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This is to certify that the
thesis entitled

ANTIBODY RESPONSE INDUCED AFTER INTRAOCULAR VIRAL
GENE ADDITION THERAPY USING ADENO—ASSOCIATED VIRUS.
LENTIVIRUS, AND ADENOVIRUS VECTORS WITH THE GFP
TRANSGENE IN DOGS

presented by

Jennifer Vander Kooi Den Houter

has been accepted towards fulfillment
of the requirements for the

MS. degree in Comparative Medicine and
Integrative Biology

 

 

 

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Date

 

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5/08 K:/PrqlAcc&Pres/ClRC/DateDue indd

ANTIBODY RESPONSE INDUCED AFTER INTRAOOULAR VIRAL GENE
ADDITION THERAPY USING ADENO-ASSOCIATED VIRUS, LENTIVIRUS,
AND ADENOVIRUS VECTORS WITH THE GFP TRANSGENE IN DOGS
By

Jennifer Vander Kooi Den Houter

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Comparative Medicine and Integrative Biology

2008

ABSTRACT
ANTIBODY RESPONSE INDUCED AFTER INTRAOCULAR VIRAL GENE
ADDITION THERAPY USING ADENO-ASSOCIATED VIRUS, LENTIVIRUS,
AND ADENOVIRUS VECTORS WITH THE GFP TRANSGENE IN DOGS
By

Jennifer Vander Kooi Den Houter

A humoral immune response induced after ocular gene addition therapy using
viral vectors, to either the transgene or the viral vector, can impact therapy safety
and efficacy. Three vectors with the green fluorescent protein transgene (GFP):
adenO-associated virus (AAV), lentivirus, and adenovirus (Av-1), were studied in
order to monitor the Circulatory antibody response after intraocular treatment in
dogs. Aqueous samples were taken both pre- and post-treatment from AAV
treated dogs to monitor the local humoral response. In order to monitor the
antibody response, two dog-specific enzyme-linked immunosorbent assays
(ELISAS) were designed: a GFP ELISA and an AAV ELISA. In dogs treated with
AAV-GFP, a high level of Circulating anti-GFP antibodies were detected in 4 out
of 22 dogs. The anti-GFP antibody response was observed to be influenced by
using the chicken-beta actin promoter (CBA) and treatment age of 3 weeks.
Anti-GF P antibodies were also found in aqueous samples up to 96 weeks post-
treatment, and a significant anti-AAV antibody response was also detected. Anti-
GFP antibodies were also found for lentivirus-GFP and Av-1-GFP treated dogs.
These results Show that despite the immune—privileged nature of the eye, an
antibody response to both the transgene protein and the viral vector occurs and

should remain a concern in future intraocular gene therapy studies.

Dedicated to my father, Leonard H. Den Houter

ACKNOWLEDGEMENTS

I would like to thank my committee: Dr. Simon Petersen-Jones, Dr. Vilma
Yuzbasiyan-Gurkan, Dr. Jeanne Burton, Dr. Art Weber, Dr. Patrick Venta, and

Dr. Matti Kiupel for your continued support, empathy, and flexibility.

Friends from the Comparative Ophthalmology Laboratory, Dr. Nalinee
Tuntivanich, Michelle Curcio, Gillian Shaw, Dr. Simon Petersen-Jones, and

Janice Forcier, thank you for being such a blessing over these past two years.

Thank you to the members of the Immunogenetics Laboratory for your patience
and support. Dr. Jeanne Burton, Dr. Patricia Weber, and Dr. Patricia Almeida,
thank you for helping me through the ELISA development and statistical analysis

processes.

My family and friends, thank you for your support as well as a listening ear
through this experience. Mom, thank you for your continued persistence. Jon,
Jessica, and Ben, thank you for being the greatest siblings anyone could ever
have. Last but not least, thank you to my father, who always believed in me and

Challenged me until the end.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................ viii
LIST OF FIGURES ...................................................................................... ix
LIST OF ABBREVIATIONS ........................................................................ xii

CHAPTER 1. INTRODUCTION
1.1. Somatic Cell Gene Addition Therapy for Inherited Retinal

Degenerations ............................................................................ 1
1.2. Literature Review ........................................................................ 3
1.2.1. Overview Of Canine Ocular Anatomy .................................. 3
1.2.2. The Retina ........................................................................... 5
1.2.3. Phototransduction and the Visual Cycle .............................. 9
1.2.4 The Central Visual Pathway ................................................ 13
1.2.5. Inherited Retinal Dystrophies .............................................. 14
1.2.5.a. Small Animal Models of Retinal Degenerations ............. 16
1.2.5.b. Large Animal Models of Retinal Degenerations ............. 18
1.2.6. Gene Therapy Viral Vectors ................................................ 19
1.2.6.a. Recombinant Adeno-Associated Virus (AAV) ............... 19
1.2.6.b. Recombinant Lentivirus ................................................ 22
1.2.6.C. Recombinant Adenoviruses (Av-1) ................................ 25
1.2.7. Summary of Successful Retinal Gene Therapy Treatments. 28
1.2.7.a. AAV Preliminary Studies in the Eye ............................. 28
1.2.7.b. Successful AAV Gene Addition Therapy for Retinal
Degenerations .............................................................. 29
1.2.7.c. Lentivirus Preliminary Studies in the Eye ..................... 29
1.2.7.d. Successful Lentivirus Gene Addition Therapy for
Retinal Degenerations ................................................. 30
1.2.7.e. Adenovirus Preliminary Studies in the Eye .................. 31
1.2.7.f. Successful Adenovirus Gene Addition Therapy for
Retinal Degenerations ................................................. 32
1.2.8. Safety Concerns that Arise from Viral Gene Therapy .......... 33
1.2.8.a. Host Immune Response .............................................. 34
1.2.8.b. Adeno-Associated Vectors- Safety and Host Immune
Response ..................................................................... 38
1.2.8.c. Safety of Lentivirus Vectors .......................................... 41
1.2.8.d. Adenovirus Vectors in the Eye- Safety and Host
Immune Response ....................................................... 42
1.2.8.e. Host Immune Response to Expressed Transgene in
the Eye .......................................................................... 44
1.2.8.f. Biodistribution of the Viral Vector within the Host
After Intraocular Treatment .......................................... 44

1.2.8.9. Genotoxicity ................................................................. 46

1.3. Objectives, Hypotheses, and Experimental Design ...................... 48
1.3.1. Objectives ............................................................................ 48
1.3.2. Hypotheses .......................................................................... 48
1.3.3. Experimental Design ............................................................ 49

CHAPTER 2. DEVELOPMENT OF ELISA ASSAYS

2.1. Introduction .................................................................................... 52

2.2. Materials and Methods ................................................................... 52
2.2.1. Immunization of Dogs ........................................................... 52
2.2.2. Development Of GFP ELISA .................................................. 54

2.2.2.a. Reagents ....................................................................... 55
2.2.2.b. Detailed GFP ELISA Protocol ....................................... 56
2.2.2.c. Results of GFP ELISA Development ............................. 58
2.2.3. Development of AAV ELISA .................................................. 59
2.2.3.a. Reagents ....................................................................... 60
2.2.3.b. Detailed AAV ELISA Protocol ....................................... 60
2.2.3.c. Results of AAV ELISA Development ............................. 61

CHAPTER 3. HUMORAL IMMUNE RESPONSES DETECTED IN DOGS
GIVEN INTRAOCULAR TREATMENT OF VIRAL VECTOR

3.1. Introduction .................................................................................... 67
3.1.1. Intraocular Injections .............................................................. 67
3.1.2. Anesthesia ............................................................................ 68
3.1.3. rAAV2/5, Lentivirus, and Av-1 ............................................... 68
3.1.4. Injection Technique Used in Puppies Where the Eyelids

Have Opened ........................................................................ 69
3.1.5. Injection Technique Used in Neonates .................................. 71
3.1.6. Dogs Used in Study and Sample Collection .......................... 72
3.1.7. Monitoring GFP Expression .................................................. 75
3.1.8. Data Analysis using SAS ....................................................... 76

3.2. Results ......................................................................................... 79

3.2.1. Presence of Circulating Anti-GFP Antibodies after

Intraocular Treatment with AAV-GFP .................................... 79
3.2.2. Presence of Anti-GFP Antibodies in Aqueous after

Intraocular Treatment with AAV-GFP ..................................... 81
3.2.3 Correlation between Anti-GFP Antibody Responses in

Serum and Aqueous Samples .............................................. 90
3.2.4. Presence of Circulating Anti-AAV Antibodies after

Intraocular Treatment with AAV-GFP .................................... 90
3.2.5. Correlation between Anti-AAV and Anti-GFP Antibodies

in the Serum .......................................................................... 91
3.2.6. Presence of Circulating Anti-GFP Antibodies after

Intraocular Treatment with Lentivirus .................................... 91
3.2.7. Presence of Circulating Anti—GFP Antibodies after

Intraocular Treatment with Av-1 ............................................ 92

vi

3.3. Discussion and Future Work .......................................................... 100

3.3.1. Discussion and Summary of Results ..................................... 100
3.3.1 .a. Anti-GFP Antibody Response after Treatment with
AAV-GFP ....................................................................... 1 00
3.3.1.b. Anti-AAV Antibody Response after Treatment with
AAV-GFP ...................................................................... 1 00
3.3.1.0. Discussion of Results from AAV Treated Dogs ............ 101
3.3.1.d. Correlation Analyses .................................................... 103
3.3.1.e. Anti-GFP Antibody Response after Treatment with
Lentivirus ...................................................................... 1 03
3.3.1.f. Anti-GFP Antibody Response after Treatment with
Av-1 ............................................................................. 1 04
3.3.1.9. AAV-GFP, Lentivirus-GFP, and Av-1 Antibody
Responses Compared ................................................. 104
3.3.2. Future Research .................................................................... 105
3.3.3. Conclusion ............................................................................ 107
APPENDIX
Appendix A. ELISA GFP Laboratory Protocol .......................................... 109
Appendix B. Anti-AAV ELISA Protocol and Summary of References ........ 113
Appendix C. AAV2/5-GFP Vector Construction ......................................... 116
Bibliography ................................................................................................ 124

vii

LIST OF TABLES

Table 2.1. lmmunized dogs ........................................................................ 54

Table 3.1. Colony dogs injected with AAV2/5-CBA-GFP or
AAV2/5-mOPS-GFP vectors for the GFP ELISA protocol ........ 73

Table 3.2. Colony dogs injected with AAV2/5 vectors that were used
for the Anti-AAV ELISA protocol ............................................... 74

Table 3.3. Least Squares Means of GFP ELISA OD Values from Dog
Serum Samples (1 :5) for Independent Variables ...................... 86

Table 3.4. Least Squares Means of GFP ELISA OD Values from Dog
Aqueous Samples (1 :20) for Independent Variables ................. 89

Table 3.5. Least Squares Means of AAV ELISA OD Values for Dog
Serum Samples (1:5) for Independent Variables ...................... 96

viii

Figure 1.1.

Figure 1.2.
Figure 1.3.
Figure 1.4.
Figure 1.5.
Figure 1.6.
Figure 1.7.
Figure 1.8.

Figure 1.9.

Figure 1.10.
Figure 1.11.
Figure 1.12.

Figure 1.13.

Figure 1.14.

Figure 1.15.

Figure 1.16.
Figure 2.1.

Figure 2.2.

LIST OF FIGURES

Images in this thesis/dissertation are presented in color.

Image of the vertebrate eye with an expanded view of the

retina ....................................................................................... 4
Schematic of the retinal layers ................................................ 6
Diagram of a rod photoreceptor from the vertebrate retina ..... 7
Phototransduction within a rod photoreceptor cycle ................ 10
Diagram of the visual cycle ..................................................... 12
An illustration of the central visual pathway ............................ 14
Fundus pictures from two different human patients ................ 15
A representation of the adeno-associated virus genome ........ 20
A diagram of the infection pathway of adeno-associated

virus ....................................................................................... 22
Diagram of the HIV provirus .................................................... 23
A representation of an HIV particle ......................................... 24
A representation of the adenovirus genome ........................... 26

Immunohistochemistry of the dog retina transduced by an
Av-1 vector after subretinal injection ...................................... 33

An overview of the host immune response to a foreign
invader ................................................................................... 37

The typical host immune response to recombinant
adenovirus vectors ................................................................. 43

Flow Chart of experimental design for the AAV-treated dogs. 50
GFP ELISA Protocol Flow Chart ............................................. 56

GFP ELISA Development: Anti-GFP Rabbit Serum Used to
Determine the Optimal GF P Concentration Range ................ 63

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 3.1.

Figure 3.2.

Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 3.11.

Figure 3.12.

GFP ELISA Development: Anti-GFP Dog Serum Used to
Optimize the Dog-Specific GFP ELISA .................................. 64

GFP ELISA Development: Variation in Control Dog Serum
Dilutions Using 1:500 GFP in Order to Determine the

Positive and Negative Control Sera for the GFP ELISA ......... 65
AAV2/5 ELISA Development: Variations in Dog Sera and AAV
to Determine the Optimal Anti-AAV Assay .............................. 66
Path taken by the injector for the subretinal injection

procedure ................................................................................ 70
Subretinal injector (RetinaJect Injector, Surrnodics, Irvine, CA)
used to perform the subretinal injections ................................. 70
Before and after subretinal injection in a dog’s eye ................. 71
Injection site in neonatal puppies ............................................ 72
Serum and Aqueous Sampling Time Line ............................... 75
GFP expression was monitored using a RetCam ................... 76

Least Squares Mean ODS for Anti-GFP Antibody Levels in
Serum for dilutions 1:5, 1:10, and 1:20 compared after

AAV-GFP Intraocular Injection Collected on Weeks 0, 2, 4,

6, 8, and >8 .............................................................................. 82

Individual Dog ODS for Anti-GFP Antibody Levels in Semm
Pre- and Post-Treatment (1:5 dilution shown) ......................... 83

Least Squares Mean (+/- SEM) ODS for Anti-GFP Antibody
Levels in Serum after AAV-GFP Intraocular Injection
Collected Pre and Post Treatment (1 :5 serum shown) ........... 84

Least Squares Mean (+/- SEM) ODS for Anti-GFP Antibody
Levels in Serum after AAV-GFP Intraocular Injection
Collected on Weeks 0, 2, 4, 6, 8, and >8 (1 :5 serum Shown) . 85

Least Squares Mean (+/-SEM) ODS for Anti-GFP Antibody
Levels in Aqueous Collected Pre- and Post-Injection
(1 :20 dilution of aqueous) ....................................................... 87

Individual Dog Aqueous GFP ELISA OD Values at Sacrifice .. 88

Figure 3.13. Correlation of Anti-GFP ELISA OD Values in Aqueous and
Serum Samples ...................................................................... 93

Figure 3.14. Least Squares Mean (+/-SEM) ODS for Anti-AAV Antibody
Levels in Serum Collected Pre- and Post-Injection after
Intraocular Treatment with AAV-GFP (1 :5 dilution of
serum shown) ........................................................................ 94

Figure 3.15. Least Squares Mean (+/-SEM) ODS for Anti-AAV Antibody
Levels in Serum Collected 0, 2, and 4 Weeks Post-
Treatment with AAV-GFP (1 :5 dilution of serum shown) ........ 95

Figure 3.16. Correlation of Anti-AAV and Anti-GFP ELISA OD Values
from Serum Samples ............................................................. 97

Figure 3.17. Least Squares Mean (+/-SEM) ODS for Anti-GFP Antibody
Levels in Serum Collected Pre and Post Treatment with
Lentivirus-GFP (1:5 dilution Of serum shown) ........................ 98

Figure 3.18. Least Squares Mean (+/-SEM) ODS for Anti-GFP Antibody
Levels in Serum Collected on Weeks 0, 2, 4, 6, and 8 Post
Treatment with Lentivirus-GFP (1 :5 dilution Of
serum shown) ........................................................................ 99

Figure 3.19. AAV-GFP, Av-1, and Lentivirus-GFP ODS for Anti-GFP
Antibody Levels in Serum after Intraocular Treatment (1 :5
serum shown for AAV and Lentivirus-GFP, 1:20 Shown for

Av-1 ) ...................................................................................... 108
Figure A. The CBA promoter sequence ..................................................... 117
Figure B. The mOPSSOO promoter sequence ............................................ 117

Figure C. The GFP CDNA sequence used in the AAV viral vector
Constructs .................................................................................. 1 18

Figure D. The pTR-UF 11 plasmid with CMV enhancer, CBA promoter,
and GFP transgene ................................................................... 119

xi

LIST OF ABBREVIATIONS

AAV = adeno-associated virus

AAV2= adeno-associated virus serotype 2

AAV5 = adeno-associated virus serotype 5

AAV-CBA-GFP = adeno-assoaciated2/5 vector with CBA and GFP
AAV-MOPS-GFP = adeno-assoaciated2/5 vector with mOPS and GFP
APC = antigen presenting cells

Av—1 = adenovirus

CAR = coxsackie-adenovirus receptor

CBA = Chicken beta-actin promoter

cGMP-PDE = cyclic GMP phosphodiesterase holoenzyme

CMV = cytomegalovirus

dsDNA = double-stranded DNA

EAM = encapsidated adenovirus mini-chromosomes

ELISA = enzyme-linked immunosorbent assay

ERG = electroretinography

GC = guanylate cyclase

GFP = green fluorescent protein

HIV = human immunodeficiency virus

IU = infectious unit

LacZ = B-galactosidase

LCA = leber congenital amaurosis

xii

LGN = lateral geniculate nucleus

LSM = least squares mean

LTR = long terminal repeats

mOPS = mouse opsin promoter

NK cells = natural killer cells

OD = optical density

OTC = omithine transcarboxylase

PBS = phosphate-buffered saline

PDE= phosphodiesterase

qPCR = quantitative PCR

rAAV2/5 = recombinant AAV serotype 2/5
rAAV = recombinant AAV

RAC = NIH Recombinant DNA Advisory Committee
Rh= rhodopsin

rHIV = recombinant HIV

RK= rhodopsin kinase

RP = retinitis pigmentosa

RPE = retinal pigment epithelium

SAS = Statistical Analysis Software
SClD-X1 = severe combined immunodeficiency disease
SEM= standard error of the mean

T= transducing

VSV-G = vesicular stomatitis virus

xiii

Chapter 1

INTRODUCTION

1.1. Somatic Cell Gene Addition Therapy for Inherited Retinal
Degenerations

Gene therapy has been at the forefront of research since the 1980’s and

continues to draw considerable attention as a novel therapy. Currently,

treatments are progressing for diseases that are difficult or even impossible to

treat with contemporary medicine, such as hemophilia B (Chao and Walsh,

2004), severe combined immune deficiency (Cavazzana-Calvo et al., 2000;

Cavazzana-Calvo et al., 2005), and many forms of cancer.

