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A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells

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A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells

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A REVIEW OF MOLECULAR CONJUGATES AND THEIR USE IN
GENE THERAPY WITH THE PRESENTATION OF A MODEL
EXPERIMENT: GENE THERAPY WITH NOVEL FUSION PROTEINS
THAT TARGET BREAST CANCER CELLS
by
Elizabeth Felix-Trunnelle
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
December 1999
COPYRIGHT 1999. Elizabeth Felix-Trunnelle
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UMI Number: 1409628
Copyright 1999 by
Felix-Trunnelle, Elizabeth
All rights reserved.
__ _I ®
UMI
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UNIVERSITY O F SOUTHERN CAUFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 10007
This thesis, written by
Elizabeth Felix-Trunnelle
under the direction of h.51..— Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
Master of Science
Da/r„„.November„22^9 9 9
f
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Dedication
I dedicate this thesis to my wonderful, caring,
and compassionate husband, Gordon, who has been very
supportive throughout my graduate studies. We began
this journey with 2,200 miles between us, and we
endured many challenges. His presence, whether physical
or spiritual, was always comforting and made this
accomplishment possible.
Also, I am eternally grateful for my wonderful
family and I would also like to dedicate this thesis to
my parents Rafael and Maria, and my brothers Rafael and
Gabriel. Their companion, motivation, and inspiration
throughout the years have made this accomplishment
possible.
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Acknowledgements
I would like to thank Dr. Lali Medina-Kauwe for her
resourcefulness and guidance with my research. I would also
like to thank Dr. Noriyuki Kasahara for his patience,
attentiveness, and willingness to guide me with the process
of writing this thesis. And last but not least, Dr. Zoltan
Tokes you have been very inspirational throughout my
graduate studies and I am grateful.
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iii
Table of Contents
Dedication ii
Acknowledgments iii
List o f Figures and Tables v
Abstract vi
Chapter 1 A General Overview of Gene Therapy 1
Introduction I
Somatic Gene Therapy 3
Viral and Non-Viral Vectors 4
Chapter 2 Molecular Conjugates 12
The Structure of Molecular Conjugates 12
Gene Transfer With Molecular Conjugates 14
Studies of Successful Gene Transfer With Molecular Conjugates 14
Drawbacks of Poly-L-Lysine Molecular Conjugates
for In Vivo Gene Transfer 15
The Advantages of Using Molecular Conjugates
for Gene Therapy 18
The Disadvantages of Using Molecular Conjugates
for Gene Therapy 19
Chapter 3 Endosomal Escape: A Viral Function Mimicked in Molecular
Conjugates for Increased Gene Transfer Efficacy
Review of Endocytosis
Viral Infection and Endosomal Escape
The Use of Fusogenic Peptides in Molecular Conjugates
to Increase Gene Transfer Efficacy
Other Methods for Endosomal Escape
Chapter 4 A Model Experiment: Treatment of Breast Cancer
With a Novel Targeted Fusion Protein 33
Introduction 33
Background 34
Materials and Methods 38
Results 41
Discussion 43
References 59
23
23
24
25
28
IV
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List of Figures and Tables
Chapter 1
Table 1 . Gene Therapy Approved Clinical Trials in the USA
as of February 3, 1998 8
Figure 1 . Ex Vivo Gene Transfer 9
Figure 2. In Vivo Gene Transfer 10
Table 2. The Characteristics of Viral and Non-Viral Delivery Vehicles 1 1
Chapter 2
Figure 3. The Structure of Molecular Conjugates 21
Table 3. Molecular Conjugates Used for Gene Transfer and Reference 22
Chapter 3
Figure 4. An Illustration of the Endocytosis Pathway 30
Figure 5. Viral Infection of Cells Via the Receptor-Mediated
Endocytosis Pathway 31
Figure 6. A Picture of Lipid/DNA Conjugates Used With Vaccinia
Virus for Gene Delivery 32
Chapter 4
Figure 7. A Schematic of Breast Cancer Targeting With the Novel
Fusion Protein and Delivery of the HSTK Gene 47
Figure 8. The Structure of Protamine 48
Figure 9. A Depiction of the Novel Fusion Protein in Two
Orientations 49
Figure 10. The Structure of pBS. 50
Figure 11. The Structure of pRSETa Plasmid
51
Figure 12. The Nucleic Acid and Amino Acid Sequence of Protamine 52
Figure 13. The Nucleic Acid and Amino Acid Sequence of GALA 53
Figure 14. A Diagram of the Cloning Strategy for the Construction of
the Novel Fusion Protein Gene Sequence in the N-Terminus
to the C-Terminus Orientation 54
Figure 15. The pRSETa-GALA-Her Confirmatory 55
Electrophoretic Gel
Figure 16. The pBS-GALA-Her Confirmatory 56
Electrophoretic Gel
Figure 17. A Diagram of the Cloning Strategy With 3’ Pstl- Protamine 57
Figure 18. The Electrophoretic Gels of the Negative pRSETa-
Pro-GALA-Her Confirmatory Digests 58
V
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A bstract
In this thesis a general review of therapeutic
gene therapy is presented with a focus on molecular
conjugates. Detailed information about advancements in
the development of molecular conjugates is provided.
The three main elements of effective therapeutic gene
therapy with molecular conjugates, as with any other
delivery vehicle, are; (1) gene transfer (transgene
delivery), (2)efficacy of the transgene, and (3)
safety. These three elements of molecular conjugate
delivery vehicles are discussed in detail in this
review. A model experiment for treatment of breast
cancer with novel fusion proteins(molecular conjugates)
is also outlined in this review.
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CHAPTER 1
A General Overview of Gene Therapy
Introduction
Since the approval of the first clinical trial for
somatic gene therapy in 1990, the field has exploded
and has demonstrated a promising future that is sure to
revolutionize all branches of medicine. The initial
gene therapy protocol was used for the treatment of a
metabolic disorder, which involves a defective gene
that produces insufficient amounts of the enzyme
adenosine deaminase (ADA) (Blaese et al., 1995).
Afflicted individuals suffer from severe combined
immunodeficiency (SCID), which shortens their life span
because of a weakened immune system that fails to
defend against thriving infections that occur early in
life. This clinical trial was just the beginning for
I
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gene therapy, and just approximately nine years later,
there are numerous ongoing clinical trials that
encompass a variety of diseases (Table 1) (Anderson,
1998).
The potential applications of gene therapy are
vast and include genetic, acquired and multifactorial
diseases. Unfortunately therapeutic gene therapy has
not yet been successful as was anticipated, however it
is still in its early development and the promise
remains. Advancements in the understanding of
molecular biology and the pathophysiology of cells and
tissue will aid in developing therapeutic gene therapy
protocols. Therapeutic gene therapy seems certain to
revolutionize medicine in the new millennium. In the
near future, perhaps even within the next decade,
therapeutic gene therapy will be able to treat
detrimental diseases such as cancer (glioblastoma,
melanoma, breast, ovarian, lung, esophageal, leukemias,
hepatoma, colon, and prostate), AIDS, cystic fibrosis,
Duchenne muscular dystrophy, familial
hypercholesterolemia, diabetes mellitus, epilepsy, and
arthritis. This list by no means entails all of the
diseases that have the potential to be treated by gene
therapy, and therefore demonstrates how powerful this
new medicine is going to be when perfected. Due to its
2
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promising future, therapeutic gene therapy
understandably has captured the attention of many
scientists, researchers, and biotechnology companies.
