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UTILIZING AAV GENE THERAPY TO PREVENT AND CORRECT AUTOPHAGY

DYSREGULATION IN CARDIAC MUSCLE OF A POMPE DISEASE MOUSE MODEL

BY: SYLVIA G. STANKOV

SENIOR UNDERGRADUATE THESIS

MICROBIOLOGY AND CELL SCIENCE

COLLEGE OF LIBERAL ARTS AND SCIENCES

UNIVERSITY OF FLORIDA

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Abstract

Pompe disease (PD) is a fatal metabolic disorder arising annually in 1 in 40,000 births. PD

is caused by mutations in the GAA gene, which encodes the enzyme acid α-glucosidase

responsible for the degradation of glycogen in lysosomes. Due to lack of enzyme, glycogen

accumulates in lysosomes leading to a cascade of autophagic dysregulation, muscle atrophy, and

a decrease in overall muscle function. Furthermore, cardiac muscle hypertrophy and skeletal

muscle weakness together lead to cardiorespiratory failure and, without treatment, a life

expectancy of 1 year with increasing disability over time. Currently, the only FDA-approved

treatment for PD is enzyme replacement therapy (ERT), which relies on functional vesicular

trafficking that becomes dysregulated and is unable to be corrected in this disease. However, PD

is being investigated for recombinant adeno-associated virus (rAAV)-mediated gene therapy as

an alternative treatment. Although preclinical studies and a clinical trial of an AAV-mediated gene

therapy treatment have been conducted, its effect on vesicular dysregulation has not been

characterized.

This project evaluated a gene therapy-based approach to prevent and correct vesicular

dysregulation, specifically autophagy, in cardiac muscle of the PD mouse model (Gaa-/-). To

complete this task, we examined GAA gene expression from the vector via anti-GAA staining,

anti-GAA immunoblots, enzyme activity assays, and PAS staining. Autophagic flux was analyzed

by H&E staining and anti-autophagy marker immunoblots such as LAMP1 and LC3. We have

found that AAV-mediated gene therapy successfully reduces total vacuolarization, including

primary pathology of lysosome accumulation, and somewhat ameliorates secondary pathology of

autophagosome accumulation. These characteristics demonstrate that gene therapy can prevent

and correct autophagic dysregulation in PD and allow us to move forward in providing an effective

treatment for these patients.

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Acknowledgements

I would like to express my thanks to my mentor Dr. Barry Byrne. His support and

knowledge have been key to this project, and I am grateful for the opportunity to work in his

laboratory. I extend my deepest gratitude to Dr. Angela McCall, without whom none of this work

would be possible. It is due to your guidance, motivation and example that I have learned nearly

all my laboratory and scientific critical-thinking skills. Your dedication to teaching me has been

invaluable, and I will carry these lessons with me as I pursue a career in the Biomedical Sciences.

My sincere thanks also goes to the other members of the Byrne Lab: Denise Cloutier, Lochlin

Cravey, Jayakrishnan Nair, Matthew Boothe, Monica Lee Tschosik, Blake Meyer and Cristina

Villena, who have given me advice and supported me throughout my years in the lab. I would also

like to thank Dr. Jennifer Lyles for sparking my interest in AAV and taking the time to teach me

skills from the ground up. I thank Dr. Darin Falk, Lauren Vaught and Kirsten Coleman for their

guidance and their humor. I would like to thank my parents, Mr. Georgi and Mrs. Svetlana

Stankov, who have always been my greatest supporters and encourage me to be relentlessly

ambitious. I thank my boyfriend Joseph, who never fails to lift my spirits. Thank you for always

being there for me. Lastly, I would like to acknowledge the Howard Hughes Medical Institute,

Science for Life for its Undergraduate Research Award, which funded me through the 2015-2016

academic year and the University Scholar’s Program, College of Medicine, which funded me

through the 2016-2017 academic year.

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Table of Contents

Title Page ................................................................................................................................... 1

Abstract ..................................................................................................................................... 2

Acknowledgements .................................................................................................................. 3

I. Introduction ............................................................................................................................ 5

a. Pompe Disease ................................................................................................................... 5

b. Autophagy Dysregulation in Pompe Disease ....................................................................... 6

c. Enzyme Replacement Therapy ............................................................................................ 7

d. Adeno-Associated Virus-mediated Gene Therapy ............................................................... 8

II. Methods ............................................................................................................................... 12

a. Study Design ..................................................................................................................... 12

b. Formalin Fixation ............................................................................................................... 13

c. Histological Analysis: GAA Staining ................................................................................... 13

d. Histological Analysis: PAS Staining ................................................................................... 14

e. Histological Analysis: H&E Staining ................................................................................... 14

f. Immunoblot ......................................................................................................................... 14

g. Biochemical Analysis: GAA Concentration ......................................................................... 14

h. Biochemical Analysis: Anti-autophagy Marker Antibody Immunoblot ................................. 15

i. Biochemical Analysis: GAA Enzyme Activity ....................................................................... 16

j. Statistical Analysis .............................................................................................................. 16

III. Results & Discussion......................................................................................................... 20

a. Vector construct and study design were modeled after previous investigations ................. 20

b. Treated mice exhibit GAA staining. .................................................................................... 21

c. GAA enzyme concentration exceeds wildtype with gene delivery ...................................... 21

d. GAA enzyme activity is restored with gene delivery ........................................................... 23

e. Treated mice exhibit a decrease in glycogen detected by PAS staining ............................. 25

f. Hearts of treated mice exhibit decreased vacuolization ...................................................... 26

g. Autophagy-related proteins decrease with gene delivery ................................................... 26

IV. Summary ............................................................................................................................ 39

V. References .......................................................................................................................... 40

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I. Introduction

a. Pompe Disease

Pompe disease (PD) is a fatal metabolic disorder arising annually in 1 in 40,000 births and

classified by infantile or late-onset forms [1-3]. The disease was first reported by pathologist J.C.

Pompe in 1932 after observing idiopathic cardiac hypertrophy in a deceased infant [4]. PD is

caused by at least 1 of the nearly 200 reported mutations in the GAA gene [5] on human

chromosome 17, which encodes the enzyme acid α-glucosidase (GAA) [6, 7]. GAA is found in

human heart, skeletal muscle, motoneurons, and liver and is responsible for the degradation of

glycogen to glucose in lysosomes [8]. Specifically, GAA catalyzes the cleavage of α-1,4 and α-

1,6 glucosidic linkages [6]. In severe infantile cases, less than 3% of normal GAA activity is found,

while less-severe late-onset forms may exhibit 3-30% normal GAA activity [9].

Without active GAA enzyme, glycogen accumulates in lysosomes, leading to a cascade

of autophagic dysregulation, disruption of muscle fibers, muscle atrophy, and a decrease in

overall muscle function [10]. PD patient tissues exhibit large vacuoles of glycogen that displace

normal cytoplasm and the organelles within [8]. Due to the presence of these vacuoles, cardiac

muscle hypertrophy and skeletal muscle weakness develop and together, lead to

cardiorespiratory failure – the predominant cause of death in untreated patients at less than 1

year of age [2, 8]. PD is considered the most severe type of glycogenosis due to death of the

patient within the first years of life [8].

Previously, to study PD in vivo, a Gaa knockout (Gaa-/-) mouse model was generated by

disrupting the Gaa gene such that no functional Gaa enzyme could be produced. On both cellular

and physiological levels, symptoms and signs of the mouse model are similar to those seen in

PD patients. These characteristics include glycogen accumulation, downstream pathway

disruption, cardiac muscle hypertrophy, skeletal muscle weakness, and respiratory distress [11].

This advancement has allowed detailed studying of PD pathogenesis and therapeutic strategies.

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b. Autophagy Dysregulation in Pompe Disease

While the first cellular consequence of PD involves accumulation of glycogen within

lysosomes, the secondary pathology consists of autophagic dysregulation. Autophagy is triggered

by nutrient deprivation and is the pathway through which long-lived proteins and damaged

organelles are degraded [10]. Mechanistically, an initiating membrane (phagophore) develops

around cellular material destined for degradation, and matures into a fully-formed

autophagosome. The autophagosome may fuse with an endosome to form an amphisome, but

ultimately fuses with a lysosome to form an autolysosome, in which the contents are degraded by

hydrolase activity [10]. Generally, movement within this system is referred to as autophagic flux,

as diagramed in Figure 1.