Gene therapy has been considered only for fatal, Chronic, or severely debilitating
diseases in the past. Recently though, diseases of the special sense organs,
such as the ear and eye, have been considered for gene therapy. The eye is a
particularly interesting organ for gene therapy research since it is a relatively
isolated part of the body and also an immune-privileged Site and thus gene
therapies would theoretically allow for long-term treatment success with leSS risk

of adverse immune responses.

Examples of gene therapy in the eye include somatic cell gene addition therapies
for autosomal recessive diseases in which the disease results from a lack of

gene product (Auricchio and Rolling, 2005); antisense RNA and ribozyme

treatments for autosomal dominant diseases in which the disease results from
accumulation of the mutated gene product (Lewin et al., 1998); and generic
treatments such as introduction of neurotrophic factors to protect and sustain the
photoreceptors as long as possible (Bessant et al., 2001 ). Unfortunately, each
approach has its own challenges. For instance, when using gene addition
therapy in an attempt to produce the missing protein for an individual with a null
mutation, the vector may be expressing a protein for which the body has not had
previous exposure, thus increasing the potential for a host immune response

against the therapeutic gene’s protein (Tripathy et al., 1996).

Since gene therapy is a relatively new field, much research needs to be done to
determine the most efficacious and safest methods for treatment of a variety of
inherited, debilitating eye diseases. Currently, little research has been performed
to investigate in detail the immune response, either within the eye or
systemically, after intraocular injection with a vector that expresses a foreign
transgene. With the potential for gene therapy currently under scrutiny, the need
to learn more about the immune response after intraocular gene therapy is
necessary to determine the safety and efficacy of this form of treatment for future

research and Clinical trials.

1.2. Literature Review

1.2.1. Overview of Canine Ocular Anatomy

The anatomy of the eye is quite complex with each region or tissue having its
own specific function. Sequentially, from the cornea to the optic nerve, a brief

description will be given.

Overall, there are three important layers of the eye as shown in Figure 1.1: 1) the
fibrous, outermost layer consists of the cornea and SClera, deemed the
comeoscleral layer with the junction between cornea and sclera termed the
limbus, 2) the middle layer consists of the uveal tract which is composed of the
iris, the Ciliary body, and the choroid, and 3) the innermost retinal layer which
consists of the retinal pigment epithelium (RPE) and neurosensory retina which

are continuous with the Ciliary epithelium anteriorly.

The cornea is the roughly circular, transparent area at the front of the eye that
allows light to enter the globe. The majority of light refraction occurs at the
corneal surface. Light then passes through the anterior chamber, which is filled
with aqueous, and then continues through the lens where it is further refracted.
The lens is a transparent, crystalin structure that is held in place by the lens
zonules and its shape is modified by the action of the Ciliary body musculature.
Just in front of the lens is the iris which acts aS a diaphragm to control the

amount of light that enters the lens. Along the periphery Of the iris lies the

iridocomeal drainage apparatus that allows fluid drainage from the anterior

chamber to the blood stream and thus controls the pressure in the globe.

Sclera _ __ Choroid

   

 

Optic nerve

 
 

Ciliary body

Figure 1.1. Image of the vertebrate eye with an expanded view of the retina.
(http://www.webvision.med.utah.edu, 2-24-07).

Light continues through the lens and through the vitreous to be focused onto the

retina. The retina, which lines the back of the eye, is composed of several

layers, and acts to convert light into an electric signal which is modified within the

inner retina and then travels to the brain initially via the optic nerve and allows

the organism to visualize its environment.

1.2.2. The Retina

The retina lines the back of the eye globe as shown in Figure 1.2. It ends
anteriorly at the ora Ciliaris retinae which is positioned just posterior of the Ciliary
body. The outer layer of the retina is known as the retinal pigment epithelium
(RPE) and is a monolayer of cells that is in intimate contact with the
photoreceptors of the neurosensory retina and plays a vital role in their nutrition
and maintenance. The RPE lies adjacent to BrUCh’s membrane posteriorly.
Tight junctions in the RPE and the retinal vessel endothelium create the blood-
retinal barrier which limits access of molecules to the neurosensory retina. The
sub-retinal space between the RPE and the photoreceptor outer segments, a
potential space into which gene therapy injections are given, has been Shown
specifically to be immune-privileged due to the action of the blood-retinal barrier
(Hoffman et al., 1997; Wenkel and Streilein, 1998; Anand et al., 2002; Isenmann
et al., 2001). With this in mind, gene therapy of the eye may be influenced by

this deviant immune response due to this immune-plivileged environment.

The three main neuronal cells of the retina are the photoreceptors, the bipolar
cells, and the ganglion cells. Photoreceptors are slender cells responsible for the
conversion of light into an electrical signal. There are two main types of
photoreceptors: cones and rods. Approximately 95% of photoreceptors are rods
in the canine eye which is comparable to the human eye in which approximately
95% are rods and 5% are cones (Koch and Rubin, 1972; Forrester et al., 1996).

In contrast, the distribution of rod and cones across the retina varies between the

human and canine eye. In the human eye, the macula, which is superotemporal
to the optic disc, contains a clustering of cone photoreceptors while in the canine
there is a region of high cone density in the area centralis which forms a streak

above the optic disc.

Rod photoreceptors as Shown in Figure 1.3 are responsible for vision in dim light
while cone photoreceptors provide for color, bright light vision. The number of
cone types vary between species in mammals. Two or three types of cones are
typically present: long and short wavelength or blue, green, and red (short,

medium, and long wavelength), respectively.

Choroid
Retinal Pigment Epithelium
(RPE)

Photoreceptor Layer

External Limiting Membrane

Outer Nuclear Layer(ONL)

194" :0. i
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Ganglion Cell Layer(GCL)

 

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Vitreous Humor \ Inner Limiting Membrane(lLM)

 

Light

Figure 1.2. Schematic of the retinal layers.

(http://webvision.med.utah.edu/20030lb.pdf#How°/o20the°/o20Retina%20Works,
2-8-07)

Discs

Outer
sfigment

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Cilium

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Figure 1.3. Diagram of a rod photoreceptor from the vertebrate retina. The
rod photoreceptor consists of four main regions: the outer segment complete with
discs where phototransduction occurs, the inner segment which is connected to
the outer segment via the Cilium and creates the energy for the cell, the cell body
which holds the nucleus of the cell, and finally the synaptic terminal which
contains synaptic vesicles that transmit the message to the next neuron
MEN/www.csulb.edu/~cwallis/482/visualsvstem/cwrod.ipg , 7-20-06).

The photoreceptor layer is made up of the inner and outer segments of the
photoreceptors, the outer nuclear layer is made up of the nuclei of the
photoreceptors, and the outer plexiform layer is made up of the photoreceptor
termini. The outer segment of the photoreceptor, made up of stacks of discs for
the rods and a comb-like structure for the cones, is connected to the inner
segment via a Cilium. Within the inner segments of the photoreceptor, metabolic
processes occur to keep the cell alive (Baldridge and Kurennvi., 1998). The
outer and inner segments are made distinct by the thin outer-limiting membrane

which creates a layer across the retina at this level (Buntmilam et al., 1985).

The termini of the photoreceptors contain synaptic vesicles with the
neurotransmitter glutamate and form synapses with both bipolar and horizontal
cells of the inner nuclear layer. Bipolar cells are second order neurons that
bridge between 20-60 photoreceptors to the ganglion cells. Rod ON-bipolar cells
connect to the ganglion cells via amacrine cells but may also contact them via
cone OFF-bipolar cells (Soucy et al., 1998; Hack et al., 1999). Horizontal cells,
which are responsible for horizontal Signaling across the retina, also reside in the
inner nuclear layer and send dendrites out to synapse with the termini of the

photoreceptors.

The inner plexiform layer is formed by the connections of the inner nuclear layer

cells, such as amacrine cells and bipolar cells, with ganglion cells and others.

Amacrine cells help the communication between the inner and outer retina

through dopaminergic pathways between the bipolar cells and ganglion cells.

The ganglion cell layer contains the cell bodies of the ganglion cells as well as
those of some amacrine and astroglial cells. Ganglion cells receive input from
many photoreceptors within their receptive field and then send this electrical
Signal in the form of action potentials through the unmyelinated nerve fiber layer
to the myelinated optic nerve. There are two classes of ganglion cells: 1) ON-
center which depolarize and 2) OFF-center which hyperpolarize in response to

light stimulation.

The innermost layer Of the retina is the inner limiting membrane that is composed
of Miller cell end feet and astrocytes. Miiller cells are retinal glial cells that
originate from hemopoietic cells. Their cell body is in the inner nuclear layer
while their cell processes spread out to form the outer and inner limiting

membranes.

1.2.3. Phototransduction and the Visual Cycle
Phototransduction (Figure 1.4.) is the process by which the photoreceptors
convert photons of light into an electrical Signal which is then transmitted through

inner retina to the ganglion cell and via ganglion cell axons to the brain. Rod

 

 
   
    

 

 

 

 

     
 

 

 
   
   
 

 

 

 

 

 

 

 

 

 

, Light
ROD [
' Pf Plasma membrane
: i _isk membrane
If: Na+
'T‘T‘i cGMP
. . .. i . Mg
1-... InthIts ‘
== CNG
. Channel
Rh*~P inhibits Mg2 . y
' GTP ; 9
i?
ll
........ 1 K+
4, Ca2
Na/Ca-K
Exchanger
To Na+
pump in
inner
‘. segment

 

Figure 1.4. Phototransduction within a rod photoreceptor.
htt ://www.biol.tsukuba.ac.' /~nakatani/ hototransduction-scheme-1.' 2-7-07)
light to be converted to an electrical message through the Stimulation of the
photopigment rhodopsin. Fi= rhodopsin, T= transducin, PDE=
phosphodiesterase, Gc= guanylate cyclase, and BK: rhodopsin kinase.

 

phototransduction has been studied extensively, and it initiates with the activation
of the photopigment rhodopsin (see Pugh and Lamb, 2005 for a review).
Rhodopsin molecules are formed by the combination of 11-ciS retinal with the
transmembrane protein rod opsin. Rhodopsin is activated by the absorption-of
photons within the outer segment of the photoreceptor. The 11-cis retinal of the
rhodopsin molecule photoisomerizes to all-trans retinal and is released from the
rhodopsin complex. This all-trans retinal is cycled back into all-trans retinol
which is then transported to the RPE where it is converted back to 11-ciS retinal
via the visual cycle involving rpe65 isomerase (Figure 1.5) and returned to the
outer segments to once again recombine with opsin to form rhodopsin (Redmond
et al., 1998; Mata et al., 2004). Following light-activation, activated-rhodopsin
then forms a complex with transducin, the rhodopsin-transducin binding complex.
Transducin is a trimeric G-protein consisting of: To which is the active subunit,
and TB and Ty which are the inhibitory subunits. The light-activated rhodopsin
changes the GDP of the transducin complex to a GTP and TBy then dissociate to
release To and GTP. The active transducin then stimulates the cyclic GMP
phosphodiesterase holoenzyme (cGMP-PDE). cGMP-PDE iS a heterotrimeric
complex with two catalytic subunits, PDEa and PDEB that are inhibited by the
two PDEv subunits (see Stryer, 1991 for a review). Activated Ta removes the
inhibitory gamma subunits from this complex leaving the active PDEq/B complex.
PDEa/B hydrolyzes cGMP to GMP resulting in closure of the cGMP-gated

Channels due to the decreased concentration of cytosolic cGMP. The Closure of

11

these channels results in the hyperpolarization of the photoreceptor cell
membrane.

 

' . all-trans retinyl ester l

1 all-trans retinol

  

 

 

     
 

 

 

all-trans retinol

Figure 1.5. Diagram of the visual cycle. The cycling of the compounds all-
trans retinal to 11-cis retinol occurs through a series of steps with various
isomerases and through transport via lRBP (interphotoreceptor binding protein)
between the RPE and photoreceptor layers. Rh is the molecule rhodopsin which
is the photopigment involved in phototransduction

(http://www.bumc.bu.edu/www/busm/by/images/carter.jpg, 2-7-07)

The closed cGMP-gated Channels consist of two subunits and one glutamate
residue which block the cation flow into the cell when cytosolic cGMP is in low

concentrations. Consequently, cations such as calcium are not able to flow into

the cell and at the same time the Na/Ca-K ion pumps are continuously pumping
Ca2+ out of the cell which then results in hyperpolarization of the cell (Haase et
al., 1990). This results in a discontinuation of the ‘dark current’ and consequently

inhibits glutamate release at the photoreceptor synaptic terminus.

1.2.4. The Central Visual Pathway

When the layers of the retina are working in harmony, the retina turns light into
an electric signal which is then sent to the brain for perception of the image
(Figure 1.6.). This Signal is sent through the ganglion cell axons via the optic
nerve. The two optic nerves, from the left and right eyes, meet at the optic
chiasm and fibers either continue to the ipsilateral optic tract or decussate to the
contralateral optic tract. If the impulse originated from the nasal portion of the
retina, then the Signal travels to the opposite side of the brain. Those from the
temporal side of the retina project to the ipsilateral Optic tract. Fibers travel to the
lateral geniculate nucleus (LGN) for conscious perception of vision, the
pretectum, and the superior colliculus for reflex pathways. Ganglion cell axons
synapse in the LGN, and then axons pass via the optic radiation to the visual
cortex. The visual cortex is divided into regions specific, also known as

retinotopic, to the many areas of the visual field (Vanduffel et al., 2002).

13

i Optic nerves l

 

 

 

 

Optic chiasm i

 

 

Optic tract l

 

 

 

 

 

./ synaps
{BK RE?) i Lateral geniculate nuclei i

 

Visual cortex l

 

 

Figure 1.6. An illustration of the central visual pathway. This picture shows
the visual field in relationship to the path that the information portraying the
image follows along the Optic nerve, through the optic chiasm, and via a synapse
in the lateral geniculate nucleus to the visual cortex (Forrester et al., 1996).

1.2.5. Inherited Retinal Dystrophies

At the time of writing, 185 different genes have been linked to retinal dystrophies

(summarized in RetNet, http://www.sph.uth. tmc.edu/Retnet/sum-dis.htm#A-

genes, 2007). Retinal dystrophies are divided into many forms including Leber

Congenital Amaurosis and retinitis pigmentosa (RP).

RP, which was first described in 1857, is a subset of these blinding hetergenous,
inherited diseases that is estimated to affect 1 in 3,000 humans (Bundey and
Crews, 1984). This bilateral disease is described as a rod-cone dystrophy since
the rods degenerate initially and are often followed by cone degeneration (Zeiss
et al., 2004; Huang et al., 1995). The inheritance of the disease can be
autosomal dominant, autosomal recessive, or X-linked. RP can present either as
a slow or fast onset disease with the most common symptoms being tunnel
vision, night blindness, headaches, and unexplained light flashes (Heckenlively
et al., 1988). Secondary to these common symptoms are cataract formation,
pigment changes in the retinal layers, a decreased ERG rod response initially

followed by a decreased cone response, and retinal thinning (Figure 1.7).

 

Figure 1.7. Fundus pictures from two different human patients. The retina
on the left is normal while the retina on the right is affected with retinitis
pigmentosa. The diseased fundus has bone-spicule pigmentation as well as
vascular attenuation.

There are four distinct categories into which most of the RP mutations fall: 1)
phototransduction cascade proteins, 2) photoreceptor structural proteins, 3)
proteins involved in photoreceptor and retinal pigment epithelial metabolism, and

4) proteins that regulate gene expression (Bessant et al., 2001).

Leber Congenital Amaurosis (LCA) is a severe early-onset condition that causes
blindness in Children (Morimura et al., 1998). Causal mutations have been
identified in several genes that are either expressed in the RPE or retinal cells.
One example is the RPE65 gene which codes for an isomerase in the RPE and
plays an important role in the visual cycle; a mutation in this gene results in a
severe vision loss but only slow degeneration of the retina and has shown
promise for gene therapy treatments (Cremers, et al., 2002). LCA is
Characterized by early onset blindness and typically include a decreased
electroretinographic (ERG) rod response initially followed by a decreased cone
response. Nystagmus, cataract formation, and impairment of the pupillary light

reflex are also features of the condition.

1.2.5.a. Small Animal Models of Retinal Degenerations

There are numerous small animal models of retinal degenerations in a variety Of
species including Drosophila, zebra fish, and many rodent models. Most of the
models are autosomal recessive while a few are inherited in an autosomal
dominant or X-linked manner. The most widely used species are the laboratory
rodents: rats and mice. Rat models include the Royal College of Surgeons rat
(RCS rat), the Rho knockout rat, and the P23H transgenic rat while the mouse
models include the spontaneously occurring rd1 mouse, the peripherin 2 (Prph2)

mouse (also known as rd2), and a knockout RPE65 mouse.

16

The RCS rat is an inbred line with an autosomal recessive mutation in the Mertk
gene which normally serves a role in RPE phagocytosis of the outer segments of
photoreceptors. In the RC8 rat, there is a build up of photoreceptor outer
segment debris in the subretinal Space and the photoreceptors eventually
degenerate as a consequence (for a review see Dejneka et al., 2003). This rat
serves as a model for the humans with Mertk gene mutation (Gal et al., 2000;

D’Cruz et al., 2000).

The P23H rat is a transgenic rat that was created to have an autosomal dominant
mutation in the rhodopsin gene due to the alteration of proline-23 to histidine
(RhoP23H) in one allele. This mutation leads to eventual blindness, and is one of
the most common rhodopsin mutations of humans (for a review see Dejneka et

al., 2003; Chader, 2002).