Somatic Gene Therapy
Therapeutic gene therapy can be applied to somatic
cells, in utero somatic cells (fetal gene therapy), and
germ line cells (zygote and embryo). Fetal and germ
line gene therapy have posed many ethical questions,
issues, and concerns thereby making them a
controversial matter (Fletcher et al., 1996). Fetal
and germ line gene therapy are not within the scope of
this paper, and in the context of this paper
therapeutic gene therapy refers only to somatic cell
gene therapy.
Somatic gene therapy in its simplest definition is
the introduction of genetic material (DNA) in the form
of genes (transgene) to a patient's cells with the
intent of the transgene to eradicate or alleviate a
disease. Strategies in therapeutic gene therapy differ
in genetic diseases and non-genetic diseases. In
genetic diseases the goal is to replace the poorly- or
non-functioning gene with the functioning transgene.
In non-genetic diseases, like cancer for example, the
goal is to deliver a transgene with cytotoxic effects.
3
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The concept involved in therapeutic gene therapy, in
which a transgene is delivered to a cell, can be
approached in many ways.
Effective therapeutic gene therapy involves three
main components: (a) gene transfer (delivery of
transgene), (b) efficacy of the transgene (gene
expression level and effects of the expressed gene) ,
and (c) safety.
Gene transfer is accomplished by ex vivo or in
vivo techniques. In ex vivo gene transfer(Figure 1) .
target cells are surgically removed and gene transfer
occurs in a cell culture and these altered cells are
subsequently re-implanted into the body. Gene transfer
in vivo (Figure 2) is accomplished by the direct
delivery of genes in situ. Both of these procedures can
involve one of many molecular delivery strategies.
Viral and Non-Viral Vectors
DNA delivery vehicles for gene therapy can be
categorized into two general groups: viral and non-
viral vectors. In viral vectors the therapeutic genes
are built right into the genetic repertoire of the
virus itself. Such viral vectors include retroviruses,
adenoviruses, and adeno-associated virus (AAV). A
retrovirus was used for gene delivery ex vivo in the
4
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first clinical trial of gene therapy, which involved
two SCID patients mentioned previously(Blaese et al.,
1995). Viral vectors are still the most common DNA
delivery vehicle and account for about 80% of the
vectors that are employed in ongoing clinical trials
(Wivel et al., 1998). About 60% of approved gene
therapy clinical protocols use retroviral vectors, 10%
use adenoviral vectors, 10% use other viral vectors,
and 20% use non-viral delivery systems (Anderson,
1998) .
Non-viral DNA delivery vehicles include either
naked plasmid DNA, or DNA bound to complex
macromolecules that aid in delivery such as lipid/DNA
complexes, solid particles, polymers, and molecular
conjugates (including protein-DNA conjugates and
peptide-based gene delivery systems). There are many
ongoing clinical trials that use non-viral delivery
systems for gene therapy. Other non-viral delivery
vehicles for gene transfer have been explored and
include hybrid/synthetic vectors, artificial
chromosomes, and transposon elements.
The numerous delivery vehicles available for gene
therapy have different characteristics, advantages and
disadvantages. Properties such as transgene size,
titers, immune response, and integration vary among
5
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delivery vehicles (Table 2). The size of the
therapeutic gene that can be delivered by viral and
non-viral vectors, is limited by the transgene size
capacity of each vector. Titers are a number that
represents the infectious units of viruses per
milliliter, and differs among viruses. Some delivery
vehicles such as adenoviruses and the herpes simplex
virus induce an immune response while others such as
retroviruses and DNA-protein conjugates do not. An
immune response is not favored because it renders gene
therapy ineffective. Integration of the transgene into
the host DNA occurs with some viral vectors such as
retroviruses. Even though transgene integration allows
for permanent expression, it also poses many risks such
as insertional oncogenesis and mutagenesis. The
various delivery vehicles encounter different physical,
cellular, and molecular limitations, and therefore
transgene efficacy in gene therapy varies. Safety
concerns also differ among the various delivery
vehicles and the greatest safety issue involves the use
of viruses. These issues of the various delivery
vehicles will not be discussed any further because the
focus of this thesis will be on non-viral molecular
conjugate delivery systems.
6
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Currently there are major shortcomings associated
with the use of molecular conjugates for gene therapy.
Many gene therapy studies that have been performed with
molecular conjugates in vitro have produced highly
promising results, but unfortunately this has not
proven true in vivo. Gene expression is found to occur
at low levels in studies that use molecular conjugates
for in vivo gene therapy. The quest for developing
effective molecular conjugates is ongoing, and their
development is in its infancy. Detailed information
about the delivery, efficacy, and safety of molecular
conjugates will be covered in chapters 2 and 3. In
chapter 4 a model experiment will be presented.
In the model experiment a novel fusion protein
(molecular conjugate) was constructed to target breast
cancer cells. The novel fusion protein was designed to
eliminate or diminish the current problems that are
associated with the use of molecular conjugates in
vivo.
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Table 1. Gene Therapy Approved Clinical Trials
in the USA as of February 3,1998.
Percentage of
Disease Targets Number Total
Cancer 138 69%
Genetic Disease 33 16.5%
Cystic Fibrosis 16 -
Monogenic Diseases 12 -
Other 5 -
AIDS 23 11.5%
Others* 6 3%
Total 200 100%
'Disease targets are peripheral artery disease, rheumatoid arthritis, arterial
rcstcnosis, cubital tunnel syndrome , and coronary artery disease.
NOTE The total number o f approved gene therapy protocols is 232. The other 32
protocols are non-thcrapeutic (2) and Marker (30).
oe
Figure 1. E x Vivo Gene Transfer
Surgical acquisition
of target cells
Re-implantation
into body
Gene transfer in cell culture
9
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Figure 2. In Vivo Gene Transfer
In vivo gene transfer makes use of natural tropism.
Localized delivery of therapeutic genes is done in situ.
10
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Table 2. The Characteristics of Viral and Non-Viral Delivery Vehicles (Wivei and Wilson, 1998)
Transgene Titers Immune
Vectors Size (kb) (lU/mL) Response Integration_________Advantages_____ Disadvantages
Viral
Retrovirus
Adenovirus
7-10
7-10
10M01
1 0 '
no
yes
yes
no
-easy to produce
-packaging cell lines available
-efficiait transfer in dividing cells only
-efficient transfer in both dividing and
non-dividing cells
-random integration into host cell
-risk o f replication
•in non-dividing cells N1LV shows no
transduction
-transits)! expression
-risk o f replication
-titers are low
Adeno-
associatcd
virus
Herpes
simplex
virus
1 0
30
10
lO M o8 yes
no
yCS (7 ) -possible site-specific integration into
host cell with wild-type virus
-efficient transfer in non-dividing cells
in vitro
HO -transduces cells in nervous system
-small packaging capacity
-risk o f contamination with adenovirus
helper
-vectors do not show site-specific
intepation
Non-viral
DNA-protein
conjugate
Liposomes
50
50
no
no
no
no
-easy to produce with uniformity
-no replication risk
-efficient transfer in dividing and
non-dividing cells
-large transgene capacity
•easy to produce with uniformity
-low immunogenicity
-no replication risk
•large transgene capacity
-transimt expression
-low gene transfer efficiency in vivo
•transient expression
CHAPTER 2
Molecular Conjugate Delivery Vehicles
The Structure of Molecular Conjugates
Molecular conjugate vectors are non-viral gene
therapy vectors in which therapeutic genes are
introduced into cells via nonspecific adsorptive
mechanisms or through receptor-mediated endocytosis.
