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RECOMBINANT CATIONIC BIOPOLYMERS FOR
NUCLEIC ACID DELIVERY
By
BRENDA F. CANINE
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
College of Pharmacy
Department of Pharmaceutical Sciences
December 2010
ii
To the faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of
BRENDA F. CANINE, find it satisfactory and recommend that it be accepted.
_____________________________
Arash Hatefi, Ph.D., Chair
_____________________________
Margaret Black, Ph.D.
_____________________________
Raymond Reeves, Ph.D.
_____________________________
ChulHee Kang, Ph.D.
_____________________________
David Koh, Ph.D.
iii
Acknowledgements
I wish to sincerely thank all of the people who have supported me during my graduate
studies. It has been a journey in not only science but also in life and personal development. A
very special thank you to those of you who lent a shoulder to lean on in times of failure and also
a smile in times of success. To those of you who helped with an experiment or a method design,
listened to a presentation, or proof-read a paper or document, I am especially thankful.
Thank you to my family, especially Mom, Dad, Kara and Katie, who have provided much
needed perspective and guidance through the years. Your wisdom and insight often made me
realize that I cannot let the outcome of my experiments dictate my frame of mind. Science is and
will continue to be both a rewarding and frustrating endeavor.
At the last graduation commencement I attended, the speaker explained the difference
between having to and getting to. I did not have to go to graduate school, I got to and I thank all
of you involved in that journey and getting me to the end. The distinction between having to and
getting to has been an enormous eye opener and a lesson I hope to carry with me in all future
endeavors.
Thank you to my mentor, Dr. Arash Hatefi who has provided guidance and support through
this process. Also, thanks go to my committee members, Dr. Black, Dr. Reeves, Dr. Kang, and
Dr. Koh for their words of encouragement and invaluable research advice. To all of my fellow
graduate students and postdocs who listened to my rants when experiments went awry , thanks
for the pep talks, and remember to hang in there when times are rough and have faith and
confidence in your abilities. Thank you to all of the Pharmaceutical Sciences faculty who have
always taken time to assist me when I need fresh ideas, expertise or reagents. Thank you to
iv
Yuhua (Al) Wang for his unwavering support and help in the lab, I could not have asked for a
better labmate.
Above all else, I wish to thank my husband, Chris, without whom I would not have
finished this chapter of my life. Thank you for all of the love and support you provide every day.
Thank you for seeing me through the struggles, for celebrating the triumphs, and most of all for
being my best friend.
v
RECOMBINANT CATIONIC BIOPOLYMERS FOR NUCLEIC ACID
DELIVERY
Brenda F. Canine, Ph.D.
Washington State University
December 2010
Chair: Dr. Arash Hatefi
Abstract
Delivery of nucleic acids in an efficient and safe manner remains an unmet need in the
biological sciences. Whether this means using gene therapy to introduce functional genes to
cells, or using RNA interference mechanisms to silence misregulated proteins, the main
hindrance in these approaches to disease mitigation, is the lack of a suitable delivery platform.
The first chapter is a literature review of the currently available technology for nucleic acid
delivery including viral vectors as well as cationic lipids and polymers. Herein the advantages
and disadvantages of both viral and non-viral delivery are discussed.
In Chapter II, a biopolymer featuring a lysine-histidine (KH) condensing region fused to a
targeting motif is discussed. The purpose of this study was to examine the effect of architecture
on transfection efficiency of plasmid DNA through a series of in vitro biological and
biochemical assays.
Chapter III describes a recombinant biopolymer which features an arginine-histidine (RH)
DNA condensing region, a targeting motif for HER2, and endosomal release fusogenic peptide,
and a nuclear localization signal. Each domain was tested for functionality through in vitro
vi
assays. The multifunctional biopolymer demonstrated selective and efficient delivery of plasmid
DNA and gene expression in SK-OV-3 cells.