The rd1 mouse has an autosomal recessive mutation in the phoshodiesterase
beta subunit (PDEB) gene which normally functions as a component of the rod
phototransduction cascade. This mutation leads to a fast retinal degeneration
and results in blindness. This was the first mouse model of retinal degeneration
discovered and has been widely used as a model to mimic phototransduction-
related disease in humans (Chader, 2002; Pittler et al., 1991).

The knockout RPE65 mouse was developed as a model for LCA. This mouse
has been used extensively for gene therapy trials Since it has a similar disease

phenotype to the human LCA patient (for a review see Dejneka et al., 2003).

17

1.2.5.b. Large Animal Models of Retinal Degenerations
Progressive Retinal Atrophy is the canine equivalent of RP and LCA and Similarly
shows genetic heterogeneity. These Spontaneous canine models are useful

because the canine eye is Similar in Size to the human eye.

Rod cone dysplasia type 1 (rcd1) affecting the Irish Setter breed is a model of
early onset autosomal recessive RP and has a nonsense mutation in the PDE6B
gene in exon 21 (Suber et al., 1993). Rcd1 has a fast onset with night blindness
occurring within 25 days after birth. The rcd1 Irish Setter is used as a model for

fast onset, phototransduction-related RP.

The Cardigan Welsh Corgi has an autosomal recessive PRA due to a 1 base pair
deletion mutation in exon 16 of the phosphodiesterase Six alpha subunit (PDE6A)
gene which results in a frameshift and downstream premature stop codon
(Petersen-Jones et al., 1999). The disease onset is comparable to the rcd1lrish
Setter and has been named ‘rod-cone dysplasia type 3’ (rcd3). This disease was

described in detail by Tuntivanich (2006).

The Briard, with a 4-bp deletion mutation in the RPE65 gene that leads to a
frameshift and downstream premature stop codon, is a canine model of LCA
(Veske et al., 1999; Aguirre et al., 1998; Wrigstad, 1994). The absence of the
rpe65 protein in the Briard results in a severe loss of vision and markedly

reduced ERG responses. However despite the early onset of vision loss,

18

structural deterioration of the photoreceptors is Slow which provides a wide

opportunity to test possible gene therapy treatments.

1.2.6. Gene Therapy Viral Vectors

Viral vectors are currently used extensively for the transport of a transgene into a
target cell. This method has been the basis for many gene addition therapy
trials, and has shown success in many studies. Three commonly used viral
vectors are adeno-associated virus, lentivirus, and adenovirus vectors. These
vectors have demonstrated beneficial treatment effects but also unexpected Side
effects. Most viral vectors are Chosen based on the disease that is to be treated.
Disease factors such as degree of transgene expression required, duration of
treatment, and target cell determine the viral vector that would be optimal. The
ultimate goals for a gene therapy vector include, but are not limited to: 1) low
immunogenicity, 2) regulated vs. unregulated expression, 3) duration of
transgene expression, 4) amount of vector needed for transduction, 5) integration

vs. no integration of the viral genome, and 6) tissue specificity (Kay et al., 2001).

1.2.6.a. Recombinant Adena-Associated Virus (AAV)

One of the first successful gene therapy trials utilized a recombinant adeno-
associated virus vector to treat hemophilia. This disease was treated using a
serotype 2 recombinant AAV vector that contained the CDNA sequence for the
coagulation factor IX gene. Not only has this vector been shown to be safe as a

gene therapy vector in a wide variety Of studies, it has also been shown to be

19

efficacious in gene delivery and long-term transgene expression (Chao and

Walsh, 2004; Flannery et al., 2005).

Adeno-associated viruses are in the family Parvoviridae and are “helper-
dependent’ viruses. Wild type AAV are typically associated with subclinical or
latent infections, and typical viral gene expression in the cell occurs during the ‘S’
phase of the cell cycle which can be activated by an adenovirus or herpesvirus
infection, for example. Adeno-associated viruses are small particles 26 nm in
diameter with an icosahedral capsid made up of viral particles VP1 and VP2.
The genome is Single-stranded DNA with flanking terminal repeat regions, the
normal genes of the virus being rep and cap, and is approximately 5,000 bases
in size which Significantly limits the space available, to approximately 2500bp, for

the transgene of interest (Figure 1.8) (Vihinen-Ranta et al., 2004).

 

ITR _ REP CAP — ITR

 

 

 

 

 

 

 

 

Figure 1.8. A representation of the adeno—associated virus genome. The
viral genome consists of the Rep and Cap genes with flanking inverted terminal
repeat regions (ITR).
Two of the many AAV serotypes are more commonly used for gene therapy
trials: serotype 2 (AAV2) and serotype 5 (AAV5). The major difference between
the two serotypes is mainly the viruses’ method of cell entry. AAV2 binds the
heparin-sulfate proteoglycan on the cell surface while AAV5 binds 02,3 or (12,6

sialic acid on the cell surface for entry through receptor-mediated endocytosis

(Vihinen-Ranta et al., 2004). Upon entry into the cell, the endosome undergoes

20

acidification which results in capsid degradation and viral escape into the
cytoplasm. Once in the cytoplasm, the virus is shuttled on microtubules to the
nuclear pore. When the virus contacts the nuclear pore at the 5—fold axis of the
virus capsid, the DNA is made into double-stranded DNA (dsDNA) by a DNA
polymerase that lies at this axis. The dsDNA is then transported into the
nucleus, through the pore at the 5-fold axis, where it is free to be transcribed into

RNA through the host cell transcription process (Figure 1.9).

AAV2 has been the primary rAAV for gene therapy studies while AAV5 also has
been studied in retinal gene therapy research in recent years. AAV2 was
efficacious in retinal gene therapy studies; it had the ability to infect ganglion
cells, photoreceptors, and the RPE of dogs (Bainbridge et al., 2003), mice (Grant
et al., 1997; Ali et al. 1998), and non-human primates (Bennett et al., 1999).
AAV5 has been Shown to be up to 1,000 times more efficient than AAV2 when
infecting retinal cells, such as photoreceptors, in the mouse and non-human
primate eye, with the average transgene activation time being between 2-4
weeks (Yang et al., 2002; Lotery et al., 2003). A further study pseudotyped
AAV2 with the capsid of AAV5 (AAV2/5) and thiS vector was found to be just as

efficient at infecting photoreceptors as AAV5 (Auricchio et al., 2001).

21

 

AAV Host Cell Entry

  

Cytoplasm

 

 

   
 

Nucleus

         
  
 

Clathrin- Early endosome
coated Endosome
vessicle

X

 

     

Perinuclear recycling
endosome

  

 

 

Figure 1.9. A diagram of the infection pathway of adeno-associated virus
(Modified from Vihinen-Ranta et al., 2004). The AAV enters the host cell via
receptor-mediated endocytosis. After escape from the endosome, the virus

makes a dsDNA version of its SSDNA genome at the 5-fold axis of the capsid.
The dsDNA then travels through the nuclear pore into the nucleus where it is

then transcribed into RNA.
1.2.6.b. Recombinant Lentivirus
Lentiviruses, such as the human immunodeficiency virus (HIV) are a kind of
retrovirus. Retroviruses, which were some of the first vectors used to transfer

cellular DNA, consist of a single stranded RNA genome, reverse transcriptase, a

viral envelope, and a complex genome (Figure 1.10.).

22

 

LTR

 

 

 

 

Figure 1.10. Diagram of the HIV provirus. The provirus is the dsDNA genome
that has been altered from a dsRNA genome via reverse transcriptase within the
host cell and is ready to be inserted into the host cell genome (modified from
Naldini, 1996). All genes of the provirus are shown in this diagram including the
accessory viral proteins: Env=vira| envelope, Rev=reverse transcriptase,
Pol=viral polymerase, SD=sp|ice donor site, Lp=packaging signal, Gag=structural
polyprotein, Vif, Tat, Nef=regu|atory proteins, and Pro and R=accessory proteins.
The two essential components needed to make a recombinant HIV (rHIV) vector

in cell culture are: 1) a modified genome with packaging signals (W), a primer-

binding site, a polyurine tract, the flanking LTRs, and the transgene in cis-acting
sequence and 2) a helper virus that can provide all the packaging materials to

create the vector (Figure 1.11.) (Lever, 1999).

Wild-type HIV are able to infect T-cells by binding helper T cell surface receptors
CD4 and CXCr4. Recombinant viruses have been pseudotyped with the
Vesicular Stomatitis Virus Glycoprotein (VSV-G) which is related to the rabies
vims and consequently allows the recombinant virus to infect neuronal cells
(Cronin et al., 2005). Once the virus has bound to the cellular receptor, the viral
envelope fuses with the host cell membrane. The viral core then travels into the
cell’s cytoplasm where reverse transcription takes place. The now double-

stranded DNA genome enters the nucleus where it integrates with the host cell

23

genome; this dsDNA genome is termed the ‘provirus’. Transcription using the

host cell machinery occurs and viral proteins are produced.

gp120
9P41 \ lipid layer

     
  
 
   

host cell proteins p17 matrix antioen

integrase

p24 core antigen HIV-RNA

reverse

D7 gag transcriptase

P9 939

Figure 1.11. A representation of an HIV particle.

(http://wwwhivmedicine.com/textbook/images/image65.ipg, 2/8/07). The HIV

particle has both a protein capsid as well as a lipid outer membrane. Within
the viral capsid, the reverse transcriptase, integrase, and dsRNA genome
reside.

One desirable trait of rHIV with respect to gene therapy applications is its
potential to infect non-dividing cells. Retinal cells do not divide after
differentiation, thus rHIV may prove beneficial as a vector for retinal gene
therapy. In retinal research, it has been found that the rHIV pseudotyped with
VSV-G is able to enter and integrate into maturing photoreceptors within the first
15 days of a mouse’s life (Pang et al., 2006a). rHIV was shown to be able to

transfect both photoreceptors and RPE with the most efficient transfection

occurring from postnatal day 1 to 3. Use of enzymes has been Shown to

24

increase photoreceptor transduction in the eye, as access of lentivirus vectors to
photoreceptors may be limited by a physical barrier within the adult retina (Gruter

et al., 2005).

1.2.6.C. Recombinant Adenoviruses (Av-1)
Recombinant adenoviruses have been used successfully as vectors for gene
therapy ranging from management of cancer to treatment of inherited systemic

diseases.

Adenovimses are non-enveloped viruses that consist of an icosahedral, Spiked
capsid that is 70-90nm in diameter. The viral genome is linear, double-stranded
DNA and iS approximately 36KB in size (Figure 1.12) (Connelly, 1999). This
virus is in the family Adenoviridae and consists of at least 50 distinct serotypes of
which group ‘C’ serotype 5 iS the most commonly used for gene therapy
purposes. Adenoviruses infect many different cell types including liver, intestine,
and even cells of the retina. Wild-type serotype 5 adenovirus can cause
respiratory infections, gastroenteritis, and even conjunctivitis, but typically little

inflammation is induced from the virus alone (Connelly, 1999).

Three main recombinant adenoviruses are available: Av-1, Av-2. and Av-3. Av-1
is the first generation recombinant adenovirus that was created by deleting the
entire region for the E1A gene and 60% of the E1 B gene which was then

replaced with the transgene of interest. Av-1 uses the cytomegalovirus (CMV)

25

promoter, a strong viral promoter that has been known to induce a strong
inflammatory response (Connelly, 1999). Av-2 vectors have one or more genes
deleted in any combination of the following genes: E1, E2, E3, and E4. The
transgene insert site is still located in the E1 gene deleted region, but these
vectors use either the CMV promoter or can use another promoter of choice

(Connelly, 1999).

Av-3, also known as ‘Helper—Dependent Ads’, are the most recent adenovirus
vectors produced. In Av-3 vectors, the entire viral genome has been deleted and
therefore the vector must be grown in the presence of ‘helper’ viruses. The DNA
within the virus is the promoter and transgene DNA along with more ‘filler’ DNA
to stabilize the structural integrity of the virus (Kumar—Singh et al., 1998;

Connelly, 1999).

 

 

 

 

 

 

E1A E1B L1 L2 L3 L4 E3 L5
——> —> > >——> ——> —> —>
4 4— ‘—
EZB E2A E4

Figure 1.12. A representation of the adenovirus genome (Modified from
http://www-ermm.cbcu.cam.ac.uk/flg005ifo.qif, 2-24-07). The adenovirus
genome is a linear dsDNA genome that is transcribed from both 5’ ends since
viral genes can be oriented in both directions. ‘E’ genes represent the early
transcribed genes whereas the ‘L’ genes represent the later transcribed genes.
Adenoviruses enter the host cell by binding a cell surface receptor, of which two

are of importance to retinal gene therapy: CAR (Coxsackie-adenovirus receptor),

which are present on most cells but not photoreceptors, and CD46, which has

26

been found on photoreceptors and binds to a fiber, F35, on the adenovirus
capsid (Mallam et al., 2004; Von Seggem et al., 2003). Integrins are also
involved in the entry of the virus and aid the cell in the process of endocytosis.
The virus can tolerate the acidic conditions within the vesicle and escapes into
the cytoplasm with the viral capsid fully intact. Once in the cytoplasm, the virus is
transported along microtubules and directed toward the nuclear pores. The
adenovirus attaches to the nuclear pore and the viral genome is transported into

the nucleus where it is then transcribed (Connelly, 1999).

When using recombinant adenoviruses for gene therapy, it has been found that
there is typically a waning of transgene expression approximately 2 weeks post
transfection. There are many theories to explain this phenomenon. One theory
suggests that antibodies generated by the humoral immune system eliminate the
transgene and virus. Another theory suggests that there is CMV-promoter
shutdown. This was eloquently demonstrated by Everett in a study which
compared the same vector in both normal mice with an intact immune system
and nude mice which lack an intact immune system (Everett et al., 2004). Both
of the mice, despite the lack of an immune response in the nude mice, Showed a
decrease in transgene expression over time, suggesting promoter shutdown

rather than immune response was the cause of loss of transgene expression.

27

1.2.7. Summary of Successful Retinal Gene Therapy Treatments

1.2.7.a. AAV Preliminary Studies in the Eye

Recombinant AAV vectors have been studied extensively for gene therapy
purposes. AAV vectors are easy to create in large quantities, have low
immunogenicity, and can transduce many different cell types. Several studies
have looked at the ability and efficiency of these vectors to infect retinal cells in
various species. Initially mouse models were studied followed later by dog and
primate models. AAV vectors have been shown to be effective and safe with
little inflammation or toxicity and to produce stable long term transgene
expression (Auricchio and Rolling, 2005; Ali et al., 1998; Bainbridge et al., 2003;

Bennett et al., 1999).

The kinetics of the AAV5 vectors was studied in the mouse eye in order to
understand the transduction and gene expression variables. Mice were given
injections of between 2x106 and 2x1010 lU/mL into the subretinal space where
the vector transduced retinal cells as soon as 3 days after injection. The time lag
between transduction of retinal cells and gene expression is believed to be due
to the requirement for dsDNA synthesis from the SSDNA viral genome. The
retinal cellS that were the main targets of the vector were located at the injection
site, but some vector diffused past the borders of the injection site for unknown
reasons. It was also determined that increasingly higher titers were able to
transduce cells more effectively and were able to yield higher gene expression

rates in retinal cells (Sarra et al., 2002).

28

1.2.7.b. Successful AAV Gene Addition Therapy for Retinal Degenerations
Attempts to treat mouse models of RP and LCA using AAV vectors have been at
least partially successful in several mouse models. Both the RPE65 mutant
mouse and the rd1 mouse have shown improvements after AAV gene addition
therapy (Pang et al., 2006b; Lai et al., 2004; Jomary et al., 1997). Further work
into the treatment of the RPE65 null mutation model of LCA has utilized a large
animal model, the Briard dog. Using an AAV vector transporting the normal
RPE65 transgene, subretinal injection was performed in an attempt to treat a
portion of the retina by gene addition therapy. These treated dogs showed
significant improvement in vision with dose—dependent, long-term results for
several years (Acland et al., 2001; Narfstrom et al., 2003a; Ford et al., 2003;

Narfstrom et al., 2003b; ACland et al., 2005; Jacobson et al., 2006).

There have also been other successful retinal treatments using an AAV vector;
the Prph2 mutant mouse showed rescue when an AAV vector with a rhodopsin
promoter driving expression of peripherin was used (Ali et al., 2000), and the

ROS rat with a mutation in the Mertk gene was rescued using an AAV vector to

deliver a normal copy of the Mertk gene as well (Smith et al., 2003).

1.2.7.e. Lentivirus Preliminary Studies in the Eye
HIV vectors pseudotyped with the VSV glycoprotein were created with the aim of
transducing differentiated, non-dividing cells such as retinal cells (Naldini et al.,

1996; Bemelmans et al., 2005). Work using these vectors in rodent models

29

Showed some successful results but with limitations. In one study, subretinal
injections of the vector in rat eyes showed sustained transgene expression 12
weeks after injection with no Signs of decrease in expression (Miyoshi et al.,

1997)

1.2.7.d. Successful Lentivirus Gene Addition Therapy for Retinal
Degenerations
Continued efforts have shown the efficiency and promise of recombinant
lentivirus vectors in delivering the missing gene responsible for fast onset retinal
degenerations. The rd1 mouse, the RPE65 knockout mouse, and the ROS rat
have been treated using a rHIV vector. Early treatment of the rd1 mouse has
Shown promise in slowing photoreceptor degeneration for up to 24 weeks
(Takahashi et al., 1999). In the RPE65 knockout mouse, early treatment of the
retina led to increased cone photoreceptor survival for up to 4 months while
adults treated with the same vector did not Show increased cone photoreceptor
survival (Bemelmans et al., 2006). Finally, lentivirus vector treatment of the RC8
rat by subretinal injection at 10 days of age resulted in survival of photoreceptors
up to 7 months (TSChemutter et al., 2005;). Unfortunately, for unknown reasons

the rescue was only temporary.

30

1.2.7.e. Adenovirus Preliminary Studies in the Eye
Adenoviruses were among the first viral vectors that were studied for gene
addition therapy in the retina. Today, this vector is still being researched as a

potential vector for treatment of many types of ocular disease.