Molecular conjugates currently being developed include
cationic polypeptides, targeted molecular conjugates,
and virosomes (Figure 3). Cationic polypeptides are
the simplest form of molecular conjugates. They bind
plasmid DNA (therapeutic genes) to form the delivery
vehicle. Targeted molecular conjugates differ from
cationic polypeptides by the addition of a ligand that
serves to target the delivery of therapeutic genes to
specific cell surface features. Virosomes are more
12
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complex. In the context of this paper targeted
virosomes will be defined as follows: delivery
vehicles for gene therapy that are composed of at least
three elements; (l)a DNA-binding domain, (2)a fusogenic
protein, and (3)a targeted ligand domain. This
nomenclature has been selected because virosomes are
capable of mimicking the behavior of natural viruses
since they too usually contain a fusogenic and ligand
domain on their capsid (coat) proteins.
The DNA-binding domain functions to bind and
condense the therapeutic gene (plasmid DNA) that will
be delivered to cells for gene therapy. The DNA-
binding domain can be composed of a polycationic
protein such as poly-L-lysine or small cationic
proteins such as protamine. The DNA binding domain is
able to bind and condense plasmid DNA (therapeutic
gene) through the electrostatic interactions between
the positively charged protein with the negatively
charged phosphate backbone of the DNA. The cationic
protein is able to condense and neutralize the plasmid
DNA (therapeutic gene)into compact structure enhancing
its entry into cells (Gao and Huang, 1996).
The use of a selective ligand domain enables the
targeting of specific cells and the therapeutic gene is
theoretically internalized only by the targeted cells
13
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via endocytosis. Ligands can also be linked to DNA
intercalating agents such as ethidium dimers (Wagner et
al., 1991) and bis-acridine (Haensler et al., 1993).
Fusogenic peptides are a crucial component of
virosomes. Fusogenic peptides are amphipathic, pH
sensitive, and capable of forming a pore in endosomal
membranes. This enables the therapeutic gene to escape
from the endosome preventing its degradation in the
lysosome.
Gene Transfer With Molecular Conjugates
Gene transfer is the introduction of a therapeutic
gene (transgene) into somatic cells and is the first
step that must occur in gene therapy. The introduction
of transgenes into cells does not mediate effective
gene therapy. In this chapter aspects of gene transfer
with molecular conjugates will be discussed and
efficacy of therapeutic genes, which is marked by
expression of the transgene, will be discussed in
chapter 3.
Studies of Successful Gene Transfer With Molecular
Conjugates
Targeted molecular conjugate vectors were first
developed by Wu. A transferrin-poly-L-lysine targeted
molecular conjugate was used for in vitro gene transfer
14
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in hepatocytes (Wu and Wu, 1987) . Transferrin is a
protein in the blood that carries and delivers iron to
cells via receptor-mediated endocytosis. The use of
transferrin-poly-L-lysine-DNA conjugates for in vitro
gene transfer has been successful in hematopoietic
cells, pulmonary epithelial cells, and others (Table
3) .
Drawbacks of Poly-L-Lysine Molecular Conjugates for In
Vivo Gene Transfer
Poly-L-lysine is commonly used as the DNA binding
domain when constructing targeted molecular conjugates,
but there are other DNA binding proteins that can be
used such as protamine, poly-L-ornithine (Nead et al.,
1995) and the protein K8(Gottschalk et al., 1996).
Gene transfer by targeted molecular conjugates has
led to high level gene expression in vitro. However
studies indicate that in vivo gene transfer has only
resulted in low level gene expression (Perales et al.,
1994; Ferkol et al., 1995). The problem appears to be
the polycationic poly-L-lysine protein widely used as
the DNA binding domain of the targeted molecular
conjugates used for the in vitro studies.
Poly-L-lysine presents many problems to cells when
used in vivo. First, gene transfer is not as efficient
in vivo due to the instability of poly-L-lysine
15
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(Michael et al., 1994) . The poly-L-lysine domain is
targeted by humoral factors after the poly-L-lysine-DNA
conjugate enters the body. Second, commercially
available poly-L-lysine macromolecules are quite
heterogeneous in molecular size, difficult to produce
with reproducible colloidal properties, and are highly
cytotoxic at nM concentrations both in vitro and in
vivo. A solution to this problem can be to incorporate
a different DNA binding domain such as protamine or K8
(Gottschalk et al., 1996) into the targeted molecular
conjugates.
The novel protein K8 is an analog of spermine,
which is a naturally occurring DNA condensing agent. A
virosome including K8 was used for gene transfer into a
variety of cell lines including HepG2, and proved to be
highly effective (Gottschalk et al., 1996). K8-
virosomes were 1000-fold less toxic than poly-L-lysine-
based molecular conjugates. Although this study was
done in vitro, K8-virosomes are a feasible alternative
to poly-L-lysine-based molecular conjugates. The K8-
virosome was used in vivo in preliminary studies, in
which mice were repeatedly administered intraperitoneal
injections of the K8-synthetic vector and over a 90 day
period did not show any signs of a humoral response
(Rolland, 1998) .
16
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Poly-L-lysine molecular conjugates have proved
ineffective when used in vivo, not only due to the
inherent properties of poly-L-lysine but mainly because
they become degraded in the lysosomes. Poly-L-lysine
molecular conjugates might be more effective if a
fusogenic peptide was linked to the poly-L-lysine
domain. Molecular conjugates are now being constructed
as virosomes (Wagner et al., 1992), in which a
fusogenic peptide is coupled to the DNA binding domain
(poly-L-lysine) to prevent lysosomal degradation.
In chapter 4, the model experiment presented
involves the design of a virosome to target breast
cancer cells in vitro and in vivo. In the model
experiment the drawbacks of using a poly-L-lysine
molecular conjugate are avoided by constructing a
virosome that incorporates protamine as the DNA-binding
domain and GALA as the fusogenic domain. Protamine is
a naturally occurring DNA binding protein that, unlike
poly-L-lysine, is not cytotoxic. GALA is a short
peptide (30 amino acids) that is derived from
endosomolytic viral proteins. GALA provides the
fusogenic domain to prevent lysosomal degradation of
the transgene once it is delivered to the cell. The
role of fusogenic peptides in augmentation of gene
17
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transfer efficiency will be further discussed in
chapter 3.
The Advantages of Using Molecular Conjugates for Gene
Therapy
The use of molecular conjugates for gene therapy
offer many advantages over conventional viral vectors.