In PD, an initial accumulation of glycogen that is unable to be degraded within lysosomes

triggers a state of nutrient deprivation and activates autophagy. Concurrently, these glycogen-

laden lysosomes are incapable of fusing with autophagosomes, resulting in an overall halt of

autophagic flux. The cell continues to attempt amelioration of its state of nutrient deprivation by

activating autophagy, further stimulating the production of autophagosomes, which culminates in

the gross accumulation of both vesicles [12, 13].

Physically, autophagosome accumulation interrupts skeletal muscle fiber contractile

proteins, likely affecting muscle contractions [10, 14]. Data also show that inhibited autophagy in

muscle – such as that seen in PD - causes muscle weakness and features of myofiber

degeneration [15]. Moreover, both human PD and animal model studies have demonstrated that

autophagy failure causes skeletal muscle damage [10]. These observations speak to the

substantial, negative effect of autophagic dysregulation in PD patients, and the need for PD

therapy to address secondary pathology. To this end, Shea et al. suggest that “successful

treatment of patients with Pompe disease will require consideration of the dramatic failure of

autophagy that occurs in this disease” [10].

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c. Enzyme Replacement Therapy

Currently, the only FDA approved treatment for PD is enzyme replacement therapy (ERT)

[16]. Patients receive protein infusions of recombinant human GAA (rhGAA), known as

alglucosidase alfa and commercially sold as Myozyme® (recently replaced with Lumizyme®) [17].

rhGAA enters the cell in clathrin-coated vesicles through receptor-mediated endocytosis using

the mannose 6-phosphate receptor [10]. rhGAA then undergoes proteolytic activation while

passing through the endocytic pathway, as early endosomes mature into late endosomes through

acidification [18]. The receptor-enzyme complex dissociates at an acidic pH, allowing the

immature GAA to be delivered to the lysosome while the receptor is returned to the plasma

membrane [18]. rhGAA is converted to its mature form in the lysosome where it then degrades

glycogen [18].

While ERT is able to deliver GAA to the lysosome which clears glycogen,

autophagosomes remain [14]. Over time, this abundance of autophagosomes impedes ERT

efficacy by obstructing delivery of rhGAA to lysosomes [10]. In Gaa-/- mice, most of the rhGAA

accumulates in a centralized vesicular area, with very little reaching the lysosome [14]. In contrast,

autophagy-deficient Pompe mouse strains lack autophagic buildup and have been shown to

respond well to ERT [19]. For these reasons, PD ERT is most effective before autophagic buildup

occurs [14]. In the clinical setting, however, this window can prove elusive.

Furthermore, PD ERT only addresses symptoms in striated muscle. While clinical

observations of PD patients on ERT have shown improvement in cardiac function and increased

average life expectancy, at best only modest improvement has been demonstrated in skeletal

muscle, and no effect on CNS-related pathologies [20]. In skeletal muscle, this discrepancy is

observed because it is composed of 2 fiber types: oxidative type I and glycolytic type II [10]. While

ERT clears glycogen in type I fibers, it has been shown that type II skeletal muscle fibers respond

poorly to ERT [10]. Notably, lower levels of trafficking proteins (clathrin and adaptor protein-2)

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were observed in type II-rich muscle than in type I-rich muscle [10]. Type I fibers also have a

greater abundance of mannose 6-phosphate receptors, facilitating more receptor-mediated

endocytosis of rhGAA than in type II fibers [21]. These findings contribute to the disproportionate

efficacy of ERT between fiber types.

Finally, ERT poorly addresses PD neural pathology due to the inability of protein to cross

the blood-brain barrier [22]. The inability of ERT to correct skeletal muscle and neural pathology

in PD has led to a search for additional therapeutic strategies [23] [24].

d. Adeno-Associated Virus-mediated Gene Therapy

Currently, PD is being investigated for recombinant adeno-associated virus (rAAV)-

mediated gene therapy. Gene therapy is an experimental technique that uses genes to treat

disease [25]. Ideally, this therapy will provide long-term and sustainable treatment delivered at

the molecular DNA level, and has proven to do so in mouse, dog, non-human primate, and human

studies [26-29]. Gene therapy can be delivered via a viral vector, and recombinant Adeno-

associated virus (rAAV) is at the forefront of vector options.

AAV is a small, nonpathogenic virus in the Parvovirus family. The AAV genome is ssDNA

and 4.7kb in length [30]. The wildtype vector carries a packaging open reading frame (ORF) (rep)

and structural ORF (cap) flanked by inverted terminal repeats (ITR) used for self-priming in

second-strand synthesis [31]. To produce rAAV vectors for gene therapy, only the ITRs of the

original AAV genome are required. A therapeutic transgene is inserted between the 2 ITRs,

replacing rep and cap [32]. Importantly, it has been shown that rAAVs infect both dividing and

non-dividing cells, are replication deficient, and do not integrate into the host genome [31].

Together, these qualities make it an attractive gene therapy vector. rAAV vectors have been used

in clinical trials for diseases including hemophilia B, Leber congenital amaurosis, Parkinson’s

disease, and Duchenne Muscular Dystrophy [33].

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In rAAV-mediated gene therapy, the gene of interest is packaged into an AAV capsid and

delivered to a patient where, on the cellular level, the virus is trafficked through endosomes and

DNA is delivered to the nucleus [31]. In contrast to ERT, a gene therapy approach expresses the

protein of interest endogenously, allowing the therapy to circumvent PD ERT’s reliance on the

mannose 6-phosphate receptor [31]. This freedom may reduce or even eliminate the difference

in therapeutic efficacy between muscle fiber types that is observed in ERT. Furthermore, by

producing the protein endogenously, rAAV-mediated gene therapy allows a more stable

expression of the transgene and its protein product instead of the fluctuations associated with

ERT protein infusions.

Although preclinical studies and a clinical trial of an AAV-mediated gene therapy treatment

for PD have been completed, the effect on autophagy has not been characterized following gene

therapy [34-37]. Furthermore, while concurrent studies are investigating success of AAV in

skeletal muscle, it is also imperative to study the therapy’s efficacy in cardiac muscle. PD patients

readily experience cardiorespiratory complications and general cardiomyocyte autophagy has

been implicated in nearly all forms of heart disease [38]. Moreover, while autophagic accumulation

in skeletal muscle of the PD mouse model is well documented, here, we define autophagy

dysregulation in cardiac muscle of the PD mouse model at multiple time points. We also propose

a single therapeutic vector to address both cardiac and skeletal muscle autophagy dysregulation.

This project aimed to evaluate an rAAV9-DES-coGAA vector to prevent and correct

autophagy dysregulation in cardiac muscle of the PD mouse model (Gaa-/-). A tissue-restricted

Desmin (DES) promoter was included to direct transgene expression to the areas of interest [38].

Additionally, the modified DES promoter’s small size (~300bp) provided ample room for the large

GAA transgene (~3000bp) within the AAV vector, which is limited to ~4.7kb. Codon optimization

provided additional enhancement of gene expression by using codons that are most commonly

found in highly expressed genes, thus using the most abundant charged tRNAs in the cell [39].

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Finally, an AAV serotype 9 capsid was selected because this serotype readily transduces

myocardium [38], skeletal muscle, and the CNS [40]. The Gaa-/- mouse model was selected for

therapeutic investigation as it confers a complete lack of GAA production and is widely used in

the PD research community [11].

The gene therapy was administered via systemic injections to examine PD secondary

pathology throughout the body, with the expectation that by replacing the defective gene

responsible for PD (GAA), primary and secondary pathology will be inhibited in the preventive

treatment group, and possibly reversed in the corrective treatment group.

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Figure 1. Autophagic flux facilitates degradation of cellular contents in the lysosome. Autophagy is triggered by nutrient deprivation. A phagophore develops around the contents to be degraded by selective and non-selective mechanisms, forming an autophagosome decorated with LC3-II. When a lysosome, decorated with LAMP1, fuses with an autophagosome, an autolysosome is formed. The inner autophagosome membrane and cellular contents are degraded by acid hydrolases.

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II. Methods

a. Study Design

A Desmin (DES) promoter driving human codon-optimized GAA (coGAA) packaged in an

AAV9 capsid (rAAV9-DES-coGAA) vector was utilized for experimentation. rAAV vectors were

produced by the University of Pennsylvania Vector Core and purified by the University of Florida

Powell Gene Therapy Center Vector Core (UF-PGTC-VC) by traditional double transfection

methods as described previously [41].