As stated earlier, adenoviruses enter the host cell by binding a cell surface
receptor, either CAR or CD46, depending on the viral capsid (Von Seggem et al.,
2003). Importantly, CAR receptors do not exist in large numbers on the surface
of photoreceptors and therefore these cells are not well transduced by the

commonly used AdV5 vectors for gene therapy research (Mallam et al., 2004).

The first study using adenoviruses for retinal gene therapy were done using Av-1
vectors with the beta-galactosidase (LacZ) transgene in mice. Within 24-48
hours after subretinal injection, transgene expression was evident in the retinal
layer (Bennett et al., 1994). Transduction by the recombinant virus and also
transgene expression was found to be dose-dependent in that study. Two weeks
after the injection, the transgene expression had waned completely.

Another study looking to determine the length of transgene expression used the
same vector type as the above experiment and found that the adenovirus-
delivered transgene could be expressed for almost 16 months in rat retinas, with
most of the infected cells being RPE with only a few cells of the neurosensory

retina infected (Loewen et al., 2004).

31

1 .2.7.f. Successful Adenovirus Gene Addition Therapy for Retinal
Degenerations
One of the first successes using Av-1 for treatment of a retinal disease involved
treatment of rd1 mice. This study demonstrated photoreceptor survival up to six
weeks post injection. The reporter transgene slowly waned for a period of two
weeks and then became completely undetectable thereafter (Bennett et al.,
1996). This study established the potential usefulness of this vector, so other
researchers have attempted to use this vector for other models Of retinal
degeneration. (See Figure 1.13. for an image of a dog retina transduced by an

Av-2 vector.)

Since the RPE is transduced well by Av-1 vectors, several retinal degeneration
models were suitable for treatment using this vector. For instance, the ROS rat
showed retinal saving after subretinal injection of the vector carrying the Mertk
transgene (Vollrath et al., 2001). Treatment of the RPE65 knockout mouse
resulted in the presence of functional rpe65 and preservation of photoreceptors

(Chen et al., 2006).

Av-3 vectors have also been investigated for retinal degeneration treatments. An

EAM with a B-pde transgene and B-pde promoter was injected into the subretinal

space of the rd1 mouse eye. This vector resulted in improved photoreceptor

32

 

 

Figure 1.13. ' L' ‘ ‘ ' ‘ y of the dog retina transduced by an
Av-1 vector after subretinal injection. The red fluorescence indicates the
green fluorescent protein (GFP) within the RPE layer of the retina and the blue
fluorescence indicates the nuclei of individual cells.
survival compared with the results using an Av-1 vector (Kumar-Singh and

Farber., 1998).

1.2.8. Safety Concerns that Arise from Viral Gene Therapy

On September 17th, 1999, Jesse Gelsinger, an 18 year old omithine
transcarboxylase (OTC) deficient patient who was a voluntary participant in a
gene therapy trial died 4 days after the treatment due to multi-organ-system
failure (Stolberg, 1999; Hartogs, 1999; Recombinant DNA Advisory Committee,
2002). Adenovirus vectors were used in the trial in an attempt to treat the OTC
deficiency. Gelsinger received the highest dose used in the trial of 3.8 x 1013
viral particles in an effort to determine the dose-dependent nature of the
treatment. It was determined on autopsy that the extreme reaction to the vector
was to the viral capsid. In response to this unfortunate event, the NIH

Recombinant DNA Advisory Committee (RAC) made recommendations for the

33

protocol and monitoring of human Clinical trials in order to lessen the chance of

future adverse events (RAC, 2002).

Another serious adverse event occurred during Clinical trials using gene therapy
to treat X-Iinked severe combined immunodeficiency disease (SCID—X1). In this
study, retroviruses were used to infect T cells ex vivo that were then injected into
the blood stream. Two of the youngest patients contracted leukemia due to
insertional oncogenesis of the retrovirus near the LMO2 promoter, which is a
proto-oncogene (Hacein-Bey—Abina et al., 2003; ASGT, 2002). The NIH RAC
has produced statements in regards to these concerns and has warned
researchers of this rare but serious consequence that is possible when using

retroviral vectors for gene therapy (Sadelain, 2004).

The safety and efficacy of gene addition therapy is a serious and legitimate
concern. Issues encompass several areas including: the concern of viral
biodistribution, insertional oncogenesis, host immune responses to both the
transgene and viral vector, and the potential for genotoxicity of the transduced

cells.

1.2.8.a. Host Immune Response
The host immune system is stimulated to eradicate foreign invaders as quickly
and as efficiently as possible (Figure 1.14). Initially, the host tries to eradicate

the invader through the innate immune response such as through phagocytosis,

34

neutralization, and complement (Janeway et al., 2005). If the innate immune
system is unable to inhibit or destroy the Wars, host cells are then infected. This
is where the life cycle of the vian begins and the eventual death of the cell
ensues. The host’s immune cells, such as antigen presenting cells (APCS);
macrophages, neutrophils, and dendritiC cells, and also natural killer cells (NK
cells), recognize the infected cells and thus the antigen. The host also mounts a
second attack against the antigen known as the adaptive immune response.
This response takes 4-7 days for elimination of the antigen after infection, which
involves both the cell-mediated and humoral pathways, separately and

collaboratively (Janeway et al., 2005).

The cell-mediated immune response is through activation of T cells by antigen
presentation by APCS within lymph nodes. Cytotoxic T cells, also known as
CD8+ T cells, and helper T cells, CD4+ Th1 and CD4+ Th2 cells, are activated
by antigens presented by APCS. CD8+ T cells, are the effector cells that destroy
infected cells by way of a pathway that induces apoptosis (Janeway et al., 2005).
CD4+ Th1 and Th2 cells each have their own unique role. Th1 cells leave the
lymph node after activation to stimulate cells, such as macrophages, into action.

Th2 cells activate B cells within the lymph node (Janeway et al., 2005).

The humoral immune response is activated immediately to any antigen upon

entry into the host. The humoral immune response is initiated by Th2 cell

activation of B cells and also B cell presentation of antigens to Th2 cells through

35

the MHCll receptor. These immature B cells produce antibodies and they are
dispersed into the blood. lgM antibodies are the first antibodies produced by
immature B cells within the first few days of an infection (Janeway et al., 2005).
Some of the B cells travel to the germinal centers within the lymph nodes where
they mature through isotype switching, which allows for a variety of antibodies to
be produced such as lgG, thus creating plasma cells. The peak lgG production
occurs between 7-10 days post-infection. The antibodies bind to the antigens
and allow for neutralization of the antigen, induction of complement, or promotion

of phagocytosis (Janeway et al., 2005).

Memory T and B cells are formed after an initial exposure to an antigen. Both
memory T and B cells are thought to be sustained through constant cytokine
stimulation. Upon a repeat exposure to the same antigen, such as a viral vector,
B cells undergo clonal expansion and are able to produce high levels of IgG
within a few days after infection. T cells also undergo proliferation after
encountering the same antigen and create effector cells that travel throughout

the host killing infected cells.

For the viruses that some of the recombinant vectors are derived from, the
details of the host response have not been elucidated, but for others, such as
adenoviruses, extensive work has been done in order to understand the immune

response (for a review see Schaack, 2005).

36

 

Virus enters host

Host Immediate Immune
Responses

Pre- -existing neutralizing body Inflammatory response
if at *T— 00

Macrophages and neutrophils0
phagocytize virus and then alert host

*v
,
/ I
/
/’ I
I

 

     
 

Cell- mediated
Immune Response ----- * Humoral Immune
Response

Secondary Host Immune Response

Flgure 1 14 An overVIew of the host immune response to a forelgn

 

 

 

invader.
NK_ natural killer cells, T: T cell and B_ B cell

37

1.2.8.b. Adena-Associated Vectors- Safety and Host Immune Response
Wild-type adeno-associated viruses are known for their low immunogenicity
during infections. Because of this trait, they have proven useful as gene therapy
vectors with the potential for repeated injection without adverse effects (Anand et
al. 2002, Anand et al., 2000). Studies to determine the safety of AAV vectors
have included the systemic safety, the possibility of gerrnline transmission, the
immune response to the viral capsid after intraocular injection, and the effect of

the immune response on the repeated administration of the vector.

AAV vectors have been found to be safe and efficacious for systemic use (Chao
and Walsh, 2004). While this may not be a major concern for ocular therapy, the
potential for spread of the vector from the eye to other parts of the body does
remain a concern. In order to determine the overall safety of the AAV vector
systemically, intramuscular injections of an AAV vector were performed on
nonhuman primates (Favre et al., 2001). Within 48-72 hours after the injection,
the viral genome was detected in the serum and up to 6 days post-injection the
vector was detected in various body fluids. No tissue abnormalities were found
at time of necropsy performed 8-18 months post-injection, but the vector was

found in the lymph nodes and liver (Favre et al., 2001).

Gerrnline transmission has been a concern after an AAV vector was used to treat

hemophilia in human patients and the AAV vector DNA was found in the semen

of treated males (Manno et al., 2003; Arruda et al., 2001). Schuettrumpf et al.

38

designed a study to determine if the germ cells were infected or if the viral vector
was only in the semen transiently. Using rabbits to study the gerrnline
transmission after a systemic injection of AAV, vector sequences were found in
the semen in a dose-dependent manner and disappeared also in a dose-
dependent manner. No infection of the germ cells was evident when monitored
over a period of 18 months when analyzed using a quantitative real-time

polymerase Chain reaction (qPCR) (Schuettrumpf et al., 2006).

One issue of remaining controversy over this vector is whether or not rAAV has
the ability to insert itself into the host cell’s genome. Random insertion into the
genome could potentially create insertional oncogenesis. In humans, it has been
Shown that the wild-type virus has the ability to integrate into the genome at a
specific place on chromosome 19 (Kotin et al., 1990). Others contest that the
viral DNA, once inside the nucleus, does not integrate into the host genome
(Song et al., 2004). AS this continues to be debated, the possibility of integration

remains a concern for the safety of gene therapy trials using AAV vectors.

Immune responses to the AAV capsid have been previously studied. After
intraocular injection of the vector, mild to moderate inflammation has been
Observed in Briard dogs, but this resolved over a period of 3 months post-
injection (Jacobson et al., 2006). An adaptive immune response has been
detected after intraocular injection of the vector. A slight qualitative anti-AAV

humoral response was detected in mice and dogs after intravitreal and subretinal

39

injection in both pre- and post-injection (time not specified) serum and aqueous
samples (Dudus et al., 1999). Further work into the adaptive immune response
was studied in mice; subretinal injections were performed in mice and a deviant
immune response was found in the eye. A footpad thickness test performed after
intraocular injection (which demonstrates the cell-mediated response) the results
of which indicated there was a minimal response against the viral capsid (Anand
et al., 2002). Bennett et al. detected a Th2-dependent humoral response to the
viral capsid (by ELISA) and found that individual primates had up to a 16-fold
increase in IgG and Dudus et al. showed a statistically Significant increase in
mice and dogs of lgG antibodies to the viral capsid (Bennett et al, 1999; Dudus et

aL,1999)

Readministration studies have been done in order to determine if a systemic
humoral response would interfere with transgene expression in the eye. In a
study by Bennett et al., they found that after initial injection neutralizing
antibodies were formed (Bennett et al., 1999). When the same AAV-GFP vector
was injected into the contralateral eye 7 months after the initial injection, no
inflammatory response was detected and transgene expression was evident,
despite the presence of neutralizing antibodies and a 128-fold increase in serum
anti-AAV antibody after readministration (Bennett et al., 1999). In a similar study,
mouse eyes were given subretinal injections of the vector, at repeat
administration transgene expression was still observed even though there was a

significant anti-AAV humoral response present in the mice (Anand et al., 2000).

40

1.2.8.c. Safety of Lentivirus Vectors
The two main concerns when using Ientiviruses for gene therapy are: 1) the
potential for insertional oncogenesis and 2) spontaneous recombination

occurring creating a wild-type virus (Lever, 1999).

The phenomenon of insertional oncogenesis is pertinent, though rare, because
the random insertion of the viral genome next to active genes of the host cell
genome can interfere with the normal cellular functioning (Recchia et al., 2006).
If near a promoter of a tumor-suppressor gene or oncogene, oncogenesis and
consequential disease could result for the patient. New vectors are being
designed that will help alleviate this issue by directing viral genome insertion to a
consistent and safe location within the genome. Non-integrating HIV vectors are
also being designed that will remain in the nucleus to allow transcription to

proceed but will not undergo genome insertion (Yanez-Munoz, 2006).

The issue of recombination occurring and leading to self-sufficient wild-type virus
regeneration can be easily overcome by designing a plasmid system that plans
for this potential hazard. Several methods have been created that are
specifically designed to inhibit insertional complications (Lever, 1999; Lu et al.,

2004).

41

1.2.8.d. Adenovirus Vectors in the Eye- Safety and Host Immune Response
Although adenovirus vectors have been shown to be efficient for gene therapy,
the recombinant adenovirus vectors have induced inflammatory and other
immune responses. It is theorized that inflammation is induced for several
reasons: size of the virus, capsid structure, and transgenes such as LacZ and

GFP.

Three distinct phases in recombinant adenovirus induced inflammation have
been described (Figure 1.15). The first phase is a Short-lived response to the
capsid, which induces cytokine release. The second phase occurs after
internalization of the virus and is thought to be due to the adenovirus genes. The
third phase is immune recognition of the adenovims gene products and also
possibly the transgene product. Antibodies are then formed which might result in

elimination of the transgene and viral vector (Schaak, 2005).

While the first and second generation adenoviruses Show a substantial immune
response complete with inflammation and a secondary immune response, the

Av-3, also known as encapsidated adenovirus mini-Chromosomes (EAMS), has
Shown a significantly lower secondary immune response since they lack all viral

genes (Muruve, 2004).

42

@ Monocytes

Granuloc es \ , .

Chemokines/ CytokInes
@ NK Cells

Leukocyte recruitment

Tissue Macropahge/ DC

__.————->

Chemokines/ Cy10kines M3531:

x}\ Target Tissue
3‘43}

 

 

 

 

 

 

 

Adhesion molecules
I l

I I I J

Endothelium
Figure 1.15. The typical host immune response to recombinant adenovirus
vectors.
NK= natural killer cells, 00: dendritic cells,
In the eye after subretinal injection of an adenovirus (Av-2) vector, there seems
to be a deviant immune response (Hoffman et al., 1997). Subretinal injection of
the vector has shown to induce a systemic humoral response, yet after
readministration of an adenovirus vector, transgene expression did not appear to
be inhibited by the host immune response (Isenmann et al., 2001). Interestingly,
in another study, vaccination of the animals in order to create an immune
response and then followed by adenovirus vector intraocular administration every
two weeks thereafter showed to have little if any effect on transgene expression

within the eye (Hamilton et al., 2006).

43

1.2.8.e. Host Immune Response to Expressed Transgene in the Eye

Few studies have focused on the immune response to the transgene after
intraocular injection. In a study done by Isenmann et al., there was a difference
in immune response based on injection site. Following intravitreal injections, an
immune response to the transgene was detectable where as following subretinal

injection an immune response was not detected (Isenmann et al., 2001).

Two studies have looked Specifically at the humoral immune response to GFP
after intraocular injection of a viral vector carrying the GFP transgene. Both of
the studies qualitatively analyzed the presence of anti-GF P antibodies via
Western Blot. Dudus et al. used pre- and 7 week post-injection serum samples
and found that 1 out of 3 mice had an antibody response to GFP (Dudus et al.,
1999). Another study showed similar results with a high Circulating anti-GFP
antibody level detected in 2 out of 4 mice after subretinal injection of an AAV-

GFP vector (Bennett et al., 1999).

1.2.8.f. Biodistribution of the Viral Vector within the Host after Intraocular
Treatment

AAV delivered by subretinal or intravitreal injection has been shown to spread

along the optic nerve to the optic tract in several species: dog, primate, and

mouse (LeMeur et al., 2005; Jacobson et al 2006; Dudus et al.1999; Shen et al.,

2003; Provost et al., 2005). Both gross investigation using reporter genes as well

44

as quantitative PCR (qPCR) have been used in order to demonstrate the

distribution of the vector in the host after intraocular injection.

In the mouse, GFP fluorescence has been observed in the layers of the
neuroretina and also along the optic nerve after subretinal injection. GFP was
observed in the retinal ganglion cells, the optic nerve fibers, and the optic disc.
Along the visual pathway, GFP was observed in the optic nerve, lateral
geniculate body, optic radiation all the way to the occipital lobe (Shen et al.,
2003). Interestingly, the type of injection performed had an effect on the
distribution of the vector. Comparing intravitreal and subretinal injections in both
dogs and mice, GFP fluorescence was detected along the visual pathway to a
greater extent after intravitreal injection than after subretinal injection as might be

expected (Dudus et al., 1999).

PCR has also been used to determine the relative amount and location of the
vector after spread from an intraocular injection in the rat, dog, and nonhuman
primate. Two studies have worked to address this issue, but with different
results. Provost et al. injected, via intravitreal or subretinal routes, an AAV-GFP
vector into the eyes of rats, dogs, and nonhuman primates; PCR for the GFP
transgene sequence was performed to detect the sequence within many body
tissues including retina, Optic nerve, Optic chiasm, optic tract, lateral geniculate
nuclei, optic radiation, visual cortex, superior colliculus, inferior colliculus,

temporal lobe, thalamus, cerebellum, spinal cord, cerebrospinal fluid, pituitary

45

gland, lachrymal gland, amygdala, salivary gland, submandibular lymph node,
preauricular lymph node, thymus, axillary lymph nodes, gonads, liver, and
peripheral blood mononuclear cells (Provost et al., 2005). Sequence was
detected occasionally in peripheral blood mononuclear cells. As expected,
vector sequence was found in the optic nerve, and along the visUal pathway after
intravitreal injection, but interestingly the vector sequence was also found in the

optic nerve after subretinal injection (Provost et al., 2005).