Molecular conjugates are safer because the use of
biologically active virus particles is eliminated and
therefore there is no potential risk of producing a
recombinant virus. Other toxic effects and immune
responses are also avoided. Currently there has not
been any finding of adverse effects caused by
incorporating plasmid DNA into cells, and there has
been no report of plasmid integration into the host
chromosomes, which eliminates the risk of insertional
oncogenesis and mutagenesis that is always present with
viral vectors. The plasmid DNA exists as an episome
and has a finite period of gene expression. Molecular
conjugates have a larger transgene capacity (50kb
compared to 4.5-30kb of different viral vectors)(Wivel
and Wilson, 1998), which offers the potential use to
treat many diseases that involve a large defective gene
such as Duchenne muscular dystrophy. Another appealing
advantage of molecular conjugates involves their
production. It is possible to produce them in a
18
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uniform manner that is not possible with viral vectors.
Viral vectors are biological entities and hence exact
replication of a vector would not be expected 100% of
the time. This may allow less expensive quality
control comparable to the production costs of
conventional therapeutic drugs. Molecular conjugates
can be used for gene transfer in both dividing and non
dividing cells and since they deliver therapeutic genes
(plasmid DNA) that have a finite period of expression,
varying doses may be administered to optimize treatment
in different patients. Therefore, molecular conjugates
have the potential to be used in the same manner as
conventional prescription drugs.
The Disadvantages of Using Molecular Conjugates for
Gene Therapy
The use of molecular conjugates is still at an
early stage even in comparison to the use of viral
vectors. Viral vectors are still the most commonly
used vehicle for gene therapy and account for 80% of
USA approved gene therapy protocols. There has been
more research involving viral vectors and hence more
data than compared to molecular conjugates. There is
still much to learn and discover about the requirements
for designing effective molecular conjugates.
Currently virosomes are being designed in which
19
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inherent properties of viruses are being incorporated
with some success, but reproducing viral properties is
difficult because sometimes not enough is known about
all the elements that are involved in viral function.
Another disadvantage of molecular conjugates is their
low efficiency when used in vivo. Transient expression
of transgenes delivered via molecular conjugates
becomes a drawback when treating monogenic diseases or
other inborn metabolic diseases in which permanent
replacement of a gene is required. The properties of
plasmid DNA also present a concern when delivered by
way of molecular conjugates. Since plasmid DNA is
highly hydrophilic, negatively charged, and has
different hydrodynamic sizes it is unable to transverse
cellular membranes on its own. Therefore, the DNA-
binding domain of molecular conjugates must be able to
condense and neutralize plasmid DNA for it to be
effectively delivered to the cell nucleus. Lastly, The
overall charge and size of the molecular conjugate must
be within very specific ranges for optimal gene
transfer, so in some situations large molecular poly-L-
lysine can be a hindrance to endocytotic
internalization.
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Figure 3. The Structure of Molecular Conjugates
A. Cationic Polypeptides
Molecular conjugates that are composed of
only a cationic DNA binding polypeptide
DNA WMttag
■" . Poaate ■ ’
++++++
Flumid ^ C XO
DNA
B. Targeted Molecular Conjugates
Molecular conjugates that are composed of
a cationic DNA Binding polypeptide and
a ligand. The ligand allows for specifc cell
targeting
DNABfcdiag
f Doiahi
++++++
c. Targeted Virosomes
Molecular conjugates that are composed of a
cationic DNA binding polypeptides, ligand
for specific cell targeting, ana a fusogenic
peptide to enable endosomal escape.
DNABtedtag Fusogenic
Donate Peptide
++++++
Plum ld ^
DNA
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Table 3. Molecular Conjugates Used for Gene Transfer and Reference
Molecular Conjugate___ DNA Delivered Efficiency Transduced Cells Reference
EGF-PLL*-DNA Reporter Gene ' In Vitro H460, H322, H358, Cristiano, 1998
pCMV-p-Gal other NSCLC cells
K8-JTS-1-DNA Reporter G ate
pCMV-Luc,
pCM V -p-G al
In Vitro Hep G2, various cell
lines
Gottsdialk, et al., 1996
surfactant B-PLUDNA
galactosylated-PLl^DNA
transferrin-PI.L-DNA
anti-thrombomodulin-PLL-DNA
transferrin-PPl^DNA-HA-2
transferrin-PLI^DNA
transferrin-protamine-DNA
transferrin-PLUDNA
astaloorosomuooid-PLL-DNA
asialoorosomucoid-PLL-DNA
Reporter Gene
pCPA-RSV CAT
pPEPCK-hFIX
foreign plasmid
DNA
Reporter Gene
pCMV-Luc,
pCMV-p-Gal
Reporter Gene
pRSVl.
Reporter Gene
pRSVL
Reporter Gene
pSV2 CAT
Reporter Gene
pSV2 CAT
In Vitro
In Vivo
In Vitro
In Vitro/
In Vivo
In Vitro
In Vitro
11441, pulmonary
adenocarcinoma cells
hepatocytes
epithelial airway cells
lung endothelial cells
K 5 6 2 , H eL a, BNL
C1..2 hepatocytes
HD-3, erythroblasts
In Vitro Hematopoietic cells
In Vivo hepatocytes
In Vitro Hep G2
B aatz, et al., 1994
Perales, et al., 1994
Harris, et al., 1993
Trubetskoy, er at., 1992
Wagner, et al., 1992
Wagner, et al., 1990
Zenke, et al., 1990
Wu, et al., 1988
Wu, el al., 1987
*PLL- Poly-L-lysine
CHAPTER 3
Endosomal Escape: A Viral Function Mimicked in
Molecular Conjugates for Increased Gene Transfer
Efficacy
Review o£ Endocytosis
Transport of extracellular macromolecules from the
plasma membrane into eukaryotic cells occurs via the
endocytosis pathway (Figure 4). Endocytosis involves
the internalization of macromolecules by invagination
of the plasma membrane which pinches off to form an
intracellular vesicle that contains the ingested
macromolecules. The intracellular vesicle is called an
endosome which has an acidic pH that is maintained by a
H+-ATPase pump. The contents of the endosome are then
delivered to the lysosomes. Lysosomes are membrane
enclosed cellular organelles that also maintain an
acidic pH by way of a H+-ATPase pump. The maintenance
of an acidic pH in the lysosomes is required for the
23
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optimal activity of its hydrolytic enzymes, which
function to digest macromolecules. The end product of
digested macromolecules are sugars, amino acids,
nucleotides, and fatty acids which are released into
the cytosol to be utilized or excreted by the cell.
Receptor-mediated endocytosis is a specialized form of
endocytosis that involves specific binding to cell
surface receptors and internalization of those specific
macromolecules that contain the ligand for the
receptors. Examples of receptor-mediated endocytosis
that occur in most animal cells are uptake of
cholesterol, transferrin (iron-binding protein in
blood),and EGF (epidermal growth factor). The problem
facing gene therapy researchers is to take advantage of
the endocytotic pathway to deliver potentially
therapeutic DNA while at the same time avoiding
lysosomal degradation.