Briefly, HEK293 cells were transfected with 2 plasmids – AAV9 helper plasmid (a kind gift

from Dr. James Wilson, University of Pennsylvania, Philadelphia, PA) and rAAV9-DES-coGAA

plasmid – by calcium phosphate methods. After 60 hours, cells were lysed and vector was purified

by an iodixanol step gradient and concentrated in excipient buffer using an Apollo 100k centrifugal

concentrator.

The University of Florida Institutional Animal Care and Use Committee (IACUC) approved

all animal use under the protocol 201408522. Mice were housed at the University of Florida

Animal Care Services. Equal numbers of 1-day-old and 3-month-old male and female mice were

utilized and randomly assigned to an experimental group.

Vector was injected intravenously (IV) via temporal vein at birth to Gaa-/- mice (Taconic,

Hudson, NY, USA) [42] at a high dose of 11014 vector genomes/kilogram (vg/kg) or via tail vein

at 3 months of age at low (11011 vg/kg), mid (11013 vg/kg), and high (11014 vg/kg) doses. For

temporal vein injections, mice were anesthetized by placing them on ice for 1-2 minutes, then

injected and returned to the cage with the dam. For tail vein injections, mice were weighed

immediately before injection, placed under a heat-lamp to promote vasodilation, then placed in a

moderately confined restraining device. Injections were made using a 28-gauge insulin syringe.

Untreated, control Gaa-/- mice and 129SVE mice were injected with vehicle, Lactated Ringer’s

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Solution to serve as controls. Hearts were harvested 1 month (short-term) or 6 months (long-term)

post injection (p.i.) as described below. A study outline detailing injection and tissue harvest

schedule is provided in Figure 2.

Mice were anesthetized using 2-4% isofluorane inhaled through a nose cone, and

euthanized via cervical dislocation between the hours of 8:00a.m. and 12:00p.m. to minimize

fasting-induced autophagy [43]. The heart was collected and either flash-frozen in liquid nitrogen

and stored at -80°C until processing by methods detailed below, or fixed in formalin, also detailed

below. A flow-chart to visualize all biochemical and histological analyses performed is provided in

Figure 3.

b. Formalin Fixation

Hearts were cut so that a 5mm cross-section from the median of the muscle was obtained.

Sections were fixed in formalin for 24 hours at 4°C, then exchanged into phosphate buffered

saline (PBS). Cardiac muscle sections were processed on an automated Sakura Tissue Tek VIP

tissue processor. Following dehydration through a gradient of alcohols and xylene, sections were

infiltrated with wax. The wax-filled tissues were oriented in embedding molds, wax was added to

surround the tissues, and the blocks were allowed to harden. Finally, cross-sectioned hearts were

serially sectioned into 3 sets using a microtome (Microm HM 330) to 6 microns, mounted to

Superfrost Plus Microscope Slides and air-dried overnight at room temperature.

c. Histological Analysis: GAA Staining

Serially sectioned cross-sections of formalin-fixed hearts were stained with a rabbit

polyclonal anti-hGAA antibody. The sections were blocked for biotin, avidin, and peroxidase prior

to incubation with the primary antibody. DAB was used to detect the peroxidase-conjugated

secondary antibody. Images were taken using an upright light microscope (Olympus BX43) and

cellSens software (Olympus Standard).

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d. Histological Analysis: PAS Staining

A second set of serially sectioned cross-sections of formalin-fixed hearts were stained with

Periodic Acid Schiff (PAS) (Sigma-Aldrich: 395B-1KT) in accordance with manufacturer’s

instructions. Image analysis was performed on the same microscope and using the same software

detailed above.

e. Histological Analysis: Hematoxalin and Eosin Staining

A third set of serially sectioned cross-sections of formalin-fixed hearts were stained with

Hematoxalin and Eosin (H&E) according to manufacturer’s instructions (Leica). Coverslips were

mounted with cytoseal. Image analysis was performed on the same microscope and using the

same software detailed under Histological Analysis: GAA staining.

f. Immunoblot

Protein samples were separated on SDS-PAGE (30 minutes at 90 Volts followed by 60

minutes at 120 Volts) then transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon-

FL: IPFL00010) for 90 minutes at 200mAmps. The membrane was subsequently blocked for 1

hour at room temperature, followed by overnight incubation at 4°C with primary antibody.

Membranes were washed 3 times for 5 minutes then incubated with secondary antibody for 1

hour at room temperature. Following incubation, membranes were washed again 3 times for 5

minutes then scanned using LI-COR Odyssey infrared detection system (model 9120).

Integrated intensity (band densitometry) of all experimental bands were determined with

coordinating software and normalized to the respective, sample-specific GAPDH band (anti-

GAPDH, 1:2500, Cell Signaling Technology: #2118).

g. Biochemical Analysis: GAA Concentration

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50 milligrams of tissue from each experimental heart was homogenized in 200μL Water

for Injection (WFI) Quality Water (HyClone: SH30221.10) with protease inhibitor (Roche: 04-693-

124-001) and Lysing Matrix D ceramic spheres (MP Biomedicals: 116540434). Samples were

physically sheared 3 times using FastPrep24 (MP Biomedicals:116004500) with alternating 1

minute homogenization and 5 minutes rest, then subjected to 3 freeze-thaw cycles. Supernatant

(water-soluble fraction) was separated via centrifugation (13.2k rpm for 10 minutes) from

remaining water-insoluble pellet and stored at -80°C.

Protein was quantified (Bio-Rad: 500-0111) using a bovine serum albumin (BSA) standard

curve and according to manufacturer’s instructions.15μg total protein aliquots of the water-soluble

homogenate fraction (with Laemeli Buffer, then heated to 100°C for 10 minutes) were prepared.

Serial dilutions of 5mg/ml rhGAA (alglucosidase alfa, Myozyme®) [44] were performed to generate

250μg, 125μg, 62.5μg and 31.25μg positive control samples for a standard curve. The water-

soluble homogenate aliquots were separated on 8% SDS-PAGE alongside the rhGAA positive

control samples. After electrophoretic separation, general immunoblot protocol was followed

using: Odyssey Blocking Buffer (PBS) (also used as antibody diluent) (Odyssey: 927-40000),

mouse anti-GAA primary antibody (3A6-IF2, gift from Jonathon Lebowitz, BioMarin, 1:100,000),

PBS-T (Gentrox: 40-028) and anti-mouse LI-COR secondary antibody.

Integrated intensity of rhGAA (110kDa band) serial dilutions were plotted against

nanogram quantities to generate a standard curve correlating amount of GAA to integrated

intensity. Integrated intensity of experimental GAA (76kDa band, normalized to GAPDH integrated

intensity) was plotted on the line, describing amount of GAA to estimate GAA concentration in

experimental samples. GAA concentration is represented as nanograms (ng) of GAA per 15μg

total protein from the respective whole tissue lysate.

h. Biochemical Analysis: Anti-autophagy Marker Antibody Immunoblot

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The water-insoluble pellet remaining from the water-soluble homogenate preparation was

homogenized in 200μl 0.5%SDS in PBS with protease inhibitor and Lysing Matrix D ceramic

spheres as previously described, followed by 1 freeze-thaw cycle. Protein was quantified by Bio-

Rad DC Protein Assay and 50μg total protein aliquots prepared (with Laemeli Buffer, then heated

to 100°C for 10 minutes). Samples were separated on 15% SDS-PAGE and the general

immunoblot protocol was followed.

For membranes probed with anti-LC3A (1:1000) (Cell Signaling Technology: #4599),

membranes were treated in accordance to manufacturing suggestions for fluorescent immunoblot

detection with modifications using 5% non-fat milk in Tris-buffered saline (TBS) as blocking buffer,

5% BSA in TBS-T (Gentrox: 40-059) as primary antibody dilution buffer, and 5% non-fat milk in

TBS-T for secondary antibody diluent. For membranes probed with anti-LAMP1 (1:250)

(Developmental Studies Hybridoma Bank: 1D4B), Odyssey Blocking Buffer was used in all steps

(Odyssey: 927-40000) with intermittent washes with PBS-T (Gentrox: 40-028). Animal-

appropriate LI-COR secondary antibodies were applied.

i. Biochemical Analysis: GAA Enzyme Activity

GAA enzyme activity was determined with modifications to methods previously described

[37]. Protein from the water-soluble homogenate fraction was assayed for activity by measuring

fluorescence after 1-hour incubation at 37°C with 3mM 4-methylumbelliferyl-α-D-glucoside

(Sigma-Aldrich: M9766) in 200mM sodium acetate buffer pH 4.3. To stop the reaction, 500mM

sodium carbonate pH 10.7 was added. A standard curve was generated using 4-

methylumbelliferone (Sigma-Aldrich: M1381). Substrate-cleavage fluorescence was detected

using Biotek Synergy HTX Multimode Reader and normalized to protein quantification values

obtained by Bio-Rad DC Protein Assay.

j. Statistical Analysis

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Figures were drawn and Ordinary One-Way ANOVA statistical analysis was performed

using GraphPad Prism [45].

