron morphology were counted to estimate

the percentage of AAV9-positive, Gaa-deficient cells in comparison to the total number

of NeuN-stained motor neurons. In R26-stop-eYFP mice, the NeuN/GFP-expressing

cells with normal motor neuron morphology were counted to determine the ratio of GFP-

expressing motor neurons in comparison to all NeuN-positive motor neurons.

Findings

AAV9 Transduction Diminishes Gaa Expression

Bilateral TA intramuscular administration of AAV9-CMV-Cre into Gaa Flox/Flox mice

knocked down Gaa expression in vector-containing motor neurons. The

immunohistochemistry performed in the Gaa Flox/Flox mice demonstrated that there is

decreased Gaa activity with altered morphology in the spinal cord after the Gaa gene is

inactivated by Cre recombinase, further supporting the notion of an underlying Pompe

disease neural pathology. One-month post-bilateral TA injections, 592 GAA-deficient

motor neurons could be detected out of 2,980 NeuN-positive neurons, yielding 19.8%

AAV9 neural transduction efficiency.

Figure 2-2 demonstrates the differences of GAA expression between Gaa Flox/Flox

motor neurons one-month post injection versus control motor neurons. Cre expression

29

due to AAV9-CMV-Cre transduction in Gaa Flox/Flox mice causes little to no GAA protein

expression detected by the anti-GAA antibody. R26-stop-lac; Chat-cre Z mice, as

functional Gaa controls, expressed Gaa with robust fluorescent detection by the anti-

GAA antibody. As shown in the figure, motor neurons that received AAV9 lack Gaa,

contained less visible cellular processes, and were significantly larger than the

surrounding cells. Gaa deficiency compromised the physiology of the cells and caused

the nuclei to move off-center to accommodate the inferred lysosomal glycogen

accumulation. AAV activity has been detected as early as two weeks post-

administration in previous reports; therefore this diminished level of Gaa protein

occurred within a maximum of two weeks before harvesting (Pacak et al., 2008). As

evidenced in Figure 2-2, Gaa deficiency induced a significant change in motor neuron

morphology within a relatively short time span.

AAV9 neural transduction to -motor neurons was observed typically within close

vicinity of one another. This raises the question of whether cells are secreting AAV to

neighboring cells, an issue briefly discussed in other reports (ElMallah et al., 2013). The

issue of cross-secretion is not addressed in this study; however, it warrants exploration

in future studies as AAV activity upon reaching the target tissues is not well understood.

Motor Neuron Morphology Associated with Pompe Disease

Induction of Gaa deficiency in Gaa Flox/Flox mice receiving the vector to knockdown

Gaa expression provides further support of the neurological pathology of Pompe

disease. Perikaryon diameters observed in Gaa Flox/Flox were up to 10 μm larger than

control motor neuron size and exhibited morphology similar to the previously reported

motor neurons experiencing glycogen accumulation (ElMallah et al., 2012). Motor

30

neurons showed signs of glycogen accumulation via distended vacuoles and enlarged

somas. As seen in Figure 2-3, motor neurons of Gaa Flox/Flox mice after AAV injection

exhibit the same swollen appearance as Gaa-/- motor neurons. Detection of Gaa in Gaa

Flox/Flox-post injection and Gaa -/- cells showed decreased, if not absent, Gaa enzyme

activity.

Interestingly, the anti-NeuN antibody did not as effectively target Gaa-deficient

motor neurons. As demonstrated by the R26-stop-lacZ; Chat-Cre motor neurons, NeuN

effectively targets neuronal nuclear protein with robust fluorescent signal (Figure 2-3).

Gaa Flox/Flox-post injection and Gaa -/- mice, however, exhibited less NeuN activity, as

based upon the diminished intensity of Alexa Fluor 488 in the fluorescent images. The

decreased production of neuronal nuclear protein could be attributed to significant

neuroinflammation and glycogen accumulation initiating a downstream cascade for

decreased nuclear material production and the beginning of apoptosis.

Glycogen storage-dependent neuroinflammation could prevent sufficient signal

transduction to innervated muscles. Most likely the majority of cells are dysfunctional

due to changes in morphology (Bellettato and Scarpa, 2010). However, there may also

be a loss of motor neurons over time as the signaling capabilities of the cells become

compromised. Neuronal loss can lead to untreatable motor insufficiency, and is

therefore a priority for therapeutic treatment. Early treatment of the motor unit via

intramuscular TA administration of AAV9-GAA into Gaa-/- mice has been shown to

increase muscle force production, “innervation status,” and overall endplate status.