Viral Infection and Endosomal Escape
Enveloped viruses such as adenovirus, retrovirus,
and influenza virus exploit the receptor-mediated
endocytotic mechanism to infect cells. The viral
infection mechanisms have been well studied and a key
factor necessary for infection involves endosomal
escape (Figure 5). Once viruses are endocytosed by
24
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host cells they are able to escape from the endosome by
way of fusogenic peptides, which prevents their
degradation by the lysosomes. Fusogenic peptides are
amphipathic and when exposed to an acidic pH
environment they are capable of disrupting the
endosomal membrane. Fusogenic peptides undergo
conformational changes into a-helices when exposed to
an acidic pH in which their hydrophobic regions become
exposed. The mechanisms of membrane disruption by
fusogenic peptides has not been fully elucidated, but
it is believed to occur in two ways: (l) the a-helices
insert vertically into the lipid membrane and form a
pore, and (2) the exposed hydrophobic regions of the a-
helices line up horizontally with the lipid membrane
inducing fusion of the membrane and the fusogenic
peptide. Both of these mechanism enable the emptying
of endosomal contents into the cytosol. The viral
genetic material can then be free to reach the nucleus
where it is expressed along with host cell DNA. viral
genetic material can be integrated into the host cell
DNA or exist in an episomal state if it is of the non
integrating kind.
Viruses have essentially evolved into mobile
genetic elements with highly specialized machinery for
efficient gene transfer into host cells (Figure 6).
25
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The fusogenic peptides of viruses are now being used to
design molecular conjugates that are more efficient in
gene transfer.
The Use of Fusogenic Peptides in Molecular Conjugates
to Increase Gene Transfer Efficacy
Cellular internalization of plasmid DNA
(therapeutic genes) delivered by molecular conjugates
via the receptor-mediated endocytosis pathway is
successful. However it is apparent that low gene
transfer efficiency observed in cells transduced with
molecular conjugates is attributed to their inability
to escape from the endosomes, and subsequent
degradation in the lysosomes. To address this problem,
molecular conjugates are now being used with
replication-incompetent viruses or are being designed
to incorporate a fusogenic peptide (virosomes).
Replication-incompetent viruses are viruses that have
been stripped of the elements necessary for
replication. Both strategies incorporate a viral
mechanism for gene transfer, enabling them to be more
effective.
Molecular conjugates have been used for gene
transfer in combination with replication-incompetent
adenovirus and has proved highly effective. A study
showed that EGF-poly-L-lysine-DNA molecular conjugates
26
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alone were not capable of transducing non-small-lung
cancer cells(NSLC), but when co-transfected with
replication-incompetent adenovirus, transduction of 66%
of NSCLC cells was observed (Cristiano, 1998). In a
similar study, transferrin-poly-L-lysine-DNA molecular
conjugates were also coupled with replication-
incompetent adenovirus and resulted in a 2000 fold
augmentation of gene transfer when compared to
transduction with the molecular conjugates alone
(Curiel et al., 1991).
Studies with virosomes have also had positive
results. A virosome was constructed by using a novel
DNA binding protein (K8) and a novel fusogenic peptide
(JTS-1) (Gottschalk, et al., (1996). The K8-JTS-1-DNA
virosome was used for gene transfer in Hep G2 cells and
had higher transfection efficiency compared to gene
transfer with K8-DNA conjugates. However, Hep G2 cells
were also co-transfected with K8-JTS-1-DNA virosomes
and replication incompetent adenovirus, which resulted
in a 10-50 fold increase in gene transfer efficiency
compared to transduction with K8-JTS-1-DNA virosomes
alone. Another study designed a virosome (N-terminal-
HA-2-transferrin-poly-L-lysine-DNA) that incorporated
the influenza virus hemagglutinin HA-2 fusogenic
peptide. Transfection of HeLa cells with the HA-2-
27
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virosome resulted in augmented gene transfer compared
to transduction with transferrin-poly-L-lysine-DNA
molecular conjugates. But, once again co-transfection
with replication-incompetent adenovirus resulted in
more effective gene transfer compared to the efficacy
of HA-2-virosomes.
The incorporation of fusogenic peptides into
molecular conjugates enhances gene transfer efficacy,
but in comparison replication-incompetent viruses have
greater gene transfer efficacy. The construction of
virosomes brings us a step closer toward designing the
perfect delivery vehicle for gene therapy, but studies
indicate that there is still more to be learned about
viral elements in order to mimic their highly effective
gene transfer mechanisms.
In the model experiment that is presented in
chapter 4, the construction of a novel fusion protein
that targets breast cancer cells incorporates the
fusogenic peptide GALA.
Other Methods for Endosomal Escape
The use of lysomotrophic compounds have also been
used to enhance endosomal escape. In vitro studies
have demonstrated that compounds such as chloroquine,
Brefeldin A, ammonium chloride, and monensin increase
28
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gene transfer (Rolland, 1998). These agents function
by altering the endosomal pH.
29
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Figure 4. An Illustration of the Endocytosis Pathway (See text for details.]
Macromolecules
Plasma Membrane (PM)
0 1 Invagination of PM
Endosome
pH 6
Nucleus
Lysosomes
pit 5
Fatty Acids
©
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Figure 5. Viral Infection of Cells Via the Receptor-Mediated Endocytosis Pathway
Enveloped viruses contain ligands that can bind specific receptors found on the cell surface of host cells. Once the
virus binds to the receptor, it is internalized by the endocytosis pathway. A fusogenic peptide in the protein envelope
is capable of forming a pore in the endosome, which allows the viral DNA or RNA to leak out into the cytosol. This
endososmal escape mechanism prevents the viral genetic material from being degraded in the lysosome.
- Genetic Material
(DNA or RNA) S
Envelope Protein —
(WMi UfMrt Tar C«* Sarfan Rmflan)
Cell Surface Receptor
Plasma Membrane
Virus
Endosome
Nucleus
Figure 6. A Picture of Lipid/DNA Conjugates Used With
Vaccinia Virus for Gene Delivery
DNA must cross two barriers, the endosomal membrane and the
nuclear membrane. The vaccinia virus is used for its fusogenic
properties to allow for the lipid/DNA conjugate to escape from
the lysosome. The small arrow are vaccinia virus and the large
arrow is the endosome with the lipid/DNA conjugate inside
(Zabner, et al., 1995).
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CHAPTER 4
A Model Experiment: Treatment of Breast Cancer With
a Novel Targeted Fusion Protein
Introduction
In a model experiment I built a novel fusion
protein virosome designed to target breast cancer
cells. The novel fusion protein that I constructed is
composed of several independent domains including a
targeting domain (heregulin), a DNA Binding domain
(protamine), and a translocation domain (GALA). This
novel fusion protein will be used to deliver
specifically to breast cancer cells a plasmid encoding
the potentially therapeutic herpes simplex thymidine
kinase (HSTK) gene. The breast cancer cells will then
be treated with the drug ganciclovir which will induce
cell death in those cells expressing HSTK (Figure 7).
33
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Studies to characterize the properties of the
novel fusion protein are in progress. The properties
that are being studied are as follows:(a)its DNA
binding capability, (b)its ability to selectively bind
to breast cancer cells, and (c) its ability for
internalization and cellular endosomolysis.
Background
Herecrulin
Heregulin is a membrane-bound protein that is
initially synthesized as a 645 amino acid protein.