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Figure 2. Study Outline. There were 3 arms of this project: prevention long-term, correction short-term, and correction long-term. For the preventative treatment group, mice were injected at birth and analyzed in the long-term (6 months p.i.). rAAV9-DES-coGAA vector was injected

intravenously (IV) via temporal vein only at the high (11014 vg/kg) dose. For the correction treatment group, mice were injected at 3 months of age and analyzed either in the short-term (1 month p.i.) or in the long-term (6 months p.i.). The same vector was IV injected via the tail vein at 3 experimental doses: low (11011 vg/kg), mid (11013 vg/kg), and high (11014 vg/kg). Mice were weighed immediately before injection. Untreated, control Gaa-/- mice and 129SVE mice (wildtype equivalent of the Gaa-/- strain) were injected with vehicle, Lactated Ringer’s Solution to serve as

controls.

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Figure 3. Simplified flow chart of methods. Hearts were either fresh frozen in liquid nitrogen and stored at -80°C or fixed in formalin and serially sectioned for histological analyses. 3 sets of formalin-fixed hearts were prepared for GAA, PAS, and H&E staining. Fresh-frozen hearts were homogenized, GAA activity assay performed, GAA concentration quantitatively estimated, and autophagy markers assessed. Analyses were combined to examine transgene expression and autophagic flux.

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III. Results & Discussion

a. Study design was modeled after previous investigations.

To investigate the therapeutic efficacy of an AAV-mediated gene therapy for PD, an

rAAV9-DES-coGAA vector was constructed. This vector conferred tissue-restricted expression of

the codon optimized human GAA gene, delivered in an AAV9 capsid.

Overall, there were 3 arms of this project: prevention long-term, correction short-term, and

correction long-term. For the preventative treatment group, mice were injected at birth and

analyzed in the long-term (6 months p.i.). The aim was to investigate prevention of autophagy

dysregulation by initiating therapy before the onset of dysregulation. For the correction treatment

group, mice were injected at 3 months of age and analyzed either in the short-term (1 month p.i.)

or in the long-term (6 months p.i.). Here, the aim was to investigate correction of autophagy

dysregulation in mature mice because their cellular pathology mimics the pathology found in

infantile-onset patients at time of diagnosis and typical initiation of ERT. The 1 month time-point

was selected because it takes approximately 3 weeks for protein expression from an AAV vector

to reach a maximum [46]. The 6 month time-point was selected because ultimately, the goal of

our therapy is to provide PD patients with a long-term solution that increases their quality of life.

Currently, PD ERT requires re-administration every 2 weeks [47], while gene therapy has been

reported to persist for years [48]. A better understanding of its potential as an alternative long-

term treatment is necessary.

A total of 3 experimental doses: low (11011vg/kg), mid (11013 vg/kg), and high (11014

vg/kg) of rAAV9-DES-coGAA were selected for therapeutic investigation. The preventative

treatment mice were only injected with the high dose of vector. The corrective treatment mice

were injected with the low-, mid-, and high-doses of vector. These doses were selected because

a previous study found 11011 total vector particles of AAV2 or AAV9 was ineffective dose for

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clearing glycogen storage from the heart [49]. Furthermore, Falk et al demonstrated that 11011

vg of AAV9 does reduce glycogen in the heart and diaphragm, but does not reach statistical

significance [50]. Finally, 51010 total vector particles of AAV1 was reported to completely restore

GAA activity in the heart but only partially restore GAA activity in other skeletal muscles [51].

Therefore, for this study, the mid-dose was designed to be equivalent to 2.51011 total vector

genomes on average to compensate for the previously reported therapeutic shortcomings. Based

on these previous findings, it was predicted that the low dose would have little-to-no effect, and

the high-dose would yield supra-physiological levels of GAA specifically in the hearts of treated

animals.

b. Treated mice exhibit GAA staining.

Formalin-fixed hearts were embedded in paraffin, sectioned, then stained with a polyclonal

anti-GAA antibody specific for human GAA. This analysis was used to visualize presence of gene

therapy-derived GAA in corrective treatment mice examined in the short-term.

No GAA staining was observed for the wildtype mice because the antibody used was

specific for human GAA, encoded within our vector and expressed only in treated animals. The

129SVE animals express mouse Gaa, encoded by the mouse Gaa gene. The hearts of affected,

untreated mice exhibited no GAA staining, as expected. Animals treated with the high dose of

vector demonstrated staining of GAA, confirming presence of the vector-derived protein (Figure

4). Staining appeared in pockets distributed throughout the cross-section, and was particularly

concentrated in the periphery of the muscle. Approximately 80% of cardiac muscle fibers

appeared to be transduced and producing GAA enzyme.

c. GAA enzyme concentration is dose-dependent.

To verify presence of GAA and estimate the enzyme’s concentration in the experimental

samples, integrated intensity of the GAA 76kDa band was measured. The 76kDa band was

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chosen because while all forms of GAA have some catalytic function, the 76kDa form is most

abundant and most active [52]. GAA concentration was determined as nanograms of GAA per

15μg total protein from the respective whole tissue lysate.

Preventative treatment mice evaluated in the long-term demonstrated high concentrations

of GAA enzyme with an average of 10.84ng GAA per 15μg total protein (Figure 5). As above, a

human GAA antibody was also utilized in this analysis, thus no GAA bands were present in

wildtype lanes.

Corrective treatment mice evaluated in the short-term also demonstrated high and dose-

dependent concentrations of GAA enzyme (Figure 6). The mice treated with the mid-dose

exhibited an average of 47.58ng GAA per 15μg total protein, while the mice treated with the high-

dose exhibited an average of 90.98ng GAA per 15μg total protein. Overall, the high-dose mice

demonstrated nearly 2 times the GAA concentration (191%) of the mid-dose mice. The detection

of GAA by immunoblot supported the GAA staining pattern observed in this groups’ hearts.

Finally, corrective treatment mice evaluated in the long-term also demonstrated dose-

dependent concentrations of GAA enzyme (Figure 7). The mice treated with the mid-dose

exhibited an average of 7.96ng GAA per 15μg total protein and those that received the high-dose

exhibited an average of 15.56ng GAA per 15μg total protein. Again, the high-dose mice

demonstrated nearly 2 times the GAA concentration (195%) of the mid-dose mice.

In all treated animals, only the 76kDa form of GAA was found. This was a positive

indication that the gene therapy-derived GAA was proteolytically activated and fully-matured in

the lysosome. Conversely, studies of ERT rhGAA processing in vivo have shown other forms of

GAA in addition to the 76kDa species [53]. This finding suggests that gene therapy-derived GAA

may be more effectively matured into its most abundant and active form than ERT-delivered

rhGAA.

23

Additionally, while the high-dosed mice consistently exhibited approximately 2 times the

GAA concentration of the mid-dosed mice for both time points, the concentration of GAA

decreased between the short-term and long-term time points.

d. GAA enzyme activity is restored with gene delivery.

To further investigate the product of coGAA transgene expression, GAA enzyme activity

assays were performed. Preventative treatment mice evaluated in the long-term demonstrated

supra-physiological levels of GAA activity in the heart at 2872.76% of wildtype (Figure 8).

Corrective treatment mice evaluated in the short-term also demonstrated supra-physiological

levels of activity with both the mid- and high-dose at 332.03% and 3538.49% of wildtype,

respectively. Finally, supra-physiological levels of activity were observed in corrective treatment

mice evaluated in the long-term. The mid-dose and high-dose hearts were at 596.82% and

938.70% of wildtype GAA activity, respectively (Figure 9).

In all hearts analyzed, activity from the low-dose animals was not significantly above

untreated knockout levels. For this reason, it was determined that the low-dose group would not

experience any downstream therapeutic effects on autophagy; thus, only the mid- and high-dosed

animals were investigated in subsequent biochemical and histological analyses.