Pathology of the disease remained evident in the neuromuscular junction after

31

treatment corrected muscular glycogen accumulation, thus showing the impact of the

underlying neuronal pathology on the overall outcome (Todd et al., 2015).

AAV9-Mediated eYFP Expression

Administration of AAV9 bilaterally into each TA muscle mediated gene

expression in the spinal cord. AAV9-CMV-Cre floxed out the loxP sites and triggered

eYFP expression in motor neurons of R26-stop-eYFP mice. As seen in Figure 2-4, the

presence of AAV caused robust expression of eYFP protein in cell somas. One-month

post-injections, 606 GFP-positive -motor neurons could be detected out of 3,008

NeuN-positive neurons, yielding 20.1% AAV9 neural transduction efficiency in R26-

stop-eYFP mice.

Although AAV9 clearly transduces α-motor neurons, there is widespread AAV

expression throughout the spinal cord. This indicates that there may other means of

vector transport. In R26-stop-eYFP mice injected with AAV9, every section of spinal

cord contained fluorescent GFP staining, indicating the sporadic presence of the vector.

Additionally, eYFP expression was not limited to NeuN-positive neurons, but rather to

presumed γ-motor neurons and non-neuronal masses throughout the spinal cord.

Similar AAV9 off targeting has been reported previously and remains a possible

limitation of AAV9-mediate gene therapy (Benkhelifa-Ziyyat et al., 2013). Small cells

with neuronal morphology expressing eYFP were detected via the anti-GFP antibody,

which indicates that perhaps the vector utilized retrograde transport to transduce γ-

motor neurons. This would be relevant as NeuN is absent in γ-motor neurons and the

cells diameters are typically 6-17 μm in size (Friese et al., 2009). AAV9 transduction to

γ-motor neurons is critical for full correction of the motor unit.

32

Retrograde Transport of AAV9

It has been widely accepted that AAV9 vector travels via retrograde transport. As

evidenced by fluorescent staining of Gaa and eYFP, Cre recombinase expression was

highest in lateral columns of motor neurons, known to be associated with TA muscles. It

can thusly be assumed that the vector utilized retrograde transport to travel from the

injected muscle to the innervating motor neuron nucleus by dynein and microtubule-

mediated movement across the synapse and along the axon. AAV9 activity is not

limited to the connected motor neurons, as Cre expression is evident throughout the

lumbar region of the spinal cord. AAV9 transduction is, however, highest in the

suspected lamina IX, with a maximum of 40% -motor neuron transduction in the

sections containing the motor columns.

Other mechanisms for AAV transport have been proposed that are not well

understood. Recent reports indicate that AAV9 may transduce motor neurons via

bloodstream transport (Benkhelifa-Ziyyat et al., 2013). GFP-positive non-neural tissues

were positive for anti-CD31 antibody, which targets endothelial cells and monocytes

(Figure 2-5). GFP expression in endothelial cells could be indicative of AAV9

transduction via vascular tissues. The anti-CD31 antibody was fluorescently labeled

with Alexa Fluor 647, and emission at this wavelength is above the range for auto-

fluorescence. This indicates that this GFP expression throughout the spinal cord is due

to vector localization in vascular tissue, rather than via auto-fluorescence. However, this

finding requires further investigation to be better understood. Additionally, hindlimb

injections of AAV9 have resulted in off-target viral expression throughout the spinal

cord. This further confirms that there may another mechanism for viral delivery

33

(Benkhelifa-Ziyyat et al., 2013). It has been proposed that AAV9 may travel by

paravascular cerebrospinal fluid (CSF) within the CNS (Murlidharan et al., 2014). CSF

transport would explain the observed presence of AAV9 in regions outside of the ventral

horn. Although cells directly connected to lower limb muscles received more AAV,

another mechanism for vector transport may be responsible for the AAV activity

elsewhere in the spinal cord.

Discussion

The most significant finding is that an AAV9 vector can effectively transduce a

significant portion of the lumbar region -motor neurons after bilateral TA intramuscular

injections. TA-mediated delivery AAV9 should therefore be explored as a means for

motor neuron-directed therapy.