Proteolytic cleavage then produces the mature 45-kDa
glycoprotein that is composed of 240 amino acids, and
it is not membrane-bound. Heregulin contains an
immunoglobulin-like domain and an epidermal growth
factor (EGF)-like domain. Heregulin is naturally
occurring, and alternative splicing gives rise to
different isoforms (a, ( 5 1 , 02, and 03) found in normal
tissue including brain, breast, ovary, prostate,
skeletal muscle, small intestine, and testis (Han et
al.,1995).
Heregulin was first identified by Holmes et al. in
1992, but its physiological functions are not well
understood. Heregulin is the human homologue of the neu
differentiation factor (NDF), which is found in
34
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neuronal tissues of mouse embryos (Bacus et al., 1993).
We have chosen heregulin as a targeting domain because
it is a ligand for a family of human epidermal growth
factor (EGF) receptors, that include HER-2, HER-3, and
HER-4 which are also called ERBB2, ERBB3, and ERBB4,
respectively (Han et al., 1995). HER-2 is an 185-
kilodalton transmembrane tyrosine kinase that is
activated by heregulin (Holmes et al., 1992). HER-2
and HER-4 are overexpressed on the cell surface of many
breast cancer epithelia. HER-2 is overexpressed in 20%
of breast cancers, and HER-4 is overexpressed in 40% of
human breast cancer cell lines and is coexpressed with
HER-2. HER-2 is not expressed at high levels in normal
breast tissue or in benign breast diseases. It is this
molecular difference of HER-2 expression that I hope to
exploit by making it the target of the novel fusion
protein that contains a heregulin domain to deliver the
herpes simplex thymidine kinase gene selectively breast
cancer cells in gene therapy (Figure 7).
Protamine
Protamines are naturally occurring proteins that
are found in fish spermatozoa. Newly synthesized
protamine can be found in the cytoplasm where
subsequently it is phosphorylated by ATP for targeting
35
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to the cell nucleus. Once in the cell nucleus
protamine displaces histones and binds to DNA.
Protamines are small (4,200 Da), basic, and exist
as a random coil in solution in the absence of double
stranded DNA or RNA (Warrant et al., 1978). Their
primary structure is composed of 31-34 amino acid
residues that contain four to five clusters of four or
five consecutive arginine residues that are usually
separated by proline, glycine, or tyrosine residues
(Figure 8). Single crystal X-ray diffraction and
circular dichroism measurements show that in the
presence of double stranded DNA or RNA, protamines
undergo a conformational change in which the random
coil structure changes to one or more a-helical
segments (Warrant et al., 1978). The a-helical
segments in protamine interact with the major and minor
groove of DNA. The a-helical segments are rich in
arginine thereby making them positively charged and
able to attract the negatively charged sugar-phosphate
backbone of DNA and RNA.
We have chosen to incorporate protamine into our
fusion protein because it tightly binds non-
specifically to DNA, condenses the DNA, and neutralizes
the DNA. Condensed and neutralized DNA enhances
translocation of DNA across cell membranes (Gao and
36
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Huang, 1996). We are using the sequence of salmon
sperm protamine, in our virosome. This protamine
consists of 32 amino acid residues. The protamine
domain of our fusion protein will bind therapeutic DNA
that is to be delivered to breast cancer cells.
GALA
GALA is a synthetic fusogenic protein with similar
endosomolytic properties as some viral proteins (Nicol
et al., 1996; Prchla et al., 1995; Puyal et al., 1994) .
GALA is a small peptide that is composed of 30 amino
acid residues with GLU-ALA-LEU-ALA repeats
(WEAALAEALAEALAEHLAEALAEALEALAA) . GALA contains
several glutamic acid residues which make it a pH-
sensitive peptide. At neutral pH GALA has a low
affinity for membranes and has a secondary structure of
a random coil. At lower acidic pH the glutamic acid
residues become neutralized and GALA undergoes a
conformational change into an amphipathic a-helix that
has high affinity for negatively charged and neutral
membranes. The conformational change that GALA
undergoes in a pH-dependent manner allows it to form
pores in membranes. Therefore when GALA is endocytosed
into an acidic endosome it is capable of forming a pore
which results in the release of the endosomal contents.
37
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GALA is being incorporated into the novel fusion
protein because we are depending upon endocytosis for
efficient therapeutic gene transfer and gene
expression. This non-viral DNA delivery system will
depend on endosomal escape of the therapeutic gene for
successful gene delivery to the nucleus. GALA will
allow endosomal escape which will prevent lysosomal
degradation of the therapeutic gene, and therefore
improve gene delivery and efficacy.
Thus the novel fusion protein will contain
heregulin, protamine, and GALA components. We have
constructed them in two orientations (Figure 9). They
will be tested for DNA binding , selective binding to
breast cancer cells, cell internalization,
endosomolysis, and efficiency of therapeutic gene
delivery to breast cancer cells.
Materials and Methods
Plasmids
The plasmids used include Bluescript SK +/-
(pBS)(Figure 10), which was used as an intermediate
vector, and pRSETa (Invitrogen Corp.)(Figure 11), which
was used to clone the gene sequence of the final novel
fusion protein construct.
38
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01 icfpn ucleoti des
Oligonucleotides with the gene sequence that
encodes protamine and GALA were purchased in both
5 '->3' and 3 '*>5' single strands at the USC/Norris
Comprehensive Cancer Center Microchemical Core
Facility. The protamine oligonucleotide sequence is as
follows (116 nucleotides): 5' ACCT GAG CTC CCG CGT CGC
CGT CGT AGC TCC AGC CGC CCG GTG CGT CGC CGT CGC CGT CCG
CGT GTG AGC CGT CGC CGT CGC CGT CGC GGC GGT CGT CGC CGT
CGC CTC GAG ACAT 3'(Figure 12). The GALA
oligonucleotide sequence is as follows (133
nucleotides) : 5' ATCT CTG CAG GGT GGC TGG GAG GCG GCC
CTG GCG GAA GCC CTC CGC GAG GCC CTG GCG GAA CAT CTC GCC
GAG GCG CTG GCC GAA GCG CTG GAG GCC CTG GCG GCC GGC GGT
AGA TCT GGT ACG GTA ACC ACT 3' (Figure 13) . The
oligonucleotides were annealed in a reaction mixture of
the single strands with 0.5 M KC1. The reaction
mixture was boiled for 5 minutes and then allowed to
cool to room temperature.
Restriction Divests
Restriction enzyme digestions were generally
performed for l hour at optimal temperatures for each
restriction enzyme. The following restriction enzymes
were used: BamHI, Bgllll, Bstell, EcoRI, PstI, SacI,
and Xhol.
39
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Ligation Reactions
Ligation reactions were performed with T4 DNA
ligase. The ligation reactions were standard reactions
containing: 50 ng of restriction digested/gel purified
vector, 0.5 ng restriction digested insert, IX ligase
buffer (50 mM Tris-HCL (pH 7.6), 10 mM MgCl2, 1 mM ATP,
and 1 mM DTT in total volume of 5 f j . L ) . The ligation
reactions were incubated overnight in a water bath at
16 °C.