In another study done by Jacobson, subretinal injection of a vector carrying the
RPE65 transgene led to distributionand thus viral sequence detection by qPCR
in various organs. Tissues that were analyzed included: optic nerve, optic
chiasm, optic tract, Iatertal geniculate nucleus, optic radiation, visual cortex,
superior colliculus, mandibular lymph node, parotid lymph node, heart, lung,
diaphragm, liver, pancreas, spleen, kidney, jejunum, gonad, and Skeletal muscle.
The spread of the vector was found not to be dose-dependent (Jacobson etal.,
2006). Vector sequence assayed by amplifying a portion of the CMV enhancer/
CBA promoter was found at a level of 100 copies/pg or higher of DNA in the

mandibular lymph nodes, heart, diaphragm, optic nerve, and optic chiasm.

1.2.8.9. Genotoxicity
Issues of genotoxocity remain a concern when introducing a vector that has a
ubiquitous promoter or when a foreign protein such as GFP, LacZ, or in gene

addition therapy if the transgenic protein is introduced into an animal that is

46

deficient for the particular protein. For example, very high doses of the AAV
vector carrying the RPE65 transgene has Shown to produce toxic effects in the
retina when the transgene is under the control of a ubiquitous promoter
(Jacobson et al., 2006). Thus, dosage toxicity studies are necessary before

Clinical trials begin to determine the safe dosage range.

In most studies, there is little systemic toxicity and retinal damage after
intraocular injection of a vector (Jacobson et al., 2006; LeMeur et al., 2005). For
example, subretinal injection of AAV-2, -4, and -5 with the GFP transgene in 14
beagles and 9 macaques showed normal retinal angiography images and ERG
amplitudes (Le Meur et al., 2005). In another study, AAV-GFP vectors were
injected into the eye and high levels of GFP in retinal cells up to 270uM Showed
no toxic effect and did not seem to affect normal photoreceptor function based on

ERG results (Rex et al. 2005; Shen et al., 2003).

One way to reduce the risk of genotoxicity is to use a tissue-specific promoter.
For the retina, a mouse opsin promoter has been developed that only expresses
the transgene in rods and cones (Glushakova et. al., 2006). Another way to
solve this issue is by using inducible promoters such as tetracycline- or
erythropoietin-inducible promoters that are activated Upon systemic
administration of the appropriate drug. These methods have even been Shown to
work in the eye despite the blood-ocular barriers (Auricchio et al., 2002; Blau et

aL,1999)

47

1.3. Objectives, Hypotheses, and Experimental Design

1.3.1. Objectives

The purpose of this work was to investigate in detail the humoral immune
response to GFP after treatment with viral vectors carrying the GFP transgene
administered intraocularly in dogs. We focused primarily on AAV constructs since
these are the most widely used vector types in retinal gene therapy. The
systemic immune response was investigated for both GFP and AAV capsid
antibodies while the local immune was investigated for GFP antibodies. The
systemic immune response to GFP after intraocular treatment with adenovirus

and lentivirus vectors was also performed.

1.3.2. Hypotheses

In previous studies, intraocular administration of AAV-GFP in mice and dogs was
reported to induce a minor, individual systemic and intraocular humoral immune
response to both the viral capsid and GFP. We hypothesize that the same
response will be observed and we wish to perform a more detailed analysis of
the timing of the humoral immune response in dogs. The systemic AAV antibody
response Should be detectable at 2 weeks post-treatment while the GFP
antibody response should be detected systemically 4 weeks post-treatment due
to slow expression of the transgene that is typical for AAV vectors. The local
GFP antibody response Should be detectable at approximately 4 weeks post-

treatment for similar reasons. Variables such as the promoter used in the

48

construct and the age of the animal at treatment are expected to influence the

immune response.

We anticipate that GFP expression resulting after intraocular treatment with
lentivirus and Av-1 vectors will also result in a detectable systemic humoral

immune response.

1.3.3. Experimental Design
Intraocular injections with vectors carrying the GFP transgene were performed:
AAV vectors were used in 22 dogs, lentivims was used in 3 dogs, and Av-1 was

used in 1 dog.

For the AAV-treated pups, one of two AAV constructs were used: AAV-CBA-GF P
or AAV-mOPS-GFP and injections performed at either ~3 days of age or ~3
weeks of age (figure 1.16). For lentivirus- and Av-1- treated dogs, subretinal
injections were performed. Aqueous samples were collected from AAV-treated
dogs pre-injection and then at the time Of euthanasia. Sera samples were
collected from each dog pre- and post-treatment. Sera samples were collected
from AAV- and lentivirus-treated dogs every two weeks up to 8 weeks for most
dogs. The Av-1-treated dog sera samples were collected at 2 and 3 weeks post-
treatment. These serum and aqueous samples were then analyzed using
canine-specific lgG ELISAS (GFP and AAV) in order to understand the systemic

and local humoral responses.

49

 

 

 

Age of Pup at Treatment

/ \

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 day treatment 3 week treatment
Injection of AAV Injection Of AAV
Aqueous/serum Aqueous/serum

sample collection pre- sample collection pre-
treatment treatment
Serum Collection 2, 4, 6, 8, Monitor GFP

 

 

 

and >8 weeks post-injection,
other further time points

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Euthanasia
Serum collection Aqueous cOllection
AAV EL'SA GFP ELISA

 

 

 

 

 

 

Figure 1.16. Flow chart of experimental design for the AAV—treated dogs.

50

Sera from AAV-treated dogs were analyzed using the AAV and GFP ELISAS
while the aqueous samples were analyzed using the GFP ELISA. Sera from the
lentivirus- and Av-1-treated dogs were analyzed using the GFP ELISA. The

optical density (OD) values from these ELISAS were then analyzed using SAS.

51

Chapter 2.

DEVELOPMENT OF ELISA ASSAYS

2.1. Introduction

Two canine-Specific enzyme-linked immunosorbent assays (ELISAS) were
developed in order to analyze the humoral immune response after intraocular
injection of viral vectors carrying the GF P transgene : rAAV2/5, Av-1, and
lentivirus. The first ELISA designed was the GFP ELISA to detect anti-GFP
antibodies and the second was the AAV ELISA to detect the anti-AAV capsid
antibodies. Previous studies have analyzed the humoral immune response in
mice and dogs based on human serum controls which could create bias and
distorted results. By creating canine- Specific ELISAS, the humoral immune

response can be more accurately assessed.

2.2. Materials and Methods

2.2.1. Immunization of Dogs

Colony dogs were immunized with the intent Of using serum samples as controls
for the GFP ELISA and AAV ELISA. Each dog was injected using a multi-
channel LectraJet injector (D’Antonio Consultants International, East Syracuse,
NY) with a vector preparation, described below, that was intended to create an
immune response to both the vector capsid as well as the GFP transgene (Carter

and Kerr, 2003).

52

The pHR’-CMV-GFP vector was created in the laboratory of Vincente Planelles
(University of Utah) and was used for immunizations because it induced a high
level of circulating anti-GFP antibodies. The protocol for creation of these
vectors has been published (Zhu et al., 2001: Planelles, 2003). The lentivirus
vector (pHR-CMV-GFP) was prepared for injection by diluting the virus to ~10"6

infectious units of the vector in sterile saline.

The AAV-GFP vector was created in the laboratory of William Hauswirth at the
University of Florida. Another member of our research group, Danielle Eifler,
prepared the UF 12 type 5 AAV vector to a concentration of ~10"13 vp/mL in

sterile saline.

A total of 4 dogs were injected with the appropriate virus and 2 negative control
dogs were injected with sterile saline as outlined in Table 2.1 below. Each dog
was sedated with 0.03-0.05 mg/kg of acepromazine (Boehringer lngelheim
Verrndica, Inc., St. Joseph, MO) and 0.2mg/kg of butorphanol (Fort Dodge
Animal Health, Fort Dodge, Iowa). The area on the medial thigh was then
shaved and scrubbed. Topical lidocaine was applied to area to be injected. Two
separate injection Sites on the medial thigh of each leg were used with a total of
four Sites per injection day. A vector preparation volume of 0.5 mL was injected
per site. After, the injection protocol was finished, each dog received 100mL
saline subcutaneously following the injection procedure. Immunizations were
first performed for all dogs on 7/21/04 and then repeated on 8/8/04 and 8/18/04.

Dogs were re-immunized on 4/20/05.

53

 

 

 

 

 

 

 

Mica Negative control dog-0.5ml of saline
Snap Negative control dog-0.5ml of saline
Pluto Lentivirus-GFP-0.5ml of virus solution
Spruce Lentivirus-GFP-0.5ml of virus solution
Red AAV-GFP- 0.5ml of virus solution
Roscoe AAV-GFP- 0.5ml of virus solution

 

 

Table 2.1. lmmunized dogs. The vector and amount of virus injected per dog
is described.
Sixty milliliters of blood was collected from each dog prior to immunization and
then serum was collected every week for the following Six weeks. After the Six
weeks, blood was collected every two weeks. The blood was placed on ice
following collection, allowed to Sit at 4°C for twenty-four hours, and then serum
was collected and stored at -20°C. After dogs were re-innoculated on 4/20/05,

serum was collected 1 and 2 weeks after innoculation.

2.2.2. Development of GFP ELISA

During the initial phases of development, GFP and antibodies were used at
varying concentrations in order to create optimal assay conditions. GFP was
used to coat the plate (BD Falcon, ref 353279, Bedford, MA) using dilutions of
1:100, 1:200, 1:500, 121,000, 1210,000, and 1:30.000 in a bicarbonate solution
(see methods) with the initial concentration GFP at 1mg/mL (Clontech, Mountain
View, CA), resulting in working concentrations of 0.01 mg/mL, 0.005mg/mL,

0.002mg/mL, 0.001 mg/mL, 0.0001 mg/mL, and 0.000033mg/ml, respectively

54

 

(Figure 2.2). The optimal GFP concentration range was found by using GFP
hyperimmune rabbit serum (HIRS) as a positive control (Abcam, Cambridge,
MA), normal rabbit serum as a negative control, and anti-rabbit-Specific HRP-
conjugated lgG as the secondary antibody (Caltag. L42007, Carlsbad, CA).
Varying dilutions of the normal and HIRS were tested: 1:500, 1:1,000, and
1:2,500, with the secondary antibody at a dilution of 120,000. The immunized
dog serum was then used to create a canine-specific assay (Figure 2.3.). The
positive control dog sera collected on 8/25 was Chosen because it produced the
highest OD values and was then used to finally optimize the assay for dog sera
samples. After analyzing serum dilutions of1:5, 1:10, 1:25, and 1:50, the optimal
positive control sera with the highest OD being ~1.2 was a 1:1 mixture of a both
positive control dog sera at a 1:25 dilution using PBS-T. The same 1:1 mixture of
negative control dog sera was used at a 1:25 dilution using PBS-T which gave an
OD of ~0.1 (Figure 2.4.). Positive and negative control sera were used for each
plate. Upon completion of the assay design, the test dog serum samples were

then analyzed using the finalized protocol described next.

2.2.2.a. Reagents

(see Appendix A for detailed instructions)
0 Phosphate-buffered solution (PBS)
0 0.05% PBS-Tween wash buffer

0 Bicarbonate solution (Pierce, Rockford, IL)

55

o GFP antigen solution for coating wells at 0.002ug/mL (Clontech, Mountain

View, CA)
. HRP-anti-Dog lgG (H+L) (Bethyl, Montgomery, TX) was made fresh for
each day of use from stock solution

0 1-Step ABTS used as substrate (Pierce, Rockford, IL)

2.2.2.b. Detailed GFP ELISA Protocol

The GFP ELISA (Figure 2.1.) was designed in order to determine if a systemic
antibody (IgG) response was induced following intraocular injection of viral

vectors [AAV2/5, Av-1, lentivirus] with a GFP transgene. (see Appendix A for

laboratory protocol).

0 O O 0 Wells washed, dog
0 O O O —'> 8 8 8 8 serum dIlutIons
O O O O

 

 

 

 

 

 

 

 

incubated for 1 hour
96-well plate coated
with 100uLof 0.002

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mg/ml GFP overnight 0 O O O
@4deg.Celsius OOQOvOOOO
/ Substrate ABTS galls wfiéhe-Ctifi
applied, incubated for n 89’ w'
. . . . 1hour @ 37C HRP’ Incubated
O O O O for1 hr
Color change \ O O O 0 Color change
occurs for pOSItIve Q O O 0 measured with
result spectrophotometer to
get OD values

 

 

 

Figure 2.1. GFP ELISA Protocol Flow Chart

56

96-well flat- bottom ELISA plates (BD Falcon, Bedford, MA) were coated
with 100pL per well of GFP antigen solution, covered with clear adhesive
Slips (Nunc, Denmark) and left for 15 hours at 4°C.
The wells were then washed and emptied three times with 200pL PBS-T
solution
Serum dilutions were prepared and applied to the wells at 100pL per well
0 Serum dilutions were prepared using a serial doubling dilution that
ranged from 1:5 to 1:320 in PBS-T down the plate column and each
dilution was run in duplicate
o Aqueous dilutions in PBS-T were done at 1:20 and were run only
once due to sample size limitations
0 Positive and negative control sera samples at 1:25 in PBS-T were
used for every plate
Plate re-covered with plastic Slip and placed on shaker, setting #5
(Wellmix, Therrno LabSystemS), and left at room temperature for one hour
The plates was removed from the shaker and the wells were then washed
and emptied three times with 200pL PBS-T solution
The secondary antibody was added at a dilution of 1:20,000, the plate re-
covered with the Slip, and left at room temperature on a Shaker (setting
#5) for one hour
The plate was removed from the Shaker and the wells were then washed

and emptied three times with 200pL PBS-T solution

57

o 125uL of 1-Step ABTS was added to each well, the plate was re-covered
with the Slip and was left at 37°C for one hour without shaking

o The plate was read at dual wavelengths of 405nm and 450nm using a
plate reader (Bio-rad microplate reader, Hercules, CA) to determine

optical density (OD) values

2.2.2.c. Results of GFP ELISA Development

The GFP ELISA was optimized through a series of steps. First, the optimal GFP
concentration was determined using anti-GFP rabbit serum (Figure 2.2). The
three best GFP dilutions for rabbit samples were found to be 1:10, 1:100, and
1:1000. Next, GFP dilutions in the range determined above were tested at 1:100,
1:200, 1:500, and 1:1000 and ELISA conditions optimized for dog samples using
immunized dog serum at dilutions of1:10, 1:100, 1:500, and 121000 (Figure 2.3).
The GFP dilution of 1:500 was Chosen from this trial for the canine GFP ELISA.
The next step involved determining the optimal canine control sera dilutions
(Figure 2.4). Sera from positive and negative control dogs as well a pool of the
control dog sera were tested at dilutions of 1:5,1 :10,1 :25,1 :50, 1:250, 1:500,
1:750, and 1:1000. The 1:25 dilution provided the best dilution tested Since it
produced the highest OD for the positive control samples with little the least

amount of background from the negative control samples.

58

2.2.3. Development of AAV ELISA

During the initial phases Of development, the AAV2/5 vector (Hauswirth
Laboratory, University of Florida, UF11 T5 #E894) was tested at several
concentrations in order to create an optimal assay. Using flat-bottom plates (BD
Falcon, ref 353279, Bedford, MA) and sterile saline (Butler, Columbus, OH) as
the diluent, the AAV2/5 virus (5.6E14Vp/ml stock) was tested at concentrations of
1,000vp/mL, 5,000vp/mL, 10,000vp/mL, 20,000vp/mL, 40,000vp/mL,
100,000vp/Ml, and at several dilutions of the virus of 1:50, 1:100, 1:200, 1:400,
1:800, and 1:1600. Sera from dogs that had been vaccinated with an AAV2/5
virus were used as positive controls for the development of the assay only.
Varying dilutions of the control dog sera were used at: 1:5, 1:10, 1:20, 1:40, 1:80,
1:160, and 1:320. The greatest OD values were Observed at the highest virus
concentrations, but were affected by the prozone effect (concentration of
antibody exceeds the concentration of antigen resulting in a false negative result)
at the highest concentrations of sera. A virus concentration of 100,000vp/mL
was Chosen for the assay Since there was a limited amount of virus available and
the OD values were comparatively high with no prozone influence (Figure 2.5). A
serum dilution of 1:5 was run in duplicate for test samples since this provided the
highest OD value per virus used. The negative control serum was not run due to
background interference observed at the 1:5 dilution, but a negative control

without sera was run per plate.

59

2.2.3.a. Reagents

Phosphate-buffered solution (PBS)

0.05% PBS-Tween wash buffer

A sterile saline solution (Butler, Columbus, OH) was used for the virus

solution preparation

The recombinant virus (made by the Hauswirth Lab at the University of
Florida, UF 11 AAV Type 5, 5.60E+13 vp/mL).

The negative control consisted of wells with all reagents except serum

HRP-anti-Dog lgG (H+L) (Bethyl, Montgomery, TX)

1-Step ABTS used as substrate (Pierce, Rockford, IL)

2.2.3.b. Detailed AAV ELISA Protocol

The AAV ELISA was designed in order to determine the systemic antibody (lgG)

response created after intraocular injection of the AAV5 vectors into the eye.

(see Appendix B for laboratory protocol)

96-well flat- bottom ELISA plates (BD Falcon, Bedford, MA) were coated

with 100uL per well of virus solution, covered with Clear adhesive slips

(Nunc, Denmark) and left for 15 hours at 4°C.

The wells were then washed with 200uL PBS-T solution and emptied

three times

Serum dilutions were prepared and applied to the wells at 100uL per well
0 Serum dilutions of 1:5, 1:10, and 1:20 in PBS-T and were run

O Positive control serum was run on each plate

60

O Negative control consisted of all reagents except sera to account
for plate variability

Plate re-covered with plastic Slip and placed on shaker, setting #5
(Wellmix, Thenno LabSystems), and left at room temperature for one hour
The plate was removed and washed with PBS-T and then emptied three
times as before
The secondary antibody was added at a dilution of 1:20.000, plate re-
covered with the Slip, and left at room temperature on a Shaker (setting
#5) for one hour without Shaking
The plate was removed and washed with PBS-T and then emptied three
times as before
125uL of 1-Step ABTS was added to each well, re-covered with the slip,
and left at 37°C for one hour
The plate was read at dual wavelengths of 405nm and 450nm using a
plate reader (Bio-rad microplate reader, Hercules, CA) to determine

optical density (OD) values

2.2.3.a. Results of AAV ELISA Development

The AAV ELISA was optimized using a range of control dog sera and virus

concentrations (Figure 2.5). The optimal virus concentration was determined

using the AAV-GFP immunized dogs. The optimal virus concentration ranged

between 2.8x1011vp/mL and 100,000vp/mL. A range of positive control serum

dilutions were used in order to determine the optimal serum dilution for the test

61

sera. The final assay design utilized 100,000vp/mL and a 1:5 serum dilution
Since this used the least amount of virus per well while resulting in high enough
OD values to analyze the anti-AAV antibody level with little wony of a prozone

effect occurring.