Overall, high-dosed mice investigated for correction in the short-term exhibited the highest

level of GAA activity (mean = 1560.54 nmol of GAA activity/μg protein/hour). Furthermore, for all

mice treated with high-dose of vector for correction, GAA activity subsequently decreased when

assayed in the long-term. This observation reflected the GAA concentration findings where

concentration is higher in tissue examined 1 month p.i. than in tissue examined 6 months p.i. The

decrease in GAA activity may indicate a decrease in transgene expression over time between 1

month p.i. and 6 months p.i. and may also reflect an immune response that is more robust in the

long-term versus the short-term. Further studies investigating intermittent time points within the

24

present study could clarify the decrease in GAA activity between 1 month p.i. and 6 months p.i.

(i.e., slow and steady decline versus sharp decline). Additionally, longer p.i. time-points (i.e. 1 and

2 year(s) p.i.) will allow us to more fully understand long-term transgene expression.

Furthermore, the fold-difference in expression between the mid- and high-dose of vector

decreased between tissues assayed in the short-term and the long-term. For tissues harvested

and analyzed in the short-term, the high-dose exhibits 1065.70% of the mid-dose GAA activity.

For tissues harvested and analyzed in the long-term, the high-dose exhibits only 157.28% of the

mid-dose GAA activity. Nevertheless, supra-physiological levels of GAA were observed with both

the mid- and high-dose and maintained 1 month and 6 months p.i. The decrease in fold-difference

between doses reflects the overall decrease in GAA activity between tissues assayed in the short-

term and those assayed in the long-term.

In addition to attenuated transgene expression, the observed fold-difference decrease

may also be attributed to the greater immune response elicited by the high-dose (greater vg/kg

delivered, therefore greater GAA presence) relative to the mid-dose. Importantly, the PD mouse

model used in this study is known to produce a rhGAA (ERT) dose-dependent immune response

[42]. Therefore, a dose-dependent immune response to our gene therapy-derived GAA is

plausible. Here, the greater amount of GAA conferred by the high-dose would elicit a more robust

immune response in comparison to the mid-dose, causing a greater fold-difference in GAA

presence and thus activity of hearts analyzed in the short-term versus the long-term. The mid-

dose still elicits an immune response, but because less GAA is produced, the response is not as

severe and the fold-difference is reduced.

The supra-physiological levels of GAA observed in the hearts of the mid- and high-dosed

animals, for both preventative and corrective treatments, may be of concern. However, GAA does

not participate in a signaling cascade; the enzyme is only active when presented with its substrate.

Therefore, increased levels of GAA should not confer toxicity associated with elevated enzymatic

25

activity at the cellular level. However, supra-physiological amounts of GAA molecules may elicit

an immune response. Falk et al. found that while anti-GAA titer is elevated in Gaa-/- animals

treated with AAV9 vector compared to untreated controls, anti-GAA titer is significantly higher in

ERT-treated groups [54]. Therefore, while overcoming the hurdle of anti-GAA titer is important in

the context of clinical PD gene therapy, fortunately, immune response is less severe when

compared to the current standard of care, ERT.

Interestingly, the fold-differences between the high- and mid-dose mice observed with

GAA activity assays (1065.70% and 157.28% of the mid-dose, 1 and 6 months p.i., respectively)

were not reflective of the fold-differences observed with GAA concentration analysis (191% and

195% of the mid-dose, 1 and 6 months p.i., respectively). GAA activity assays of hearts analyzed

in the short-term described a substantially greater difference between the doses and the p.i. time-

points. GAA concentration analysis maintained the same 2-fold difference between doses

between both time-points.

e. Treated mice exhibit a decrease in glycogen detected by PAS staining.

Formalin-fixed hearts were embedded in paraffin, sectioned, and then stained with

Periodic Acid-Schiff (PAS) to detect polysaccharides. The presence of glycogen was used to infer

in vivo activity of the GAA enzyme. Increased presence of glycogen is inversely correlated to

presence of GAA. This analysis was performed on corrective treatment mice examined in the

short-term.

In affected, untreated mice, bright pink staining of glycogen is observed in the heart,

indicating a high amount of glycogen accumulating due to lack of GAA enzyme. Wildtype mice

exhibit light and uniform pink staining, indicating moderate amounts of glycogen throughout the

tissue. Mice treated the high dose of vector demonstrate reduced glycogen with areas of light and

uniform pink staining, much like the unaffected controls (Figure 10). The decrease in glycogen

26

confirmed in vivo GAA activity. With confirmation of enzyme activity and resolution of the primary

pathology, gene therapy’s effect on PD secondary pathology could now be assessed.

f. Hearts of treated mice exhibit decreased vacuolization.

Formalin-fixed hearts were embedded in paraffin, sectioned, and stained with H&E to

observe vacuolization, which is indicative of the presence of vacuoles such as lysosomes,

endosomes, and autophagosomes. Increased vacuolization leads to muscle fiber damage, and

has been correlated to diminished clinical outcomes [55]. This analysis was performed on

corrective treatment mice examined in the short-term.

Untreated, affected mice demonstrated profuse vacuolization and fiber disruption in the

heart in comparison to wildtype hearts, as expected. Mice treated with the high dose of vector

exhibited marked improvement in pathology with decreased vacuolization and an overall

appearance that resembles the wildtype hearts (Figure 11). This improvement in pathology

provided an indication that secondary pathology may be resolving in gene therapy-treated mice.

This hypothesis was investigated quantitatively by immunoblot.

g. Autophagy-related proteins decrease with gene delivery.

To investigate autophagic flux in the heart, lysosome associated membrane protein 1

(LAMP1) and an autophagosomal membrane protein (LC3) were analyzed by antibody

immunoblots. Heart lysates were homogenized in an SDS-containing buffer to release the

membrane-bound proteins. These analyses quantified the presence of autophagy-associated

proteins to infer lysosome and autophagosome membrane presence and draw conclusions on

overall autophagic flux. LAMP1 was used as a marker for lysosome membrane. LC3-II (lipidated,

autophagosomal form) was used as a marker for autophagosome membrane while LC3-I (non-

lipidated, cytosolic form) was used as an indicator of autophagy induction. Additionally, the LC3-

27

II to LC3-I ratio was used to indicate movement through the autophagic pathway, where a lower

ratio suggests increased flux because less LC3 is occupied in the autophagosomal form.

In both preventative and corrective treatments, and at both time-points, untreated Gaa-/-

mice exhibited increased levels of LAMP1, LC3-II, LC3-I, and LC3-II/LC3-I ratios in comparison

to wildtype animals (Figures 12, 13, and 14). These findings align with the expected accumulation

of lysosomes and autophagosomes, leading to decreased autophagic flux (increased LC3-II/LC3-

I ratio) in untreated, Pompe mice.

Preventative treatment mice investigated in the long-term demonstrated significantly

decreased levels of LAMP1 and the LC3-II/LC3-I ratio when compared to untreated Gaa-/- mice

(Figure 12). Corrective treatment mice investigated in the short-term also demonstrated

significantly decreased levels of LAMP1, LC3-II and the LC3-II/LC3-I ratio when compared to Gaa-

/- mice. LC3-I levels were also markedly decreased in treated mice when compared to untreated,

affected mice (Figure 13). Finally, corrective treatment mice investigated in the long-term

exhibited significantly decreased LC3-II/LC3-I ratio (Figure 14). LAMP1, LC3-II, and LC3-I protein

level decreases did not reach significance when compared to untreated, affected mice; however,

these proteins did demonstrate a downward trend towards resolution.

Overall, preventative and corrective treatment mice, in the short-term and long-term,

showed decreased levels of autophagy-related proteins which approached wildtype levels,

indicating some resolution of lysosomes and autophagosomes. Decreased LAMP1 and LC3-II

suggested decreased lysosomal membrane and autophagasomal membrane, respectively. This

observation, paired with statistically significant decreases in LC3-II/LC3-I ratios in all arms of the

project, suggested improved autophagic flux in the hearts of our gene therapy-treated mice.

These findings are particularly significant as they define autophagy dysregulation in the cardiac

muscle of this PD mouse model at multiple time-points and simultaneously provide an effective

therapeutic strategy to resolve contributing disease pathology.

28

Figure 4. Treated mice exhibit GAA staining. Formalin-fixed hearts were embedded in paraffin,

sectioned, and stained with a polyclonal anti-GAA antibody specific for human GAA. Images are

representative of untreated Gaa-/-, untreated wildtype, and corrective treatment mice that received

a high-dose (11014 vg/kg) of vector and were examined in the short-term. Treated hearts exhibit

pockets of staining for human GAA that are particularly concentrated in the periphery of the

muscle cross-section. Approximately 80% of cardiac muscle fibers appear to be transduced and

producing GAA enzyme.