Bacterial Strains
Competent DH5a Escherichia coli (E. coli) cells
(Strategene)were used for transformations.
Transformations
The ligates were used to transform competent DH5a
E. coli cells. The transformations were performed by
the heat shock method. Transformed E. coli cells were
spread onto LB-Amp plates with 950 |iL of LB broth. The
LB-Amp plates were then incubated at 37 °C for 16
hours. Numerous colonies were then selected. Each
colony was grown separately in LB Broth (5 mL)
containing ampicillin (50 ng/mL), overnight on a shaker
at 3 7 ° c .
40
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Mini Preps
Mini preps were used to isolate the plasmid DNA
(Qiagen) . The elution of DNA was modified and
deionized water was used.
Aaarose Gel Electrophoresis
Clones carrying DNA inserts were identified by
running electrophoretic gels (0.8-1.0 % agarose) of
restriction digested plasmid DNA.
Gel Extraction
Insert DNAs were extracted from the agarose
electrophoretic gels with a gel extraction kit
(Qiagen).
Heregylin
The heregulin oligonucleotide was provided. It
was a PCR product of 600 base pairs.
Results
Plasmid Construction
The cloning strategy for the gene sequence of the
novel fusion protein is shown in figure 14. The
cloning strategy incorporated several restriction
digests and ligations so the three components of the
novel fusion protein would be a continuos gene
sequence. The final construct was the gene sequence
for expression of the novel fusion protein as a single
41
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protein in the N-protamine-GALA-heregulin-C
orientation.
Construction of pRSETa-GALA-Her
The construction of the pRSETa-GALA-Her plasmid
involved a triple ligation (Ligation A, Figure 14).
The three elements that were ligated are as follows:
(1) Pstl/Bstell digested GALA (Digest A, Figure 14),
(2) Bstell/EcoRI digested heregulin (Digest B, Figure
13), and (3) Pstl/EcoRI digested pRSETa plasmid (Digest
C, Figure 14). A confirmatory digest with PstI and
BamHI (Digest D, Figure 14)was used to identify the
pRSETa-GALA-Her plasmid. The digest was ran on an
electrophoretic gel and resulted in a GALA-Her 700 bp
insert, which is what was expected (Figure 15).
Construction of the pBS-GALA-Her Plasmid
The PstJ-GALA-Her-BamffJ fragment was extracted
from the electrophoretic gel shown in figure 15, and
inserted into a pBS plasmid to pick up the Xhol
restriction site, which was needed for the final
construct. The pBS-GALA-Her plasmid was constructed by
digesting pBS plasmid with PstI and BamHI (Digest D,
Figure 14) , and the Ps 11-GALA-Her-BeunHI fragment was
inserted into the pBS plasmid (Ligation B, Figure 14).
42
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A confirmatory digest of the pBS-GALA-Her plasmid was
done with BamHI and Xhol (Digest E, Figure 14) . The
confirmatory gel of Digest E resulted in a 700 bp
insert as was expected (Figure 16) .
Construction of the pRSETa-Pro-GALA-Her
The pRSETa-Pro-GALA-Her plasmid was the final
construct of the gene sequence encoding the novel
fusion protein. The pRSET-Pro-GALA-Her clone was not
successful. The triple ligation reaction with
SacJ/Xhol-Protamine (Digest F, Figure 14) , Sacl/Bglll-
new-pRSET (Digest G, Figure 14) , and the BamHI/XhoI-
GALA-Her insert (Digest E, Figure 14)resulted in an
unsuccessful bacterial transformation. Two
confirmatory digest were performed; (1) EcoRI which
should have resulted in a 700 bp insert, and (2) Xhol
which should have resulted in linearization of the
vector (Figure 17). Both confirmatory digest were
negative, the triple ligation was attempted two other
times with no success.
Discussion
We attempted to make the novel fusion protein in
one orientation (N-Pro-GALA-Her-C) , and the
construction of the other orientation (N-Pro-GALA-Her-
43
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C) is currently in progress. The cloning strategy that
was presented in this study was unsuccessful, and the
only accomplishment was the construction of the GALA-
Her fragment of the novel fusion protein (Ligation B,
Figure 14).
The cloning strategy that we used to construct the
novel fusion protein should have theoretically been
effective. All the ligation sequences were examined
carefully to ensure that a reading frame shift would
not occur. Among the possible explanations for the
failure of the construction of the final pRSET-Pro-
GALA-Her plasmid are: (1) the enzymatic reaction of
the ligations may have not been at optimal conditions,
(2) the ligase enzyme used for the ligation reaction
may have been damaged ie. denatured, old, low enzymatic
activity, (3) the restriction enzymes used to produce
compatible sticky ends for the 3 part ligation reaction
may have also been ineffective, and (4) the E. coli
cells used for transformations may have not been fully
competent. A control was not used. An alternative
strategy that could have been used to produce the
pRSETa-Pro-GALA-Her plasmid is by making the construct
in the following two steps: (1)ligation of the
BamHI/ XhoJ-GALA-Her pBS insert with new BamHI/ Xhol -
44
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pRSETa plasmid, and (2) ligation of SacJ/XhoJ-protamine
with SacJ/XhoJ-pRSET plasmid from step l.
The cloning strategy that was implemented in this
study was complicated, and was used as a last resort
due to an error. The protamine oligonucleotides were
accidentally purchased without a 3' PstI restriction
site (Figure 12). The incorporation of the 3' PstI
restriction site into the protamine oligonucleotides
would have eliminated the need to use the pBS plasmid
which was used to pick up a Xhol restriction site
(Figure 18).
This study is currently in progress. Since the
results of the study presented, the novel fusion
protein has currently been successfully constructed in
the N-Pro-GALA-Her-C orientation by using a different
cloning strategy that has not been mentioned in this
paper. The construction of the novel fusion protein in
the N-Her-GALA-Pro-C orientation is also still in
progress.
The N-Pro-GALA-Her-C novel fusion protein is a
virosome as defined in the context of this review. The
properties of the novel fusion protein are currently
being tested. Its ability to bind DNA (HSTK),
selectively bind to breast cancer cells, and ability
for internalization and cellur endosomolysis are all
45
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being determined. The ongoing studies include both in
vitro and in vivo models. If these studies are
conclusive, the novel fusion protein (virosome) will be
able to be used for gene therapy of breast cancer.
This is an example of the use of molecular conjugates
for gene therapy, with the application to breast
cancer.
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Figure 7. A Schematic o f Breast Cancer Targeting With the Novel Fusion
Protein and Delivery of the HSTK Gene
The shaded bar represents a fusion protein consisting of a DNA-binding. an endosomolytic.
and a targeting domain. The targeting domain binds to HER-2. and the endosomolytic domain
enables the DNA encoding herpes simplex thymidine kinase (HSTK) to be delivered to the
nucleus.
DNA
binding
Endmom olysis
Breast caiccr
^targeting
HER2
(Heregulin receptor)
Breast Cancer
Cells
Nucleus
Ganciclovir Drug Treatment
O.
Cell death occurs in the cells that
express the HSTK gene
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Figure 8. The Structure of Protamine
Protamine consists of four a-helical segments which are
arginine rich. The a-helical segments are seperated by
flexible joints that contain proline, glycine, and tyrosine
residues (not shown). Arginine residues are indicated by
(+). The a-helical segments are depicted by rectangles.