62

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66

Chapter 3
HUMORAL IMMUNE RESPONSES DETECTED IN DOGS GIVEN

INTRAOCULAR TREATMENT OF VIRAL VECTOR
3.1. Introduction
The humoral immune response induced from three different viral vectors after
intraocular administration was investigated. AAV-GFP vectors were studied
extensively since the AAV vectors have been most commonly used for
intraocular gene therapy. Systemic and intraocular immune responses to both
GFP and the AAV capsid were investigated. Lentivirus and Av-1 vectors were
being assessed as potential vectors for ocular gene therapy by using the GFP
reporter transgene. The anti-GFP response was monitored in dogs tested with

these vectors.

3.1.1. Intraocular Injections

In puppies after eyelid opening a subretinal injection was made under direct
visualization using an operating microscope via a transvitreal approach.
Subretinal injections were performed on puppies approximately 3 weeks of age
or older while transscleral injections were performed on neonatal pups 3 to 8
days of age. In neonates the eyelids are fused shut and the cornea is not yet
clear so injection under direct visualization is not possible. The neonatal pups
have immature retinas and the aim of the injection was to reach the developing
neurosensory retina to introduce the transgene at an early stage of retinal

development. This timing may be required for very early-onset retinal

67

dystrophies. The precise site of injection, subretinal, intraretinal, or intravitreal,
cannot be visualized when transscleral injections are performed prior to eyelid

opening.

3.1.2. Anesthesia

Anesthesia was induced in three day old puppies with isoflurane (Abbott
Laboratories, North Chicago, IL) delivered by oxygen mask and anesthesia was
maintained in the same manner. For three week old puppies, induction was by
masking with isoflurane; they were then intubated and maintained at a surgical
level of anesthesia with isoflurane (Abbott Laboratories, North Chicago, IL)

delivered in oxygen. For dogs older than 9 weeks of age, intravenous thiopental

sodium (PentothalQ, 6-12 mg/kg) was used to induce anesthesia; dogs were

then intubated and maintained using isoflurane (Abbott Laboratories, North
Chicago, IL) delivered in oxygen for the rest of the procedure. A pulse-oximeter
(Vet/Ox 4400, Heska Corporation, Fort Collins, CO) was used to monitor the
animal while under anesthesia, recording both blood oxygenation and pulse rate.
A heating pad was used to maintain the dog’s body temperature. Pulse rate,
oxygen saturation, and body temperature were recorded every 5 minutes during

the injection procedure.
3.1.3. rAAV2/5, Lentivirus, and Av-1

rAAV2/5 vectors with one of two different promoters driving the GFP transgene

expression were used; one promoter was the chicken-beta actin promoter (AAV-

68

CBA-GFP) which results in ubiquitous expression of the transgene and the other
was the mouse opsin promoter (AAV-mOPS-GFP) which results in
photoreceptor-specific expression of the transgene. Both vectors were used in
both neonate and 3 week old puppies. These rAAV vectors were created in the
laboratory of William Hauswirth (University of Florida). The protocol for creation

of these vectors has been published (Zolotukhin et al., 2002).

The Av-1 vector was a serotype 5 vector that was used at a concentration of

3x10"12 vp/mL (Amalfrtano Laboratory, Michigan State University).

The lentivirus vector (pHR’-CMV—GFP, Planelles, University of Utah) was used at
a concentration of 10"7 to 5x10"7 infectious units‘(Zhu et al., 2001: Planelles,

2003)

3.1.4. Injection Technique Used in Puppies Where the Eyelids Have
Opened

Subretinal injections were performed by Simon Petersen-Jones under aseptic

surgical conditions. A subretinal injector as seen in Figure 3.1. (RetinaJect

Injector, Surrnodics, Irvine, CA ), with Hamilton syringe (Hamilton Company,

Reno, Nevada) and loaded with the vector solution, was used to enter the eye

through the sclera (Figure 3.2.). The injector was positioned just anterior to the

retina in the tapetal region dorsal to the optic disc.

69

Subretinal in'e tion

./’¥\k .,

 

Figure 3.1. Path taken by the injector for the subretinal injection
procedure. The arrow indicates the area of injection, the subretinal space
between the RPE and photoreceptor layers of the retina.

Silicone tubing
399 cannula \ 7 ,
/ l,

25g needle
\

' I
l I Delrin handpiece

 

Figure 3.2. Subretinal injector (RetinaJect Injector, Surrnodics, Irvine, CA)
used to perform the subretinal injections.
(htt ://www.surmodics.com/ a eDetail.as x? a eld=93&menuld=95, 3/6/07)

 

The small internal cannula inside the injector was then advanced so that it was
pressed against the retinal surface. The vector was then injected and the force
of the injected fluid created a retinotomy allowing the fluid, ~50pL, to pass into

the subretinal space resulting in a retinal detachment (see Figure 3.3. for fundus

70

photographs showing before and after injection). The cannula was withdrawn

into the needle and the injector withdrawn from the eye.

 

Figure 3.3. Before and after subretinal injection in a dog’s eye. The top
image is prior to injection. In the bottom image, a retinal detachment is evident in
the tapetal fundus dorsal to the optic disc. This is the site of subretinal injection.
3.1.5. Injection Technique Used in Neonates
These injections were performed by Simon Petersen-Jones on pups prior to

eyelid opening. A small area on the upper eyelid was cleaned with diluted

betadine, and a small skin incision was then made. Blunt dissection of the area

71

was performed using tenotomy scissors until the sclera of the eye was identified.
A small incision was made through the sclera posterior to the ciliary body region
to expose the choroid. A 309 blunt cannula was introduced through the choroid
and an injection was made, On average, a 25uLvolume of vector was injected
into the eye (Figure 3.4.). The subcuticular tissues and skin incision were closed

using 6-0 vicryl (Ethicon, Piscataway, NJ) sutures.

 

Figure 3.4. Injection site in neonatal puppies. After incision into the eye
globe, the vector was injected into the region of the developing retina as is shown
by the arrow.

3.1.6. Dogs Used in Study and Sample Collection

The dogs used in this study were from a purpose-bred colony of mixed and
purebred (Cardigan Welsh Corgis and Briards) dogs at Michigan State
University. All animals were treated with care under IACUC specifications. See
Tables 3.1 and 3.2 for a list of dogs in the AAV anti-GFP study and in the anti-
AAV study, respectively. Adult dogs that underwent intraocular lentivirus

(Planelles, University of Utah) treatments included Haley, Cornet, and Jackie. All

dogs were treated with 50pL of the vector through subretinal injection.

72

One dog undenrvent intraocular treatment with 50uL of an Av-1 through subretinal

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

injection.
3 Weeks of Age Injections (0-8 week serum samples)
ID Eye
Dog Number Injected Vector Injection Type
Kumquat 8 Left AAV-CBA-GFP Subretinal
Lazarus 1 2 Left AAV-CBA-GFP Subretinal
Martha 1 3 Left AAV-CBA-GFP Subretinal*
Magdalene 1 4 Left AAV-CBA-GF P Subretinal*
Mary 1 5 Left AAV-CBA-GFP Subretinal
Andrew 1 6 Left AAV-CBA-GFP Subretinal
Moses 28 Right AAV-mOPS-GFP Subretinal
Barbie 29 Left AAV-mOPS-GFP Subretinal
3 Da 5 of Age Injections (0-8 week serum samples)
ID Eye
Dog Number Injected Vector
Jude 1 0 Right AAV-CBA-GFP Transscleral
Bartholomew 1 1 Right AAV-CBA-GFP Transscleral
Steve 26 Right AAV-mOPS-GFP Transscleral
Kelly 27 Right AAV-mOPS-GFP Transscleral
3 Weeks of Age Injections (0-4 weeks serum samples)
ID Eye
Dog Number Injected Vector
Chinook 1 Left AAV-CBA-GFP Subretinal
Hyde 2 Right AAV-CBA-GFP Subretinal
Aramis 3 Left AAV-CBA-GFP Subretinal
Frenchy 4 Right AAV-CBA-GFP Subretinal
Penny 5 Left AAV-CBA-GFP Subretinal
Shaila 6 Left AAV-mOPS—GFP Subretinal
Merry 7 Left AAV-CBA-GFP Subretinal
Lucy 1 7 Right AAV-mOPS-GFP Subretinal
Marty 1 9 Left AAV-CBA-GFP Subretinal
Tywyn 30 Right AAV-CBA-GF P Subretinal

 

 

 

 

 

Table 3.1. Colony dogs injected with AAV2/5-CBA-GFP or AAV2I5-mOPS-
GFP vectors for the GFP ELISA protocol.
Subretina|* = some vector went intravitreal.

73

 

 

 

 

Anti-AAV Dog Serum Samples for Anti-AAV ELISA
Eye Injection

Dog Number Injected Vector Type
Chinook 1 Left AAV-CBA-GFP Subretinal
Aramis 3 Left AAV-CBA-GFP Subretinal
Frenchy 4 Left AAV-CBA-GFP Subretinal
Penny 5 Left AAV-CBA-GFP Subretinal
Shaila 6 Left AAV-mOPS-GFP Subretinal
Merry 7 Left AAV-CBA-GFP Subretinal
Bartholomew 1 1 Right AAV-CBA-GFP Transscleral
Lazarus 1 2 Left AAV-CBA-GFP Subretinal
Martha 1 3 Left AAV-CBA-GF P Subretinal*
Magdalene 14 Left AAV-CBA-GFP Subretina|*
Mary 1 5 Left AAV-CBA-GFP Subretinal
Andrew 1 6 Left AAV-CBA-GF P Subretinal
Marty 19 Left AAV-CBA-GFP Subretinal
Steve 26 Right AAV-mOPS-GFP Transscleral
Kelly 27 Right AAV-mOPS-GFP Transscleral
Moses 28 Right AAV-mOPS-GFP Subretinal
Barbie 29 Left AAV-mOPS-GFP Subretinal
Tywyn 30 Right AAV-CBA-GF P Subretinal
Braedon 31 Both Both Subretinal

 

 

 

 

 

Table 3.2. Colony dogs injected with AAV2/5 vectors that were used for the
AAV ELISA protocol. Subretinal” = some vector went intravitreal.
Both aqueous and serum samples were taken from all dogs injected with the
GFP vectors. Aqueous samples were collected pre-injection on the day of
surgery and then post-injection at the time of euthanasia. Serum samples were
collected in two different groups of sampling. The first group of dogs had serum
collected on the day of injection and then every two weeks until four weeks post-
injection and at the time of euthanasia. The second group of dogs had serum
samples taken the day of injection and then every two weeks until eight weeks

post-injection and at the time of euthanasia. Several dogs had samples taken

74

past the eight week limit that were not ‘day of euthanasia’ serum samples.
Serum samples were centrifuged for 30 minutes in at 3500 rpm at room

temperature and then stored in 1 mL aliquots at -80°C.

Serum collection

 

 

\
Day 29 Y1 $ \L Ni 3 week treatment

 

 

3 day treatment

I
Birth \ Y J = 2 weeks

Serum collection

 

 

 

 

 

 

Figure 3.5. Serum and Aqueous Sampling Time Line.

3.1.7. Monitoring GFP Expression

GFP expression in the eye was monitored using a RetCam (Clarity Medical
Systems, Inc., Pleasanton, CA). Every two weeks post-injection until sacrifice, all
of the injected dog eye(s) were examined under regular white light and then
using the fluorescein angiography setting. This setting provides light of a
wavelength to cause fluorescein to fluoresce and a barrier filter in the viewing
pathway to enhance the visualization of the fluorescence. The images were
used to observe the GFP fluorescence after intraocular injection and were stored

for further assessment at a later date (Figure 3.6.).

75

 

Figure 3.6. GFP expression was monitored using a RetCam. In the top
image, a white light image of the fundus in which the retina appears normal with
a small trace of a subretinal injection scar. In the bottom image, the fluorescein

angiography capabilities of the RetCam are used to view GFP expression.

3.1.8. Data Analysis using SAS

Statistical Analysis Software (SAS) was used to analyze data sets. All data sets

(AAV-GFP, Av-1, and lentivirus) were analyzed separately.

The anti-GFP AAV-GFP serum and aqueous data sets were not normally
distributed but were still analyzed using a ‘mixed’ model analysis. The statistical
model to analyze this data set included fixed effects of injection age, injection
method, and promoter. The dog (based on ID number) was the random effect for
the model. The positive and negative control sera were used as covariates in the
analysis in order to account for individual plate variations. The least squares
mean (LSM) antibody level was reported for pre- and post-treatment, time points
collected, injection method, injection age, and promoter. All 3 week serum

samples were included in the 4 week serum sample category for this analysis.

76

The anti-AAV data set was not normally distributed but was still analyzed using a
‘mixed’ model analysis. The statistical model to analyze this data set included
fixed effects of injection age and injection method. The dog (based on ID
number) was the random effect for the model. The assay date was used to
account for plate variation. The LSM antibody level was reported for pre- and

post-treatment, time points collected, injection method, and injection age.

The anti-GFP lentivirus data set was normally distributed and was analyzed
using a ‘mixed’ model analysis. The dog (based on ID number) was the random
effect for the model. The positive and negative control sera were used as
covariates in the analysis in order to account for individual plate variations. The
LSM antibody level was reported for pre- and post-treatment and time points

collected.

The anti-GFP Av-1 data set included only one dog for analysis and thus was not

analyzed using SAS. All original values were reported.

A ‘PROC CORR’ analysis was used to compare the OD values of two data sets:

anti-AAV and anti-GFP AAV-GFP. The correlation value was reported.
A ‘PROC CORR’ analysis was used to compare the OD values of two data sets:

Anti-GFP AAV-GFP aqueous OD values and anti-GFP AAV-GFP serum 00s.

The correlation value was reported.

77

A p-value of <0.05 was considered significant for all statistical analyses

performed.

78

3.2. Results

Two ELISAs were developed in this work and used to determine if gene therapy
administered in the canine eye induced local and/or systemic antibody responses
either to the therapy gene and/or the virus vector. In this study, GFP was used
as a reporter gene, and AAV, Av-1, and lentivirus were the vectors for subretinal
and intravitreal injection methods. Thus, ELISAS developed utilize either GFP or
AAV viral capsid as antigens. The GFP ELISA was used for the samples
collected from each dog in order to detect a systemic humoral response to GFP
after treatment with AAV-GFP, Av-1, and lentivirus. The AAV ELISA was used
for the serum samples in order to detect a systemic humoral response to the

AAV viral capsid.

3.2.1. Presence of Circulating Anti-GFP Antibodies after Intraocular
Treatment with AAV-GFP

Three serum dilutions were tested using the GFP ELISA previously described

(Figure 3.7.). All dilutions followed a parallel pattern and therefore serum dilution

1:5 is reported. The least squares mean (LSM) was reported for each dilution;

therefore, a negative value may be reported for the pre-treatment sera since the

mean has been calculated using the positive and negative covariates from each

plate.

The anti-GFP antibody response was found to vary between individual dogs. A

high anti-GFP level, comparatively, was observed in 4 out of the 22 dogs tested

79

(Figure 3.8.). Overall, there was a statistically significant systemic humoral
response detected to GFP after intraocular injection of the AAV-GFP viral vectors
with a p-value of 0.0029 for the comparison of pre- versus all post-treatment time

points (Figure 3.9.).

Time points analyzed in this assay were: 0, 2, 4, 6, 8, and >8 weeks post—
injection (Figure 3.10.). Using paired t-tests, antibody levels for time points 4
weeks post—injection showed a significant difference from zero (p= 0.0409). The
results from samples collected at 6, 8, and >8 weeks post-injection were not
significant (p= 0.1038, p= .190, and p= 0.115, respectively) based on the LSM
compared to zero. Overall, the trend observed in dogs was a detectable immune
response at 4 weeks post-treatment and was found to decrease starting at 6
weeks post-treatment. One dog that was monitored long-term had an antibody

response that was maintained up to 26 weeks post-injection.

Additional independent variables included in the statistical model were: promoter
(CBA vs. mOPS) and injection age (3 days vs. 3 weeks). The animals treated
with the AAV-CBA-GFP vector had a significant antibody response while those
treated with the AAV-MOPS-GFP construct did not. Dogs treated at 3 weeks of
age had a significant anti-GFP antibody response while the group at 3 days of

age did not (see Table 3.3).

80

3.2.2. Presence of Anti-GFP Antibodies in Aqueous after Intraocular
Treatment with AAV-GFP

An aqueous dilution of 1:20 was used for assays for two reasons: 1) limited

sample volume and 2) higher aqueous concentrations did not result in an

increased antibody observation capability based on sample tn'als (not shown).

The anti-GFP antibody response was found to vary between individual dogs with
some dogs having an aqueous anti-GFP response while others did not. Overall,
there was not a significant local humoral response detected to GFP after
intraocular injection of the AAV-GFP viral vectors with a p-value of 0.0667 for the
comparison of pre- versus post-treatment samples (Figure 3.11.). Based on
euthanasia schedules, a range of 13 to 96 weeks post-injection aqueous
samples were analyzed with an equally wide range of individual antibody

responses observed (Figure 3.12.).

Two independent variables were analyzed including: promoter (CBA vs. mOPS)
and injection age (3 days or 3 weeks). Unlike the case for serum anti-GFP
antibody responses, none of these variables were shown to have a significant

effect on the local immune response in the eye (Table 3.4.).

81

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3.2.3. Correlation between Anti-GFP Antibody Responses in Serum and
Aqueous Samples
A correlation analysis (PROC CORR) was performed in SAS for the GFP ELISA
results between the serum and aqueous samples for 17 dogs (Figure 3.13.). A
correlation (R"2) of 0.30062 was observed based on this analysis with a p-value
of 0.2255; therefore, a relationship between the systemic and local antibody
responses against the GFP reporter gene was not determined from this sample
of dogs. One dog had a high level of both aqueous and circulating anti-GFP
antibodies at euthanasia (seen in Figure 3.13.). The data were analyzed with
and without this dog’s data, which was removed for the final analysis since they

were found to significantly bias the analysis.