29

Figure 5. GAA enzyme is present with a high-dose of rAAV9-DES-coGAA delivered at birth.

Hearts were homogenized in water and separated on SDS-PAGE, then transferred to PVDF. The blot was probed with anti-GAA antibody and average integrated intensity of each band determined. Concentration of GAA was calculated using a positive control standard curve. Treatment groups include: untreated Gaa-/-, untreated wildtype and Gaa-/- treated with the high-

(11014 vg/kg) dose of vector. Animals were treated at birth and analysis performed 6 months p.i. GAA enzyme is present in high-dose mice treated at birth. Data represented as mean ± SEM (n = 3-8 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001,

**** p ≤ 0.0001.

30

Figure 6. GAA enzyme concentration increases dose-dependently when rAAV9-DES-coGAA is delivered at 3 months of age and assayed 1 month p.i. Hearts were homogenized

in water and separated on SDS-PAGE, then transferred to PVDF. The blot was probed with anti-GAA antibody and average integrated intensity of each band determined. Concentration of GAA was calculated using a positive control standard curve. Treatment groups include: untreated Gaa-

/-, untreated wildtype and Gaa-/- treated with the mid- (11013 vg/kg) and high- (11014 vg/kg) dose

of vector. Animals were treated at 3 months of age and analysis performed 1 month p.i. GAA enzyme is present in treated mice. Also, concentration of GAA increases between the mid- and high-dose treated animals in a dose-dependent manner. Data represented as mean ± SEM (n = 3-4 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ****

p ≤ 0.0001.

31

Figure 7. GAA enzyme concentration increases dose-dependently when rAAV9-DES-coGAA is delivered at 3 months of age and assayed 6 months p.i. Hearts were homogenized in water and separated on SDS-PAGE, then transferred to PVDF. The blot was probed with anti-GAA antibody and average integrated intensity of each band determined. Concentration of GAA was calculated using a positive control standard curve. Treatment groups include: untreated Gaa-

/-, untreated wildtype and Gaa-/- treated with the mid- (11013 vg/kg) and high- (11014 vg/kg) dose of vector. Animals were treated at 3 months of age and analysis performed 6 months p.i. GAA enzyme is present in treated mice. Also, concentration of GAA in mid- and high-dose treated animals increases in a dose-dependent manner. Data represented as mean ± SEM (n = 3-4 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤

0.0001.

32

Figure 8. GAA enzyme activity is restored in the heart with a high-dose of rAAV9-DES-coGAA delivered at birth. Data represents activity of acid α-glucosidase (GAA) in the heart when tissue lysates are incubated with fluorescent substrate. Treatment groups include: untreated Gaa-

/-, untreated wildtype and Gaa-/- treated with the high dose of vector (11014 vg/kg). Animals were

treated at birth and analysis performed 6 months p.i. Supraphysiological levels of GAA activity are observed in Gaa-/- animals treated with the high-dose of vector. Data represented as mean ± SEM (n = 4-10 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p

≤ 0.001, **** p ≤ 0.0001.

33

Figure 9. GAA enzyme activity is restored in the heart with a mid- and high-dose of rAAV9-DES-coGAA delivered at 3 months of age. Data represents activity of acid α-glucosidase (GAA) in the heart when tissue lysates are incubated with fluorescent substrate. Treatment groups include: untreated Gaa-/-, untreated wildtype and Gaa-/- treated with the low- (11011 vg/kg), mid-

(11013 vg/kg) and high- (11014 vg/kg) dose of vector. Animals were treated at 3 months of age and analysis performed 1 month or 6 months p.i. Supraphysiological levels of GAA activity are observed in Gaa-/- animals treated with the mid- and high-dose of vector. GAA activity of Gaa-/-

animals treated with the low-dose of vector is approximately that of background. Data represented as mean ± SEM (n = 4-8 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤

0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

34

Figure 10. Treated mice exhibit decreased glycogen detected by PAS staining. Formalin-fixed hearts were embedded in paraffin, sectioned, and stained with Periodic Acid-Schiff (PAS) to detect polysaccharides. Images are representative of untreated Gaa-/-, untreated wildtype, and

corrective treatment mice that received a high-dose (11014 vg/kg) of vector and were examined in the short-term. Treated mice demonstrate reduced glycogen in areas of light, uniform pink staining, which resembles the unaffected controls. The decrease in glycogen confirms in vivo

GAA activity.

35

Figure 11. Treated mice exhibit decreased vacuolization. Formalin-fixed hearts were

embedded in paraffin, sectioned, and stained with H&E to observe vacuolization. Images are

representative of untreated Gaa-/-, untreated wildtype, and corrective treatment mice that received

a high-dose (11014 vg/kg) of vector and were examined in the short-term. Untreated, affected

mice demonstrate profuse vacuolization. Treated mice demonstrate reduced vacuolization and

an overall appearance that resembles wildtype hearts.

36

Figure 12. Lysosome and Autophagosome proteins decrease in the heart with a high-dose

of rAAV9-DES-coGAA delivered at birth. Hearts were homogenized in 0.5% SDS Buffer and

separated on SDS-PAGE, then transferred to PVDF. Blots were probed with anti-LAMP1 and anti-

LC3 antibodies. The average integrated intensity of each band was determined and normalized

to the integrated intensity of the respective GAPDH band. Treatment groups include: untreated

Gaa-/-, untreated wildtype and Gaa-/- treated with the high- (11014 vg/kg) dose of vector. Animals

were treated at birth and analysis performed 6 months p.i. LAMP1 and LC3-II/LC3-I ratio decrease

significantly to near-wildtype levels, suggesting autophagic flux restoration. Data represented as

mean ± SEM (n = 4-10 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01,

*** p ≤ 0.001, **** p ≤ 0.0001.

37

Figure 13. Lysosome and Autophagosome proteins decrease in the heart with a high-dose

of rAAV9-DES-coGAA delivered at 3 month of age and analyzed 1 month p.i. Hearts were

homogenized in 0.5% SDS Buffer and separated on SDS-PAGE, then transferred to PVDF. Blots

were probed with anti-LAMP1 and anti-LC3 antibodies. The average integrated intensity of each

band was determined and normalized to the integrated intensity of the respective GAPDH band.

Treatment groups include: untreated Gaa-/-, untreated wildtype and Gaa-/- treated with the mid-

(11013 vg/kg) and high- (11014 vg/kg) dose of vector. Animals were treated at 3 months of age

and analysis performed 1 month p.i. LAMP1 and LC3-II/LC3-I ratio decrease significantly to near-

wildtype levels, suggesting autophagic flux restoration. Data represented as mean ± SEM (n = 4-

6 animals per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p

≤ 0.0001.

38

Figure 14. Lysosome and Autophagosome proteins decrease in the heart with a high-dose

of rAAV9-DES-coGAA delivered at 3 month of age and analyzed 6 months p.i. Hearts were

homogenized in 0.5% SDS Buffer and separated on SDS-PAGE, then transferred to PVDF. Blots

were probed with anti-LAMP1 and anti-LC3 antibodies. The average integrated intensity of each

band was determined and normalized to the integrated intensity of the respective GAPDH band.

Treatment groups include: untreated Gaa-/-, untreated wildtype and Gaa-/- treated with the mid-

(11013 vg/kg) and high- (11014 vg/kg) dose of vector. Animals were treated at 3 months of age

and analysis performed 6 months p.i. LAMP1 and LC3-II/LC3-I ratio decrease to near-wildtype

levels, suggesting autophagic flux restoration. Data represented as mean ± SEM (n = 4-6 animals

per group). Significance is relative to Gaa-/-, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

39

IV. Summary

Here, we investigated an AAV gene therapy to prevent and correct autophagy

dysregulation in the cardiac muscle of a Pompe Disease mouse model, assessed in the short-

term (1 month p.i.) and the long-term (6 months p.i.). We used a tissue specific promoter and

codon-optimized, human GAA transgene delivered systemically in an AAV9 capsid.

For all treatment strategies, the mid- and high-doses were effective in producing GAA,

confirmed by GAA staining and immunoblot, which elicited supra-physiological levels of GAA

activity, confirmed by enzyme activity assays and decreased presence of glycogen by PAS

staining. Together, these findings indicated successful delivery of the transgene and endogenous

production of active GAA protein.