In Chapter IV the RH based biopolymers were characterized and evaluated in terms of their
ability to deliver nucleic acids to either the cytoplasm or cell nucleus. Past delivery vectors have
often emphasized extracellular barriers to entry and success meant internalization of the particle.
Intracellular trafficking, however is also a key compenent in successful delivery. The objective
of this study was to design a biopolymer that can be programmed via its amino acid sequence to
deliver siRNA specifically to cytoplasm. By modifying the amino acid sequence, the same
biopolymer can also be programmed to deliver pDNA to the cell nucleus. Intracellular
trafficking was observed through fluorescent microscopy and assays were conductd to
demonstrate either siRNA knockdown or delivery of a suicide gene/prodrug combination.
In the research presented, we describe a non-viral biopolymer system that is able to
overcome the extracellular and intracellular barriers to nucleic acid delivery.
vii
Table of Contents
Acknowledgements ...................................................................................................................... iii
Abstract .......................................................................................................................................... v
Table of Contents ........................................................................................................................ vii
Table of Figures............................................................................................................................. x
Table of Tables ............................................................................................................................ xii
Chapter I. Nucleic Acid Delivery Methods: A Review .............................................................. 1
1.1. Gene Therapy ................................................................................................................ 1
1.2 Gene Silencing .............................................................................................................. 3
1.3 Nucleic Acid Delivery ................................................................................................... 5
1.3.1 Physical delivery ................................................................................................... 7
1.3.2 Viral Vectors ......................................................................................................... 9
1.4 Non Viral Vectors ....................................................................................................... 15
1.4.1 Non-viral Synthetic Vectors ................................................................................ 16
1.4.2 Cationic Lipids .................................................................................................... 16
1.4.3 Synthetic Cationic Polymers ............................................................................... 17
1.5 Biologically Inspired Motifs for Use in Nucleic Acid delivery ................................... 19
1.5.1 Nucleic Acid Condensation ................................................................................. 19
1.5.2 Targeting and Cellular Uptake ............................................................................ 21
1.5.3 Membrane Lysis/Endosomal Escape .................................................................. 22
1.5.4 Nuclear Localization and Import......................................................................... 24
1.6 Recombinant Biopolymers for Gene Delivery ............................................................ 28
viii
1.7 Conclusions ................................................................................................................. 35
1.8 References ................................................................................................................... 36
Chapter II. Lysine-Histidine Recombinant Cationic Biopolymers ........................................ 47
2.1 Background ................................................................................................................. 47
2.2 Evaluation of the Effect of Vector Architecture on DNA Condensation and Gene
Transfer Efficiency ...................................................................................................... 48
2.2.1 Abstract ............................................................................................................... 48
2.2.2 Introduction ......................................................................................................... 49
2.2.3 Materials and Methods ........................................................................................ 51
2.2.4 Results ................................................................................................................. 55
2.2.5 Discussion ........................................................................................................... 65
2.2.6 Conclusion ........................................................................................................... 71
2.2.7 Acknowledgement ............................................................................................... 72
2.2.8 References ........................................................................................................... 72
Chapter III. Arginine Histidine Recombinant Cationic Biopolymers for Gene Delivery ... 76
3.1 Background ................................................................................................................. 76
3.2 Biosynthesis and Characterization of a Novel Genetically Engineered Polymer for
Targeted Gene Transfer to Cancer Cells .................................................................... 77
3.2.1 Abstract ............................................................................................................... 77
3.2.2 Introduction ......................................................................................................... 78
3.2.3 Materials and Methods ........................................................................................ 81
3.2.4 Results ................................................................................................................. 86
ix
3.2.5 Discussion ........................................................................................................... 98
3.2.6 Future Directions ............................................................................................... 107
3.2.7 Acknowledgements ........................................................................................... 108
3.2.8 References ......................................................................................................... 108
Chapter IV: Arginine Histidine Recombinant Cationic Biopolymers for siRNA Delivery 112
4.1 Background ............................................................................................................... 112
4.2 A Genetically Engineered Multifunctional Polymer Designed for Site-Specific Nucleic
Acid Delivery ............................................................................................................ 112
4.2.1 Abstract ............................................................................................................. 112
4.2.2 Introduction ....................................................................................................... 113
4.2.3 Experimental Methods ...................................................................................... 116
4.2.4 Results ............................................................................................................... 121
4.2.5 Discussion ......................................................................................................... 131
4.2.6 Conclusions ....................................................................................................... 136
4.2.7 References ......................................................................................................... 137
Chapter V. General Conclusions and Future Directions ..................................................... 140
5.1 General Conclusions ................................................................................................. 140
5.2 Future Directions ...................................................................................................... 142
5.3 References ................................................................................................................. 145
Appendix A: Amino Acid and DNA Sequences ..................................................................... 147
x
Table of Figures
Chapter I.