3.2.4. Presence of Circulating Anti-AAV Antibodies after Intraocular
Treatment with AAV-6F P
A single dog out of the 19 dogs tested had a high anti-AAV OD value. The data
were analyzed both with and without this dog included, and the dog was found to
bias the data set and was therefore removed. Overall, there was a statistically
significant systemic anti-AAV antibody level after intraocular injection of the AAV-
GFP viral vectors with a p-value 0f 0.0157 for the comparison of pre- versus all
post-treatment time points (Figure 3.14). Time points analyzed were: 0, 2, and 4
weeks post-treatment (Figure 3.15). Overall, the trend observed started with a
significant immune response detectable at 2 weeks post-treatment which then

tapered off slightly at 4 weeks post-treatment.

90

Additional independent variables were included in this statistical analysis which
included: promoter and injection age (3 days vs. 3 weeks). Neither of the
variables was observed to significantly influence the anti-AAV antibody response

(Table 3.5.).

3.2.5. Correlation between Anti-AAV and Anti-GFP Antibodies in the Serum
A correlation analysis (PROC CORR) was performed in SAS for the GFP ELISA

and AAV ELISA results for serum samples at time points 0, 2, and 4 weeks post-
treatment (Figure 3.16.). A correlation (R"2) of 0.07859 was observed based on
this analysis with a p-value of 0.6390; therefore, no relationship between the

systemic anti-AAV and anti-GFP was detected in this sample of dogs.

3.2.6. Presence of Circulating Anti-GFP Antibodies after Intraocular
Treatment with Lentivirus

Overall, there was a systemic humoral response detected to GFP after

intraocular injection of the lentivirus viral vectors with a p-value of 0.0200 for the

comparison of pre- versus all post-treatment time points (Figure 3.17.).

Individual time points analyzed in this assay were: 0, 2, 4, 6, and 8 weeks post-
injection (Figure 3.18.). Using paired t-tests, antibody levels for time points 2, 4,
6, and 8 weeks post-injection showed a significant difference. Overall, the trend
observed starts with the largest peak at 2 weeks which then tapers off slowly until

8 weeks post-treatment.

91

3.2.7. Presence of Circulating Anti-GFP Antibodies after Intraocular
Treatment with Av-1
One dog was treated with Av-1 in order to study the potential systemic antibody
response to GFP. Time points analyzed in this assay were 0, 2, and 3 weeks
post-injection using a 1:20 serum dilution since the 1:5 and 1:10 semm dilution
003 were influenced by a prozone. At 0 weeks, the OD value was 0.116 while at
2 and 3 weeks post-treatment the OD values were 1.055 and 1.078, respectively.
Overall, the antibody response observed was high at 2 weeks post-treatment and
increased further at 3 weeks post-treatment (at which time the trial was

terminated).

92

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3.3. Discussion and Future Work

3.3.1. Discussion and Summary of Results

Circulatory anti-GFP and anti-AAV antibodies were observed in some but not all
of the dogs treated with the AAV-GFP vector. The single dog treated with the
Av—1 vector had a high immune response to GFP and the lentivirus treated dogs
all maintained a circulatory antibody response. Variations in antibody response
differed between vectors based on time of onset and level of antibodies detected.
The AAV-GFP vectors induced an anti-GFP antibody response approximately 4
weeks post-treatment whereas the lentivirus and Av—1 vectors induced an anti-

GFP antibody response as soon as two weeks post-treatment.

3.3.1.a. Anti-GFP Antibody Response after Treatment with AAV-GFP

As we hypothesized, an anti-GFP humoral immune response was detectable in
serum as early as 4 weeks post-treatment. This delay in development of a
humoral immune response correlates with the relatively slow onset of transgene
expression typical of AAV vectors. The anti-GFP antibody response was also
detectable up to 26 weeks post-treatment (the last time point that was collected).
An anti-GFP antibody response could be detected in post-treatment aqueous

samples as late as 96 weeks post AAV-GFP treatment.

3.3.1.b. Anti-AAV Antibody Response after Treatment with AAV-GFP

The AAV ELISA detected an anti-AAV humoral immune response in serum

samples from some of the treated dogs, and this response was first detected 2

100

weeks post-injection. The antibody response was detected two weeks sooner
than for GFP, as would be expected since the antigen is present at injection and
does not require transgene expression to be occur. The antibody response was
low compared to the anti-GFP response but was still significant over the time

points analyzed.

3.3.1.c. Discussion of Results from AAV Treated Dogs
Unlike other studies, we were able to study the antibody response to GFP in
enough detail to understand the approximate onset of the anti-GFP antibody
. response and also duration of up to 26 weeks for serum and 96 weeks for
aqueous samples. One area of interest in this study was the variation in anti-
GFP antibody responses between individual dogs. Previous studies have
observed an antibody response to GFP in both serum and aqueous post-
treatment samples after intraocular treatment of AAV-GFP (Bennett et al., 1999;
Dudus et al., 1999). In those studies, 2 out of 4 and 1 out 3 mice, respectively,
had a similar circulating high level of anti-GFP antibodies after intraocular
treatment with AAV-GFP (Bennett et al., 1999; Dudus et al., 1999). Specific for
the AAV-GFP treated dogs in this study, two independent variables in the study
were found to significantly influence the anti-GFP response: the CBA promoter
led to a greater immune response compared to the mOPS promoter and
injections given at 3 weeks of age resulted in a greater response than those

given at 3 days of age.

101

Since the eye is an immune-privileged environment, antibodies would be unable
to cross the blood-retinal barrier due to the size of the molecule; therefore, an
intraocular immune response has been explored. Recently, the iris was found to
produce a homing effect which led monocytic bone marrow cells to the iris which
then participated in a local immune response (Li et al., 2006). Furthermore, the
iris monocytic cells may participate in the systemic induction of regulatory T cells;
thus, systemic viral transduction may not need to occur to induce a systemic

humoral response.

Viral escape from the eye could potentially allow for viral systemic transduction of
host organs and tissues. Virus escape could occur through many avenues which
include escape through the drainage apparatus as well as escape through a
degraded blood-retinal barrier. Upon aqueous sampling from the eye,
breakdown of the blood-retinal barrier occur this could allow virus to escape into
the bloodstream (Strubbe and Gelatt, 1998). lntravitreal or subretinal injection
can also allow viral vectors to reach the central visual pathway. The highly
significant influence of the CBA promoter on the anti-GFP response could be
because the mOPS promoter limits expression of GFP to the photoreceptor;
thus, if the viral vector construct reached other tissues, GFP expression would
not have occurred in those tissues, whereas with the CBA promoter it would.

The CBA promoter is a ubiquitously expressed promoter whereas the mOPS

promoter is photoreceptor-specific.

102

The influence of the age of 3 weeks at the time of treatment is also fascinating
since the pup’s immune system at 3 days and 3 weeks of age are different. At 3
days of age, the pup’s immune system is less mature and is more dependent on
the passive immunity from mother’s colostrum and is in the process of
recognizing “self” (T izard, 2004). By three weeks of age, the pup is starting to
lose maternal antibodies and therefore its immune system is more able to
recognize and produce antibodies to foreign antigens. Also to consider, since
only an lgG antibody response was analyzed, genetic variability between dogs

may account as well for the individual response observed between dogs.

3.3.1.d Correlation Analyses

No correlation was observed between the anti-AAV antibody response and the
anti-GF P response after intraocular treatment of AAV-GFP. The other
correlation, between the anti-GFP antibody responses in serum and aqueous
samples, also showed no significant relationship. A small number of euthanasia
serum and aqueous samples were collected from a large range of time points

post-treatment and therefore could account for the lack of correlation observed.

3.3.1.a. Anti-GFP Antibody Response after Treatment with Lentivirus
The serum samples from dogs treated with lentivirus undenrvent analysis using
the GFP ELISA. As we hypothesized, an anti-GFP systemic humoral immune
response was detectable in serum from all dogs as early as 2 weeks post-

treatment up to 8 weeks post-treatment (the last time point at which samples

103

were collected). These results showed the highest antibody level at 2 weeks
post-treatment with a slow decrease over the 8 week time period. These results
are limited since the serum samples were collected every two weeks, and thus
an antibody response may be detectable sooner than 2 weeks post-treatment
due to fast expression of the GFP transgene. Further work should be done in
order to understand in more detail the antibody response produced against the

virus as well as the transgene.

3.3.1 .f. Anti-GFP Antibody Response after Treatment with Av-1

The serum sample from the dog treated with Av-1 underwent analysis using the
GFP ELISA. This vector results in rapid, strong GFP expression within 24 hours
post-treatment and also induces some degree intraocular inflammation. As we
hypothesized, an anti-GFP systemic humoral immune response was detectable
in serum at 2 weeks post-treatment and remained high at 3 weeks post-treatment
(the last time point at which samples were collected). More work should be done
using this vector in order to understand the antibody response in dogs and it

should also be studied over a longer period of time.

3.3.1.9. AAV-GFP, Lentivirus, and Av-1 Antibody Responses Compared
Each of the vectors induced a different anti-GFP response after intraocular
treatment (Figure 3.19.). The AAV vectors, overall, seemed to produce a low
antibody level, but this response did vary between the dogs. The lentivirus

vector produced a slightly higher antibody level in the three dogs studied, and the

104

antibody levels did seem to decrease over time. Finally, one dog was studied
after intraocular treatment of Av-1 which induced the highest antibody level.
These results are important for continued research using viral vectors for gene
addition therapy within the eye. A humoral immune response could limit the
therapeutic effectiveness over time and could also inhibit success of repeated

dosing.

3.3.2. Future Research

The AAV vector ELISA results allow for the continuation of research in several
connected areas. The potential for genotoxicity and other adverse long-term
effects such as waning of the transgene or limited therapy potential remain

important issues for the feasibility and success of ocular gene addition therapy.

. The spread of the vector within the host may be correlated with the
systemic antibody response. Real-Time PCR analysis should be
performed in order to detect the spread of the vector from the treatment
site. Many filtering organs, such as kidney, liver, and lung, as well as
tissues from along the central visual pathway, should be included in this

analysis.

. Since, from previous studies, no inflammation or decreased transgene

expression was observed upon repeat administration of an AAV vector

into the eye, tolerance may be occurring (Bennett et al., 1999; Anand et

105

al., 2000; Isenmann et al., 2001). Using a constant dose of vector for
intraocular injections, the level of GFP expression after one or several
injections could be quantified for individual photoreceptors (using a
photon-counting confocal laser scanning microscope as described in Rex
et al., 2005) and averaged together at each injection time point for
individual animals. This would allow for a more detailed analysis of the
neutralizing antibody effects on the transgene expression over time and

after repeat administration.

. As mentioned previously, an antibody response may not significantly
affect the transgene expression in the eye. Since ubiquitous promoters
are used, the concern about genotoxicity within organs remains a concern.
Further studies to observe the possible cytotoxic effect of either single or
repeated administration over time would create a more thorough analysis
of the effects of using ubiquitous promoters as opposed to tissue-specific

promoters for gene therapy.

Further work into the lentivirus and Av-1 vectors could also be done in order to

further understand the antibody response in the host after intraocular treatment.

. An ELISA could be developed to study the antibody response to the

lentivirus packaging.

106

. An ELISA could be developed to study the antibody response to the Av-1

capsid.

3.3.3. Conclusion

A variable degree of humoral immune response to the transgene and AAV capsid
was observed after intraocular injection of AAV vectors. For lentivims and
adenovirus vectors, a humoral immune response was observed after intraocular
injection. This information is imperative for the safety of intraocular gene addition
therapy trials since every patient will have a unique response to the foreign

transgene.

107

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APPENDICES
Appendix A. ELISA GFP Laboratory Protocol

MATERIALS NEEDED:
For solutions:
-N32HPO4
-KH2PO4
-KCL
-NaCl
-Tween (Polyoxyethylene- Sorbitan Monolauvate)
-gelatin-
-bicarbonate packets- from Pierce
-access to milli-Q water
-pH meter
-Probind ELISA plates

For ELISA protocol:
-GFP from Clonetech
-secondary antibody-
-HRP- anti-dog lgG
-HRP- anti-rabbit lgG
-ABTS (acts as the substrate)

PREPARATION OF EQUIPMENT AND SOLUTIONS:

Cleaning of Conboy:

Rinse conboy thoroughly with tap water five times, distilled water five times, and
with milli-Q water five times.

Preparation of PBS:

- In a 1000ml beaker:
NazHPO4 — 23g
KH2PO4 — 4g
KCI - 4g
NaCl — 1609
Added 800ml milli-Q water to mix
Added ~ 200ml to make 1L
- Add solution to large conboy
- Add milIi-Q water until 20L
- Calibrate pH meter

0 Want pH to be 7.2 - add drops of HCI or NaOH solutions until
desired pH attained ‘

000000

109

Preparation of Bicarbonate solution for dilution of GFP:
- Bicarb will last a long time in cabinet- but need to watch out for
contamination!
- Add 500mL nanopure water to one packet of powdered buffer

Preparation of GFP for coating ELISA plates:
GFP concentration (from Clonetech) = 1mg/mL = 1000ug/mL
0 Aliquoted into 20uL/ per tube -) 1 tube/ 96-well plate
To dilute GFP to coat ELISA plates:
Need 100uLper well at a concentration of 1pg/mL
20uLof GFP and add Bicarb to a total volume of 10,000uL(10mL)
-Only thaw and dilute GFP to be used that day
-Do NOT pre-dilute GFP

Control serum samples
Dog serum was tested for the best results on varying concentrations of GFP.
The serum from 8/25 was combined from both dogs (either + or - control dogs) in
a 1:1 mixture and then diluted to 1:25 using wash buffer. The mixture and 1:25
control dilutions were then stored in the freezer.

+ control = Spruce and Pluto

- control = Mica and Snap

WASH/ DILUENT solution = 0.05% PBS-T

10L BULK-add 5mL of Tween-20 to 10L of PBS
1 500mL bottle- add .25mL Tween to 500 mL bottle of PBS
ELISA Procedure:
1- Coating Plate with GFP: DO THIS 15 HOURS BEFORE OTHER
STEPS!
a. 100pl/well of GFP- bicarbonate solution
b. Place clear, adhesive coverslip on plate
c. Incubate overnight at 4°C (in fridge) on a flat surface, do not tip
plate
2- Wash Plate:
a. Following ovemight incubation at 4°C, dump solution in wells and
wash plate with PBS-T
i. Pipette 200ul/well of PBS-T — repeat this for a total of 3
rinses- empty wells of solution
3- “Whack” Plate:
a. Slam plate upside down onto a stack of paper-towels until paper-
towels do not have any moisture on them (plate is essentially dry)
4- Serum Dilutions:
a. During 1 hour block, prepare serum dilutions:
i. Using a 96 round bottom well plate, pipette 115p|lwell for
doubling dilutions into second thru last rows.
ii. Pipette 230ul/well of the starting dilution of serum in the first
row of wells.

110

iii. Pipette 115ul out of the first row and pipette this into the
second row, remembering to mix well before pipetting 115p|
into the next row.

iv. Continue in this manner to the end of the plate.

v. From the last row, pipette 115ul out and discard so that all
rows end up with 115p| volume.

"Start with 1 :5 dilution“
230pLin first well: 46uLserum and 184 prash buffer

. If using pooled normal dog serum for a negative control, dilution used is
1:25. Use wash buffer to dilute.
o If using pooled hyperimmune serum, dilution used is 1:25. Use wash
buffer to dilute.
. If using rabbit Hyperimmune serum (rabbit anti-GFP from abcam), dilution
used is: 1:2500. Use PBS-T to dilute.
o If using normal rabbit serum for a negative control, dilution used is 1:
2,500. Use PBS-T to dilute.
5- Add Serum Dilutions:
a. Pipette 100pl of serum dilution from round well plate into
corresponding well of ELISA plate
6- Incubate:
a. Leave for 1 hour at room temperature with vigorous shaking
(setting #5 on shaker)
7- Preparation of Secondary Antibodies :
Prepare secondary detection antibodies while plate is incubating with
serum samples

HRP-Anti-Dog:
o 100uI/well
0 A dilution of 1/20,000 is used- use PBS-T to dilute
0 1 uU20ml PBS-T
HRP-Anti-Rabbit:
. 100ul/well
0 A dilution of 1/20,000 is used- use was buffer to
dilute.
0 1 uL/20ml wash buffer.
8- Wash and ‘Whack’ plate:
a. Following incubation, wash plate with 200ul/well of PBS-T (3X) and
‘wack’ dry on a stack of paper towels
9- Addition of secondary detection antibody:
a. Add 100ullwell of the appropriate (dog or rabbit) HRP-conjugated
secondary detection antibody
10- Incubation:
a. Incubate plate with secondary antibodies for 1 hour at room
temperature with vigorous shaking (setting #5 on shaker)

111

11- Wash and ‘Whack’ plate:
a. Following incubation, wash plate with 200ul/well of PBS-T (3X) and
‘wack’ dry on a stack of paper towels.
12-Addition of ABTS:
a. Add 125uI/well 1-step ABTS and incubate for 1 hour at 37°C
without shaking
13-Use Plate Reader to determine absorbance (set filter at 405nm and
450nm)

Storage of antibodies/substrates etc:

Goat Anti-Rabbit lgG (H+L)
a. Caltag - L42007
b. Store at 2-8 °C, dilute only what is needed at a time
HRP-Anti-Dog lgG (H+L)
c. Bethyl — A40-123P
cl. Store at 2-8 °C
Rabbit Anti-GFP (Rabbit Polyclonal to GFP)
e. Store at 4 °C short term
f. Long term: -20 or -80°C - add 1:1 glycerol for stability
i. Discussed with Dr. Burton —at 4 °C
GFP - 1 mglmL
g. Clontech #632373
h. Aliquots of 20u| at -80 °C
1-STEP ABTS for ELISA from Pierce Biotechnology
i. Store at 4 °C
Serum samples from Subretinal Injection Dogs and Positive and
Negative Control from lmmunized Dogs:
j. Stored at -80 °C
- Plates- BD Falcon 353279 sold through VWR