Furthermore, we described autophagic dysregulation in the heart of untreated, affected

Gaa-/- mice at multiple time points by quantifying increased levels of LAMP1, LC3-II, and LC3-I

proteins when compared to wildtype animals. Additionally, elevated LC3-II/LC3-I ratios indicated

decreased autophagic flux in these mice. The hearts of mid- and high-dose treated mice

demonstrated decreased levels of LAMP1, LC3-II, and LC3-I along with statistically significant

decreases in all LC3-II/LC3-I ratios. These results persisted between the short-term and long-

term time points and suggest continued restoration of autophagic flux.

Importantly, a concurrent study investigating skeletal muscle outcomes after therapeutic

administration reveals that the high-dose (11014 vg/kg) is more beneficial when examining

systemic outcomes (in preparation). Overall, rAAV9-DES-coGAA is an effective gene therapy

vector specifically when examining the prevention and correction of autophagy dysregulation in

the cardiac muscle of a PD mouse model.

40

V. References

[1] Di Rocco, M., Buzzi, D., and Tarò, M., 2007, "Glycogen storage disease type II: clinical

overview," Acta Myol, 26(1), pp. 42-44.

[2] Winkel, L. P., Hagemans, M. L., van Doorn, P. A., Loonen, M. C., Hop, W. J., Reuser, A. J.,

and van der Ploeg, A. T., 2005, "The natural course of non-classic Pompe's disease; a review of

225 published cases," J Neurol, 252(8), pp. 875-884.

[3] Manganelli, F., and Ruggiero, L., 2013, "Clinical features of Pompe disease," Acta Myol,

32(2), pp. 82-84.

[4] Pompe, J. C., 1932, "Over idiopathesche hypertrophie van het hart," Ned. Tijdschr.

Geneeskd, 76, pp. 304-312.

[5] Kroos, M., Pomponio, R. J., van Vliet, L., Palmer, R. E., Phipps, M., Van der Helm, R.,

Halley, D., and Reuser, A., 2008, "Update of the Pompe disease mutation database with 107

sequence variants and a format for severity rating," Hum Mutat, 29(6), pp. E13-26.

[6] D'Ancona, G. G., Wurm, J., and Croce, C. M., 1979, "Genetics of type II glycogenosis:

assignment of the human gene for acid alpha-glucosidase to chromosome 17," Proc Natl Acad

Sci U S A, 76(9), pp. 4526-4529.

[7] Solomon, E., Swallow, D., Burgess, S., and Evans, L., 1979, "Assignment of the human acid

alpha-glucosidase gene (alphaGLU) to chromosome 17 using somatic cell hybrids," Ann Hum

Genet, 42(3), pp. 273-281.

[8] Hers, H. G., 1963, "alpha-Glucosidase deficiency in generalized glycogenstorage disease

(Pompe's disease)," Biochem J, 86, pp. 11-16.

[9] van der Ploeg, A. T., and Reuser, A. J., 2008, "Pompe's disease," Lancet, 372(9646), pp.

1342-1353.

[10] Shea, L., and Raben, N., 2009, "Autophagy in skeletal muscle: implications for Pompe

disease," Int J Clin Pharmacol Ther, 47 Suppl 1, pp. S42-47.

[11] Raben, N., Nagaraju, K., Lee, E., Kessler, P., Byrne, B., Lee, L., LaMarca, M., King, C.,

Ward, J., Sauer, B., and Plotz, P., 1998, "Targeted disruption of the acid alpha-glucosidase

gene in mice causes an illness with critical features of both infantile and adult human glycogen

storage disease type II," J Biol Chem, 273(30), pp. 19086-19092.

[12] Cardiff, R. D., 1966, "A histochemical and electron microscopic study of skeletal muscle in a

case of Pompe's disease (glycogenosis II)," Pediatrics, 37(2), pp. 249-259.

[13] Griffin, J. L., 1984, "Infantile acid maltase deficiency. I. Muscle fiber destruction after

lysosomal rupture," Virchows Arch B Cell Pathol Incl Mol Pathol, 45(1), pp. 23-36.

[14] Fukuda, T., Roberts, A., Ahearn, M., Zaal, K., Ralston, E., Plotz, P. H., and Raben, N.,

2006, "Autophagy and lysosomes in Pompe disease," Autophagy, 2(4), pp. 318-320.

41

[15] Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., Metzger, D.,

Reggiani, C., Schiaffino, S., and Sandri, M., 2009, "Autophagy is required to maintain muscle

mass," Cell Metab, 10(6), pp. 507-515.

[16] Guo, J., Kelton, C. M., and Guo, J. J., 2012, "Recent developments, utilization, and

spending trends for pompe disease therapies," Am Health Drug Benefits, 5(3), pp. 182-189.

[17] Kishnani, P. S., Corzo, D., Nicolino, M., Byrne, B., Mandel, H., Hwu, W. L., Leslie, N.,

Levine, J., Spencer, C., McDonald, M., Li, J., Dumontier, J., Halberthal, M., Chien, Y. H.,

Hopkin, R., Vijayaraghavan, S., Gruskin, D., Bartholomew, D., van der Ploeg, A., Clancy, J. P.,

Parini, R., Morin, G., Beck, M., De la Gastine, G. S., Jokic, M., Thurberg, B., Richards, S., Bali,

D., Davison, M., Worden, M. A., Chen, Y. T., and Wraith, J. E., 2007, "Recombinant human acid

[alpha]-glucosidase: major clinical benefits in infantile-onset Pompe disease," Neurology, 68(2),

pp. 99-109.

[18] Moreland, R. J., Jin, X., Zhang, X. K., Decker, R. W., Albee, K. L., Lee, K. L., Cauthron, R.

D., Brewer, K., Edmunds, T., and Canfield, W. M., 2005, "Lysosomal acid alpha-glucosidase

consists of four different peptides processed from a single chain precursor," J Biol Chem,

280(8), pp. 6780-6791.

[19] Raben, N., Schreiner, C., Baum, R., Takikita, S., Xu, S., Xie, T., Myerowitz, R., Komatsu,

M., Van der Meulen, J. H., Nagaraju, K., Ralston, E., and Plotz, P. H., 2010, "Suppression of

autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--

murine Pompe disease," Autophagy, 6(8), pp. 1078-1089.

[20] Prater, S. N., Patel, T. T., Buckley, A. F., Mandel, H., Vlodavski, E., Banugaria, S. G.,

Feeney, E. J., Raben, N., and Kishnani, P. S., 2013, "Skeletal muscle pathology of infantile

Pompe disease during long-term enzyme replacement therapy," Orphanet J Rare Dis, 8, p. 90.

[21] Sun, B., Li, S., Bird, A., Yi, H., Kemper, A., Thurberg, B. L., and Koeberl, D. D., 2010,

"Antibody formation and mannose-6-phosphate receptor expression impact the efficacy of

muscle-specific transgene expression in murine Pompe disease," J Gene Med, 12(11), pp. 881-

891.

[22] Chien, Y.-H., Lee, N.-C., Peng, S.-F., and Hwu, W.-L., 2006, "Brain Development in

Infantile-Onset Pompe Disease Treated by Enzyme Replacement Therapy," Pediatric Research,

60(3), pp. 349-352.

[23] Raben, N., Danon, M., Gilbert, A. L., Dwivedi, S., Collins, B., Thurberg, B. L., Mattaliano, R.

J., Nagaraju, K., and Plotz, P. H., 2003, "Enzyme replacement therapy in the mouse model of

Pompe disease," Mol Genet Metab, 80(1-2), pp. 159-169.

[24] Sidman, R. L., Taksir, T., Fidler, J., Zhao, M., Dodge, J. C., Passini, M. A., Raben, N.,

Thurberg, B. L., Cheng, S. H., and Shihabuddin, L. S., 2008, "Temporal neuropathologic and

behavioral phenotype of 6neo/6neo Pompe disease mice," J Neuropathol Exp Neurol, 67(8), pp.

803-818.

[25] Reference, G. H., 2016, "What is gene therapy?," http://www.ncbi.nlm.nih.gov/pubmed/.

[26] Song, S., Morgan, M., Ellis, T., Poirier, A., Chesnut, K., Wang, J., Brantly, M., Muzyczka,

N., Byrne, B. J., Atkinson, M., and Flotte, T. R., 1998, "Sustained secretion of human alpha-1-

42

antitrypsin from murine muscle transduced with adeno-associated virus vectors," Proc Natl Acad

Sci U S A, 95(24), pp. 14384-14388.