Early CNS Gaa deficiency in Pompe patients and mouse models may contribute

to the progressive skeletal muscle weakness. Neural pathology progression into

adulthood may lead to further motor dysfunction as glycogen accumulation persists and

damages the motor unit. It is important to note that only the lower motor neuron

population appears to be affected in Pompe disease, with upper motor neuron function

remaining intact (Sidman et al., 2008). As the majority of the Pompe disease CNS

involvement appears to be limited to the brainstem and spinal cord, intramuscular

delivery of gene therapy would be ideal to easily access the associated neuronal

populations. In conclusion, our AAV9 approach was able to transduce approximately

20% of -motor neurons throughout the lumbar region of the spinal cord indicates that

the TA is an ideal tissue for widespread lumbar enlargement spinal vector delivery.

34

Clinical Significance: Early targeting of the neuromuscular junction in Gaa-/-

mice with AAV9 carrying therapeutic GAA gene has been shown to restore

neuromuscular function and prevent loss of motor neurons (Todd et al., 2015). These

findings, along with the neuronal pathology confirmed in this report, supports the notion

that correcting the muscle and associated motor neurons can more effectively treat the

underlying neuronal pathology of the disease. Additionally, the TA muscle is an ideal

delivery tissue for gene therapy as it mediates high neural transduction of AAV9 to the

spinal cord.

35

Figure 2-1. Experimental design. A) Schematic of bilateral TA intramuscular injections of

AAV9-CMV-Cre for retrograde transport to motor neurons (1.5 x 1011 vector genomes per TA injection). Gaa Flox/Flox mice will exhibit Pompe disease and R26-stop-eYFP mice will produce eYFP in cells containing AAV. B and C) AAV9-positive cells will express Cre recombinase that excises sequences flanked by loxP sites. In Gaa Flox/Flox mice, exons 4-8 in the Gaa gene will be excised, rendering the Gaa gene inactive and inducing a Gaa-deficient CNS phenotype, inferred to be Pompe disease. In R26-stop-eYFP mice, Cre recombinase activity excises the STOP sequence upstream of the eYFP gene and induces eYFP expression.

Figure2.1

C

B

A

Gaa Flox/Flox

R26-stop-eYFP

INACTIVATION OF

GAA

ACTIVATION OF

eYFP

36

Table 2-1. Experimental animals. Schematic of the experimental animals with the mice strain, vector administration, and immunohistochemistry primary and secondary antibody staining.

Mouse Strain Vector Immunohistochemistry

R26-stop-lacZ; Chat-Cre N/A NeuN (AF 488) / GAA (AF 594)

Gaa Flox/Flox AAV9-CMV-Cre NeuN (AF 488) / GAA (AF 594)

R26-stop-eYFP AAV9-CMV-Cre NeuN (AF 488) / GFP (AF 594)

37

Figure 2-2. Immunohistochemical acid α-glucosidase (GAA) staining of Gaa Flox/Flox one month post-injection and R26-stop-lacZ; Chat-Cre spinal cord sections. A) GAA staining of Gaa Flox/Flox shows very low anti-GAA antibody Alexa Fluor 594 activity. Arrows indicate motor neurons lacking Gaa expression, which are inferred to be vector-positive. B) GAA staining of control strain, R26-stop-lacZ; Chat-Cre, spinal cord sections shows wildtype Gaa expression. Scale bar represents 50 μm.

38

Figure 2-3. Immunohistochemical acid α-glucosidase (GAA) and NeuN staining of R26-stop-lacZ; Chat-Cre and post-injection Gaa Flox/Flox spinal cord sections. A-C) NeuN and Gaa staining of control strain, R26-stop-lacZ; Chat-Cre, spinal cord sections demonstrate normal NeuN and GAA expression in functional motor neurons, as well as typical motor neuron morphology. D-F) NeuN and GAA staining of Gaa-/- mouse spinal cord section displays enlarged perikaryon with little GAA antibody detection, signaling a lack of Gaa expression. NeuN is not targeting the neuronal nuclear protein as efficiently in Gaa -/- cells, possibly due to cellular disruption from glycogen accumulation. G-I) Gaa Flox/Flox spinal cord sections one month post-injection with AAV9-CMV-Cre exhibit diminished Gaa expression, with morphology and staining patterns similar to Gaa-/-. The central motor neuron has been clearly transduced with vector. The adjacent appears to be vector-negative and expressing normal levels of neuronal nuclear material and Gaa. Scale bar represents 20 μm.