112

Appendix B. Anti-AAV ELISA Protocol and Summary of References

MATERIALS NEEDED:
For solutions:
-Na2HPO4
-KH2PO4
-KCL
-NaCl
-Tween (Polyoxyethylene- Sorbitan Monolauvate)
-access to milIi-Q water
-pH meter
-Sterile saline
-Probind ELISA plates from VWR

For ELISA protocol:
-recombinant UF11 virus from Hauswirth Lab, FL
-secondary antibody-
-HRP— anti-dog lgG
-ABTS (acts as the substrate)

PREPARATION OF EQUIPMENT AND SOLUTIONS:
Cleaning of Conboy:
Rinse conboy thoroughly with tap water five times, distilled water five times, and
with milIi-Q water five times.
Preparation of PBS:

- In a 1000m| beaker:
NazHPO4 — 23g
KHzPO4 - 4g
KCI — 4g
NaCl - 160g
Added 800ml milli-Q water to mix
Added ~ 200ml to make 1L
- Add solution to large conboy
- Add milli-Q water until 20L
- Calibrate pH meter

0 Want pH to be 7.2 — add drops of HCI or NaOH solutions until
desired pH attained

OOOOOO

Virus concentration math-) 100.000 vplml Er 100uLIweII
Use saline for dilutions
5.6E+13 -) 1:1000 = 5.6E+10 -) 1:1000 = 5.6E+7
[Virus] dilute dilute
“vortex between dilutions“

(5.6E+7) X = (4.5ml)x(100,000vp/ml)
X= 8.03 vairus in 4.5 ml saline solution

113

Add 100prer each well used for Anti-AAV ELISA

WASH/ DILUENT solution = 0.05% PBS-T

BULK-add 5mL of Tween-20 to 10L of PBS
1 500mL bottle at a time- add .25mL Tween to 500 mL bottle of PBS
ELISA Procedure:
1 Coating Plate with virus: DO THIS 15 HOURS BEFORE OTHER
STEPS!
a. 100uI/well of virus-saline solution
b. Place clear, adhesive coverslip on plate
c. Incubate overnight at 4°C (in fridge) on a flat surface, do not tip
plate
Wash Plate:
a. Following ovemight incubation at 4°C, pour off solution from wells
and wash plate with PBS-T
i. Pipette 200pI/well of PBS-T - repeat this for a total of 3
rinses- empty wells of solution
“Whack” Plate:
a. Slam plate upside down onto a stack of paper-towels until paper-
towels do not have any moisture on them (plate is essentially dry)
4- Serum Dilutions:
a. During 1 hour block, prepare serum dilutions- use 1:5 dilutions for
all samples:
i. 115/5= 23 uL
ii. 23pLserum and 92uLwash buffer per well in round bottom
plate
iii. Aliquot 100uLinto each well

2

w
I

Control Dog Serum
- If using pooled normal dog serum for a negative control, dilution used is
1:25. Use wash buffer to dilute.
. If using pooled hyperimmune serum, dilution used is 1:25. Use wash
buffer to dilute.

5- Add Serum Dilutions:
a. Pipette 100u| of serum from round well plate into corresponding
well of ELISA plate
6- Incubate:
a. Leave for 1 hour at room temperature with vigorous shaking
(setting #5 on shaker)
7- Preparation of Secondary Antibodies :
Prepare secondary detection antibodies while plate is incubating with
serum samples

114

HRP-Anti-Dog:
. 100pllwe||
o A dilution of 1/20,000 is used- use PBS-T to dilute
o 1 W 20ml PBS-T

8- Wash and ‘Whack’ plate:
a. Following incubation, wash plate with 200pI/well of PBS-T (3X) and
‘wack’ dry on a stack of paper towels
9- Addition of secondary detection antibody:
a. Add 100uI/well of the appropriate HRP—conjugated secondary
detection antibody
10- Incubation:
a. Incubate plate with secondary antibodies for 1 hour at room
temperature with vigorous shaking (setting #5 on shaker)
11-Wash and ‘Whack’ plate:
a. Following incubation, wash plate with 200uI/well of PBS-T (3X) and
‘wack’ dry on a stack of paper towels.
12-Addition of ABTS:
a. Add 125ullwell 1-step ABTS and incubate for 1 hour at 37°C
without shaking
13-Use Plate Reader to determine absorbance (set filter at 405nm and
450nm)

115

Appendix C. AAV2I5-GFP Vector Construction

AAV2/5 Plasmid Construction

The AAV2 vector pseudotyped with the capsid from AAV5 was produced in the
laboratory of Dr. Hauswirth at the University of Florida. An adenovirus helper

plasmid was used to pseudotype the vector with the AAV5 capsid.

Adenovirus Helper Plasmid Production

The creation of the adenovirus helper plasmid with AAV5 capsid was performed
in a series of steps. The open reading frame (ORF) of AAV5 was amplified with
PCR and was then subcloned into the plasmid pACGZ resulting in the hybrid
plasmid pACG2R5C which contains the ORF for the AAV2 Rep proteins and the
ORF for the AAV5 capsid proteins. A fragment from the pACGZRSC plasmid
was subcloned into the pXYZ adenovirus helper plasmid to create the pXYZS

plasmid complete with the AAV5 capsid gene (Zolotukhin et al., 2002).

AAV Vector Plasmid Production

The AAV vector plasmid was constructed using the pTR-UF backbone and
consists of AAV2 flanking inverted terminal repeat (ITR) regions (Zolotukhin et
al., 2002). Two different AAV vector plasmids were used in this study to create
two different AAV2/5 vectors. The first AAV vector consisted of the chicken beta
actin promoter, CMV enhancer regions, and GFP transgene. The second AAV
vector consisted of the mouse opsin promoter and GFP transgene (Glushakova

etaL,2006)

116

Promoters

Chicken Beta Actin (CBA)

The CBA promoter is a ubiquitous promoter that allows a constant and powerful

expression of the transgene. This promoter is active in any host cell and thus

transcribes the transgene in any cell of the body.

 

 

1
51
101
151
201
251

catggtcgag
cctccccacc
gcgatggggg
9990939999
agagcggcgc
cggccctata

gtgagcccca
cccaattttg

ngggggggg

cggggcgggg
gctccgaaag

aaaagcgaag

cgttctgctt
tatttattta
gggggggcgc
cgaggcggag
tttcctttta
cgcgcggcgg

cactctcccc atctcccccc
ttttttaatt attttgtgca
gcgccaggcg gggcggggcg
aggtgcggcg gcagccaatc
tggcgaggcg gcggcggcgg

9C9

 

Figure A. The CBA promoter sequence (provided by the Hauswirth Lab). This
sequence was Blasted (RID: 1171314962-8045-14060912295.BLASTQ3,

2/12/07) with hits found only in the chicken genome.

Mouse Opsin (mOPSSOO)

The mOP8500 promoter is a tissue-specific promoter present in some of the

AAV vectors that was characterized by the Hauswirth laboratory (Glushakova et

al., 2006). The promoter was shown to be specific for photoreceptors and thus

the likelihood that the transgene would be expressed outside the eye is low.

 

 

1
51
101
151
201
251
301
351
401
451

cgtcaagtga
tcctgggaag
gagccattgt
tgaggccacc
gacaagatga
gtcaccttgg
attagcgtta
9939393399
gcgctgtggg
gtctacgaag

gccattgtca
agatgggata
ggagaagtga
agactgacat
gacacccttt
cccctctgca
gtatgatatc
tcactttata
agccgtcagt
agcccgtggg

gggcttgggg
ggtgagttca
atttagggcc
ggggaggaaa
cctttcttta
agccaattag
tcgcggatgc
agggtctggg
ggctgagctc
gcagcct

actggataag
ggaggagaca
caagggttcc
tcccagagga
cctaagggcc

gccccggtgg
tgaatcagcc

gggggtcagt
gccaagcagc

tcagggggtc
ttgtcaactg

agtcgcagcc
ctctggggca
tccacccgat
cagcagtggg
tctggcttag
gcctggagtt
cttggtctct

 

 

 

Figure B. The mOPS500 promoter sequence (provided by the Hauswirth Lab).
This sequence was blasted (RID: 1171314867-26111-185748596015.BLASTQ3,
2/12/07) with hits only found in the mouse genome on chromosome 6.

117

The Green Fluorescent Protein (GFP) Transgene

The GFP transgene is a reporter gene commonly used to determine the efficacy

of the gene transfer event. GFP originates from the jellyfish, Aequorea Victoria,

and fluoresces a bright green color when under a UV or blue light and has been

shown to be effective in many species and systems.

 

 

1
51
101
151
201
251
301
351
401
451
501
551
601
651
701

caccatgagc
tggaactgga
ggtgaaggtg
cactggaaag
atggcgtgca
tttttcaaga
tttcaaagat
gtgacaccct
gatggaaaca
tgtgtacatc
agatcagaca
caacagaaca
ttacctgtcc
accacatggt
atggacgagc

aagggcgagg
tggcgatgtg
atgccacata
ctccctgtgc
gtgcttttcc
gcgccatgcc
gacgggaact
ggtgaataga
ttctcggcca
atggccgaca
caacattgag
ctccaatcgg
acccagtctg
cctgctggag
tgtacaagtg

aactgttcac
aatgggcaca
cggaaagctc
catggccaac
agatacccag
cgagggctat
acaagacccg
atcgagctga
caagctggaa
agcaaaagaa
gatggatccg
cgacggccct
ccctgtctaa
tttgtgaccg
agc

tggcgtggtc
aattttctgt
accctgaaat
actggtcact
accatatgaa
gtgcaggaga
cgctgaagtc
agggcattga
tacaactata
tggcatcaag
tgcagctggc
gtgctcctcc
agatcccaac
ctgctgggat

ccaattctcg
cagcggagag
tcatctgcac
accctgacct
gcagcatgac
gaaccatctt
aagttcgaag
ctttaaggag
actcccacaa
gtcaacttca
cgaccattat
cagacaacca
gaaaagagag
cacacatggc

 

Figure C. The GFP cDNA sequence used in the AAV viral vector constructs
(provided by the Hauswirth Lab).

Protocol for AAV2/5 vector production

Cell transfection

293 cells (ATCC, Manassas, VA) were used for the transfection with the above

plasmid constructs. The cells were cultured with Dulbecco’s modified Eagle’s

medium (DMEM) which was supplemented with 5% fetal bovine serum (FBS).

Cells were split 1:3 one day prior to transfection and were allowed to reach ~75-

80% cell confluency. The plasmid is CsCI-purified and was then transfected

using a CaPO4 precipitate. 1.8mg of pXY25 and 0.6mg of AAV vector plasmid in

a total volume of 50mL of 0.25M CaClz followed by the addition of 50mL of 2x

118

 

HBS. pH7.05, to the DNA/CaClz (Zolotukhin et al., 2002). After 1-2 minutes of

room temperature incubation, 1100mL of warm DMEM with 5% PBS was added

   

 

 

 

 

    
   

lPstI(1)
' SrmI(87)
,TR
./
f1(+) origin , //Srm1(98)
\wmi Id <~\.;\\” AEnRI(171)
[5"‘5‘A'_I:’W;;»..::L ’- ' \\\.L\\¥\
54"” > Iqm [(181)
._ k .
/ >4 CMV Ie enhancer
\\ Chiken b-actin promoter
ApR/-, \ Exon1
\\
,5 ‘r, Intmn
r2 pTR-UF11 \
Q L I — \\\\\\ XbaI(1798)
W 72oobp ._
NKEDRI(1894)
PS? I (4370) - ,'- , i' ‘ "NotI (1922)
81111101280) \r: GFPh
TR‘ . ~’ ‘Bamm(2455)
SnuI(4269) // ~. . \ \.\ NotI(2654)
bGH po|y(A) \ z i, \ BamI-II(2663)
SaII(3960) ‘1‘ \ swo poly(A)
BamHI(3954) 1, \ .\ S011 (2861)
1 ‘ ‘1, \
‘._ i]: PYF441 enhancer
1‘ “\ EDRI(3044)
i ' ' HSV-tk
I “PstI(3141)
‘PstI(3338)

neoR

Figure D. The pTR-UF11 plasmid with CMV enhancer, CBA promoter, and
GFP transgene (Vince Chiodo, Hauswirth Lab, University of Florida).

119

The medium was removed from the cells and then the plasmid solution was
added to the cells and allowed to incubate for 60h at 37°C at 5% C02. Cells
were then washed with PBS and then harvested. The cells then undenNent
centrifugation for 10 minutes at 10009, were resuspended in 60mL of lysis
solution (150mM NaCl, 50mM Tris, pH 8.4), and then stored at -20°C (Zolotukhin

etaL,2002)

AAV2/5 Purification

Purification of the AAV5 vector occurred through a series of steps: cell lysis,
iodixanol gradients, and column chromatography. Cells were lysed using a
freeze/thaw method that involved dry ice in ethanol and a 37°C water bath.
Benzonase (Sigma, St. Louis, MO) was then added to the cell Iysate and
incubated for 30 minutes (Zolotukhin et al., 2002). The lysate was then
centrifuged for 30 minutes at 40009 in order to clarify the crude Iysate. The

supernatant was then divided 4 ways into iodixanol gradients.

Iodixanol gradients are created by using quick-seal tubes (25 x 89 mm,
Beckman) and iodixanol (5,5’-[(2-hydroxy-1,3-propanedin)bisacetyI-amino)]
bis[N,N’-bis (2,3dihydroxypropyI-2,4,6-triiodo-1,3-benzenecarboxamide] using a
60% sterile solution of OptiPrep (Nycomed) and PBS-MK buffer (1 x PBS
containing 1mM MgCIz and 2.5mM KCI). The cell lysate solution was added then

centrifuged for 1 hour at 18C at 69,000rpm (Zolotukhin et al., 2002). 5mL of the

120

60-40% step interface was aspirated with an 189 needle, and the bands of the
gradient were stored either in the freezer or refrigerator (Zolotukhin et al., 2002).
Column chromatography of the iodixanol gradient further purifies the vector. A 4-
mL HiTrap Q column (Pharmacia) was used. First, the column was equilibrated
at 5mL/min with 5 column volumes of Buffer A (20mM Tris, 15mM NaCl, pH 8.5)
followed by 25mL of Buffer A using the Pharrnacia ATKA FPLC system
(Zolotukhin et al., 2002). The iodixanol fraction was diluted 1:1 with Buffer A and
then added to the column at a rate of 3-5 ml/minute. The column was then
washed with 10 column volumes (50mL) of Buffer A. The vector was eluted with

Buffer B (Zolotukhin et al., 2002).

AAV2/5 Concentration

The vector was concentrated through a three cycle-centrifugation method
involving a Biomax 100 K concentrator (Millipore, Bedford, MA). The virus is
concentrated to 1 mL and is then stored at -8OC in 1 x PBS (Zolotukhin et al.,

2002)

AAV2/5 Quality Control Assays

Quality control measures are taken using an assay to determine the protein
purity of the AAV2/5, another assay for the infectiousness, and then a dot-blot
assay for the physical particle titer (Zolotukhin et al., 2002). The protein purity of
the AAV2/5 vector was determined by running a 10% SDS-polyacrylamide gel

with silver staining. A western blot was then done using an anti-AAV2 capsid

121

monoclonal antibody B1 (American Research Products) that recognizes AAV5
capsids at a dilution of 1:2000. A horseradish peroxidase-conjugated sheep anti-
mouse (Amersham) was used for detection at a dilution of 1:5000 and Super

Signal (Pierce, Rockford, IL) was used as the substrate (Zolotukhin et al., 2002).

The infectious center assay (ICA) was used to determine the infectiousness of
the vector. 96-well plates were coated with 2 x 104 C12 cells and infected 16
hours after coating using 10-fold dilutions of the AAV and also infected with
adenovirus 5 at a multiplicity of infection of 10 (Zolotukhin et al., 2002). The C12
cells were then harvested and suspended in 5 mL of 1 x PBS. The cells are then
vacuum filtered onto nylon membranes (0.45pm) which were then transferred to
filter paper moistened with 0.5 N NaOH/1.5 M NaCl and 1M Tris-Cl. The filter
paper was then probed for the transgene. Titer was calculated by using the
dilution factor from the infectious centers (spots) observed on the filter paper,

accurate in the range of 10-200 spots (Zolotukhin et al., 2002).

Another assay was used to determine the infectiousness of the AAV2/5 vectors
that have the GFP transgene. The single-cell fluorescence assay (SCFA)
determined the vector titer based on the expression of GFP. 2 x 104 293 or 012
cells are seeded into a 96 well plate. Serial dilutions of the AAV2/5 and Ad5
(MOI of 10) were added to wells. After thirty hours, cells infected with the GFP
vector were visually scored using a fluorescent microscope. Titer is calculated

based on the dilution factor (Zolotukhin et al., 2002).

122

The last quality assurance assay used is the dot-blot assay. This assay
determines the number of vectors with a viral genome. DNase I (Roche) was
used to digest all plasmid and unpackaged vector DNA for 1 hour at 37°C in a
volume of 200pL containing 5U of the enzyme, 10mM Tris-Cl, pH 7.5, and mM
MgCIz. Then, in order to free the viral genomes from the capsids, an equal
volume of proteinase K buffer (20mM Tris-Cl, pH 8.0, 20mM EDTA, pH 8.0, 1%
SDS) was added as well as proteinase K (30ug). This solution was incubated for
1 hour at 37°C. The DNA was then extracted using phenol and precipitated
using ethanol and a glycogen carrier (Zolotukhin et al., 2002). DNA was then
dissolved in 40pL of distilled water. 10pL of this was diluted in 400pL 0.4 N
NaOH/10mM EDTA. A twofold dilution series was prepared from the plasmid
DNA as above. The viral DNA was immobilized onto a nylon membrane. The
plasmid DNA was also immobilized onto the membrane using a dot-blot
apparatus (Bio-Rad, Hercules, CA). The membrane was then probed for the
transgene and exposed to film. Comparing the plasmid DNA standard curve to

the vector DNA allows for extrapolation of the viral titer.

123

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