[27] Herzog, R. W., Hagstrom, J. N., Kung, S. H., Tai, S. J., Wilson, J. M., Fisher, K. J., and

High, K. A., 1997, "Stable gene transfer and expression of human blood coagulation factor IX

after intramuscular injection of recombinant adeno-associated virus," Proc Natl Acad Sci U S A,

pp. 5804-5809.

[28] Nathwani, A. C., Rosales, C., McIntosh, J., Rastegarlari, G., Nathwani, D., Raj, D.,

Nawathe, S., Waddington, S. N., Bronson, R., Jackson, S., Donahue, R. E., High, K. A.,

Mingozzi, F., Ng, C. Y., Zhou, J., Spence, Y., McCarville, M. B., Valentine, M., Allay, J.,

Coleman, J., Sleep, S., Gray, J. T., Nienhuis, A. W., and Davidoff, A. M., 2011, "Long-term

safety and efficacy following systemic administration of a self-complementary AAV vector

encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins," Mol Ther, 19(5), pp.

876-885.

[29] High, K., 2002, "AAV-mediated gene transfer for hemophilia," Genetics in Medicine, 4, pp.

56S-61S.

[30] Srivastava, A., Lusby, E. W., and Berns, K. I., 1983, "Nucleotide sequence and organization

of the adeno-associated virus 2 genome," J Virol, 45(2), pp. 555-564.

[31] Daya, S., and Berns, K. I., 2008, "Gene Therapy Using Adeno-Associated Virus Vectors,"

Clin Microbiol Rev, 21(4), pp. 583-593.

[32] Daya, S., and Berns, K. I., 2008, "Gene Therapy Using Adeno-Associated Virus Vectors."

[33] Mingozzi, F., and High, K. A., 2011, "Therapeutic in vivo gene transfer for genetic disease

using AAV: progress and challenges," Nature Reviews Genetics, 12(5), pp. 341-355.

[34] Smith, B. K., Collins, S. W., Conlon, T. J., Mah, C. S., Lawson, L. A., Martin, A. D., Fuller,

D. D., Cleaver, B. D., Clement, N., Phillips, D., Islam, S., Dobjia, N., and Byrne, B. J., 2013,

"Phase I/II trial of adeno-associated virus-mediated alpha-glucosidase gene therapy to the

diaphragm for chronic respiratory failure in Pompe disease: initial safety and ventilatory

outcomes," Hum Gene Ther, 24(6), pp. 630-640.

[35] Falk, D. J., Soustek, M. S., Todd, A. G., Mah, C. S., Cloutier, D. A., Kelley, J. S., Clement,

N., Fuller, D. D., and Byrne, B. J., 2015, "Comparative impact of AAV and enzyme replacement

therapy on respiratory and cardiac function in adult Pompe mice," Molecular Therapy - Methods

& Clinical Development, 2.

[36] Doerfler, P. A., Todd, A. G., Clément, N., Falk, D. J., Nayak, S., Herzog, R. W., and Byrne,

B. J., 2016, "Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and

Phenotypic Correction of Pompe Disease," Hum Gene Ther, 27(1), pp. 43-59.

[37] Fraites, T. J., Jr., Schleissing, M. R., Shanely, R. A., Walter, G. A., Cloutier, D. A.,

Zolotukhin, I., Pauly, D. F., Raben, N., Plotz, P. H., Powers, S. K., Kessler, P. D., and Byrne, B.

J., 2002, "Correction of the enzymatic and functional deficits in a model of Pompe disease using

adeno-associated virus vectors," Mol Ther, 5(5 Pt 1), pp. 571-578.

43

[38] Pacak, C. A., Mah, C. S., Thattaliyath, B. D., Conlon, T. J., Lewis, M. A., Cloutier, D. E.,

Zolotukhin, I., Tarantal, A. F., and Byrne, B. J., 2006, "Recombinant adeno-associated virus

serotype 9 leads to preferential cardiac transduction in vivo," Circ Res, 99(4), pp. e3-9.

[39] Gray, J. T., and Zolotukhin, S., 2011, "Design and construction of functional AAV vectors,"

Methods Mol Biol, 807, pp. 25-46.

[40] Zincarelli, C., Soltys, S., Rengo, G., and Rabinowitz, J. E., 2008, "Analysis of AAV

serotypes 1-9 mediated gene expression and tropism in mice after systemic injection," Mol Ther,

16(6), pp. 1073-1080.

[41] Zolotukhin, S., Potter, M., Zolotukhin, I., Sakai, Y., Loiler, S., Fraites, T. J., Jr., Chiodo, V.

A., Phillipsberg, T., Muzyczka, N., Hauswirth, W. W., Flotte, T. R., Byrne, B. J., and Snyder, R.

O., 2002, "Production and purification of serotype 1, 2, and 5 recombinant adeno-associated

viral vectors," Methods, 28(2), pp. 158-167.

[42] Nayak, S., Doerfler, P. A., Porvasnik, S. L., Cloutier, D. D., Khanna, R., Valenzano, K. J.,

Herzog, R. W., and Byrne, B. J., 2014, "Immune responses and hypercoagulation in ERT for

Pompe disease are mutation and rhGAA dose dependent," PLoS One, 9(6), p. e98336.

[43] Ma, D., Panda, S., and Lin, J. D., 2011, "Temporal orchestration of circadian autophagy

rhythm by C/EBPβ," EMBO J, 30(22), pp. 4642-4651.

[44] 2017, "Lumizyme | Patients & Families," https://www.lumizyme.com/patients.aspx.

[45] 2017, "Prism - graphpad.com."

[46] Konkalmatt, P. R., Wang, F., Piras, B. A., Xu, Y., O’Connor, D. M., Beyers, R. J., Epstein,

F. H., Annex, B. H., Hossack, J. A., and French, B. A., 2012, "AAV9 administered systemically

after reperfusion preferentially targets cardiomyocytes in the infarct border zone with

pharmacodynamics suitable for the attenuation of left ventricular remodeling," J Gene Med,

14(0), pp. 609-620.

[47] Chien, Y. H., and Hwu, W. L., 2007, "A review of treatment of Pompe disease in infants,"

Biologics, 1(3), pp. 195-201.

[48] High, K. A., 2012, "The gene therapy journey for hemophilia: are we there yet?," Blood,

120(23), pp. 4482-4487.

[49] Han, S. O., Li, S., and Koeberl, D. D., 2016, "Salmeterol enhances the cardiac response to

gene therapy in Pompe disease," Mol Genet Metab, 118(1), pp. 35-40.

[50] Falk, D. J., Soustek, M. S., Todd, A. G., Mah, C. S., Cloutier, D. A., Kelley, J. S., Clement,

N., Fuller, D. D., and Byrne, B. J., 2015, "Comparative impact of AAV and enzyme replacement

therapy on respiratory and cardiac function in adult Pompe mice," Mol Ther Methods Clin Dev,

2, pp. 15007-.

[51] Mah, C., Cresawn, K. O., Fraites, T. J., Pacak, C. A., Lewis, M. A., Zolotukhin, I., and

Byrne, B. J., 2005, "Sustained correction of glycogen storage disease type II using adeno-

associated virus serotype 1 vectors," Gene Therapy, 12(18), pp. 1405-1409.

44

[52] Reuser, A. J., Kroos, M., Willemsen, R., Swallow, D., Tager, J. M., and Galjaard, H., 1987,

"Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-

glucosidase in mutant fibroblasts," J Clin Invest, 79(6), pp. 1689-1699.

[53] Fukuda, T., Ahearn, M., Roberts, A., Mattaliano, R. J., Zaal, K., Ralston, E., Plotz, P. H.,

and Raben, N., 2006, "Autophagy and mistargeting of therapeutic enzyme in skeletal muscle in

Pompe disease," Mol Ther, 14(6), pp. 831-839.

[54] Falk, D. J., Soustek, M. S., Todd, A. G., Mah, C. S., Cloutier, D. A., Kelley, J. S., Clement,

N., Fuller, D. D., and Byrne, B. J., 2015, "Comparative impact of AAV and enzyme replacement

therapy on respiratory and cardiac function in adult Pompe mice," Molecular Therapy - Methods

& Clinical Development, 2.

[55] van den Berg, L. E., Drost, M. R., Schaart, G., de Laat, J., van Doorn, P. A., van der Ploeg,

A. T., and Reuser, A. J., 2013, "Muscle fiber-type distribution, fiber-type-specific damage, and

the Pompe disease phenotype," J Inherit Metab Dis, 36(5), pp. 787-794.