39

Figure 2-4. Immunohistochemical Green Fluorescent Protein (GFP) and NeuN staining of spinal cord section in R26-stop-eYFP one month post-injection with AAV. A) NeuN staining targets all neurons in the spinal cord B) GFP staining shows expression of eYFP protein in R26-stop-eYFP one month post-injection with AAV9-CMV-Cre. C) Merged image of NeuN and GFP staining shows co-localization of NeuN and GFP in vector-positive motor neurons, indicative of AAV expression via vascular tissue.

40

Figure 2-5. Immunohistochemical Green Fluorescent Protein (GFP) and CD31 staining

of spinal cord section in R26-stop-eYFP one month post-injection with AAV. A) GFP staining shows expression of eYFP protein in R26-stop-eYFP one month post-injection with AAV9-CMV-Cre B) CD31 staining targets endothelial cells. C) Merged image of GFP and CD31 staining shows co-localization of GFP and CD31 expression, indicating GFP activity in endothelial cells.

41

CHAPTER 3 CONCLUSIONS

Significance

This study confirms the neuronal pathology of the disease as well as shows the

efficacy of the TA muscle as a viable tissue for gene therapy injection that enables

delivery of AAV9 to the CNS. Inhibition of Gaa expression in Gaa Flox/Flox mice by

administration of viral therapy demonstrated the altered morphology of motor neurons

lacking Gaa. The observed accumulation of glycogen in the motor neurons may alter

the function of the neuron and prevent sufficient signaling to the innervated muscle.

Induction of eYFP expression in R26-stop-eYFP demonstrates the ability of AAV9 to

penetrate the blood-brain barrier and induce gene expression wherever present. This

study indicates that the bloodstream may be implicated in the delivery of AAV9 to the

CNS after direct intramuscular delivery.

Localized 40% neural transduction to lamina XI -motor neurons enables high

efficiency targeting of the lateral motor columns. In conclusion, intramuscular delivery of

AAV9 targets the CNS and transduces sporadic -motor neurons and 20% of -motor

neurons throughout the lumbar enlargement, which makes it the ideal vector to treat

neuromuscular diseases, such as Pompe disease or Amyotrophic Lateral Sclerosis.

Future Studies

The methodology outlined in this study will be carried out in Gaa -/Flox; R26-stop-

eYFP; Chat-Cre mice to elucidate the physical manifestation of the CNS pathology of

Pompe disease alone. Gaa -/Flox; R26-stop-eYFP; Chat-Cre mice are being developed

with the goal of creating a mouse that expresses eYFP in the motor neurons and is

completely deficient of Gaa in the CNS.

42

Gaa -/Flox; R26-stop-eYFP; Chat-Cre mice will be divided into treated and

untreated groups. Treated mice will receive bilateral intramuscular TA injections of

AAV9-DES-hGAA in order to alleviate the symptoms of Pompe disease and correct for

Gaa deficiency in the CNS. One month-post injection the mice will be harvested for

histological assays and the lumbar region of the spinal cord sections will undergo

immunohistological assays with anti-GAA and anti-eYFP antibodies. It is hypothesized

that due to treatment with gene therapy, Gaa -/Flox; R26-stop-eYFP mice will have less

swollen motor neurons than untreated Gaa -/Flox; R26-stop-eYFP mice. Assuming that

neural transduction of the therapeutic vector, , will have similar CNS transduction rates

as outlined in this study, restoration of 20% of Gaa protein in the motor neurons should

provide clinical improvement and correction for the disease. These experiments will

show whether the underlying neuronal pathology of Pompe disease is responsible for

symptoms of the disease. Results from the proposed study will provide information

regarding the viability of therapeutic strategies to target the CNS in preparation for

human clinical trials.

43

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46

BIOGRAPHICAL SKETCH

Emmanuelle Lavassani received her Master of Science in medical sciences

degree from the University of Florida in December 2016. In May 2015, Emmanuelle

graduated Summa Cum Laude with a Bachelor of Science degree in biology from the

University of Florida. For the past three years she has conducted research on gene

therapy to treat Pompe disease in the Department of Pediatrics and Powell Gene

Therapy Center under the tutelage of Dr. Barry Byrne and Dr. Manuela Corti.