C Terminus
> f
N Terminus
The arginine-rich a-helical segments
of protamine interact with the major
and minor grooves of DNA.
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Figure 9. A Depiction of the Novel Fusion Protein in Two Orientations
Protamine, GALA, and heregulin are being used as the DNA-binding domain, membrane
translocation domain, and targeting domain, respectively. The amino terminus is indicated by (N)
and the carboxy terminus is indicated by (C).
N
DNA-Binding Targeting
N
Targeting DNA-Binding
Figure 10. The Structure of pBS.
The pBS plasmid was used as an intermediate vector to clone the
GALA-Heregulin gene sequence. The pBS plasmid was used to pick up a
restriction site {Xhol) ___ _____________________ _____
ft (+) origin
(6-162)
462
lacZ
2832
657
MCS
(657-759)
759
Ampicillin
(1975-2832)
Bluescript SK + /-
2.96 kb
1975
1032
1912
ColEi origin
(1032-1912)
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Figure il. The Structure of pRSETa Plasmid
T7 promoter (T7). termination sequence (Term.).origin of replication (Ori.). and
ampicillin resistance gene (Amp.). _______________________________
Amp.
11=_ f 1
a a x o a £ 2
T7
Ori
pRSETa
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Figure 12. The Nucleic Acid and Amino Acid Sequence of Protamine
The nucleic acid sequence shown is the oligonucletide encoding protamine. The lower line represents the
corresponding amino acid sequence. The restriction sites (S a d and Xhol) shown were used to clone this
sequence into the fusion protein.
5’ ACCT GAG CTC CCG CGT CGC CGT CGT AGC TCC AGC
SacI P R R R R S S S
CGC CCG GTG CGT CGC CGT CGC CGT CCG CGT GTG
RP V R R R R R P R V
AGC CGT CGC CGT CGC CGT CGC GGC GGT CGT CGC
S R R R R R R G G R R
CGT CGC CTC GAG ACAT 3’
R R Xhol
C/l
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Figure 13. The Nucleic Acid and Amino Acid Sequence of GALA
The nucleic acid sequence shown is the oligonucletide encoding GALA. The lower line represents the
corresponding amino acid sequence. The restriction sites (PstI and Bstell) shown were used to clone this
sequence into the fusion protein.
5’ ATCT CTG CAG GGT GGC TGG GAG GCG GCC CTG GCG
PstI W E A A L A
GAA GCC CTC GCG GAG GCC CTG GCG GAA CAT CTC
E A L A E A L A E H L
GCC GAG GCG CTG GCC GAA GCG CTG GAG GCC CTG
A E A L A E A L E A L
GCG GCC GGC GGT AGA TCT GGT ACG GTA ACC ACT 3’
A A G G Bstell
L/l
Ul
Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
Figure 14. A Diagram of the Cloning Strategy for the Construction of the Novel Fusion Protein
Gene Sequence in the N-Terminus to C-Terminus Orientation, (plasmids are not drawn to scale)
Bifell Bam HI EcoRl
Bam H I
DIGEST B
(Bstell/EcoRJ)
iiFnTMiJ
Patl
DIGEST A
( P s t l / B s t e ^ ^
Xhol/Sad
Bam HI
EcoR J
pRSETa
DIGEST C
(Pstl/EcoRI)
Batell
GALA
pRSETa-GALA-Her
Bam H I
EcoR J
Hindi 1 1
Sacl
LIGATION A
DIGEST D
(PstI/BamHI)
EcoRl
X hol
LIGATION C
B ate] I
Xhol
K o o R J
s.ci_^. Im pRSETa-
Bamlll
Sacl
GALA Har BamHLBilU
EcoRl
Pro-GALA-Her
DIGEST G
(SacI/BgUI)
pRSETa
DIGEST E
(Bamin/Xhol)
DIGEST F
(Sacl/Xhol)
BantHI
GALA
EcoR J
pBS-GALA-Her
X hol
LIGATION B
Ul
Figure 15. The pRSETa-GALA-Her Confirmatory
Electrophoretic Gel.
The picture of the electrophoretic gel (1% agarose) is of the
digest of pRSETa-GALA-Her with PstI and BamHI. The GALA-
Her insert is 700 bp. The marker lane is indicated by M. Lane 1 is
the digest of pRSETa-GALA-Her. Lane 2 is the digest of
pRSETa-GALA-Inv, in which the GALA-Inv is also a 700 bp
insert.
M 1 2
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Figure 16. The pBS-GALA-Her Confiruiatory Electrophoretic Gel
The picture of the electrophoretic gel (l%agarose) is of the digest of pBS-
GALA-Her with BamHI and Xhol. The GALA-Her insert is 700 bp. The
maifcer lane is indicated by M. Lanes 1 and 2 are the digests of pBS-GAL A-
Her. Lanes 3 and 4 are the digests of pBS-GALA-Inv. in which the GALA-Inv
is also a 700 bp insert.
M 1 2 3 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
Figure 17. The Electrophoretic Gels of the Negative pRSETa-Pro-GALA-Her Confimatory Digests
In gel A, M is the maiker, lane U is uncut pRSET and lanes 1-3 are different pRSETa-Pro-GALA-Her clones.
The digest with Xhol should have resulted in the linearization of the plasmid. In gel B, M is the m aricer, lanes
1-3 are the same pRSETa-Pro-GALA-Her clones used in gel A. The digest with EcoRl should have resulted in
a 800bp band if tne insert was present and in linearization in the absence of the insert.____________________
A. Xhol Digest B. EcoRl Digest
IS)
■ * 4
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Figure 18. A Diagram of the Cloning Strategy With 3* Pslf-Protamine
The dashed arrow indicates the cloning strategy with ST^/Z-Protamine.fpiasnuds arc not draw n to scale)
!li£utL3.L21BI
i
BUdl
BimHI EcoRl
Xhol/Sacl
B am H I
DIGEST B
(Bstell/EcoRI)
t i i i i i R " ; F ' i i l i H !
Pftl
DIGEST A
( P s t l / B s t e ^ ^
Xhol'Sacl
Bam HI
pRSETa
DIGEST C
(Pstl/EcoRI)
GALA
pRSETa-GALA-Her
B am H I
EcoRl
H indll I
Bam HI
Sacl
LIGATION A
EcoRJ
H indi!)
DIGEST D
(PstI/BamHI)
B ate) I
LIGATION C
Bstell
BemHIBgM GALA Her
EcoRl
pRSETa-
Pro-GALA-Her
DIGEST G
(SacI/BgUI)
pRSETa
DIGEST E
(BamHI/Xhol)
DIGEST F
(Sacl/Xhol)
BamHI
GALA
pBS-GALA-Hcr
X hol
X hol
LIGATION B
(A
0 0
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Asset Metadata

Creator Felix-Trunnelle, Elizabeth (author)

Core Title A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells

School Graduate School

Degree Master of Arts

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, oncology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Kasahara, Noriyuki (committee member), Medina-Kauwe, Lali (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-287959

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Rights Felix-Trunnelle, Elizabeth

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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