Figure 1.1 Barriers to entry for nucleic acids, DNA and siRNA pathways .............................. 6
Figure 1.2 Structure of Linear Polyethyleneimine Monomer ................................................. 18
Figure 1.3 Nuclear Import of Protein with NLS ..................................................................... 26
Chapter II.
Figure 2. 1 Cloning Strategy and Amino Acid Sequence for cKH-FGF2 .............................. 56
Figure 2. 2 SDS and Western blot of cKH-FGF2 ................................................................... 57
Figure 2. 3 Particle Size and Stability Analysis of cKH-FGF2 and dKH-FGF2 .................... 58
Figure 2. 4 Cell Proliferation and Toxicity Assays for cKH-FGF2 and FGF2 Only.............. 60
Figure 2. 5 Percentage of Transfected Cells and Fluorescent Microscopy Images ............... 62
Figure 2. 6 Inhibition Assay................................................................................................... 63
Figure 2. 7 Luciferase Activity to Compare Transfection Efficiency ................................... 64
Figure 2. 8 Effect of Chloroquine on Transfection Efficiency .............................................. 65
Chapter III.
Figure 3. 1 Schematic of Multidomain Biopolymer ............................................................... 80
Figure 3. 2 Cloning, Expression and Characterization of the Purified Biopolymer ............... 87
Figure 3. 3 Digestion of Biopolymer by Proteases ................................................................. 88
Figure 3. 4 DNA Neutralization at Two pH Values ............................................................... 89
xi
Figure 3. 5 Size, Charge and Serum Stability of Bioplymer/pEGFP Complexes ................... 90
Figure 3. 6 Transfection Efficiency and Cell Toxicity at Various NP Ratios ........................ 92
Figure 3. 7 Evaluation of the Targeting Motif ........................................................................ 94
Figure 3. 8 Evaluation of the Functionality of the Fusogenic Peptide.................................... 96
Figure 3. 9 Evaluation of the Functionality of the Nuclear Localization Signal .................... 98
Figure 3. 10 The 3D Structure of M9-NLS Predicted by SWISS-MODEL Program .......... 107
Chapter IV.
Figure 4. 1 Schematic of FDT and FDNT ............................................................................ 115
Figure 4. 2 Evaluation of pEGFP Delivery to SK-OV-3 Cells Using FDNT or FDT .......... 123
Figure 4. 3 Real Time, Live Cell Imaging of Delivery to SK-OV-3 Cells ........................... 125
Figure 4. 4 Z-stack of SK-OV-3 cells Transfected with FDNT/pDNA or FDT/pDNA ....... 127
Figure 4. 5 Evaluation of GFP or BCL2 Knockdown Using FDNT or FDT Complexed With
siRNA for GFP or BCL2 ................................................................................................ 130
Figure 4. 6 Cell Killing Efficiency ....................................................................................... 131
xii
Table of Tables
Chapter I.
Table 1. 1 Viruses Used in Gene Therapy .............................................................................. 10
Table 1. 2 Naturally Occurring Biological Motifs for Nucleic Acid Condensation ............... 21
Table 1. 3 Naturally Occurring Biological Motifs for Endosomal Escape ............................. 24
1
1. Chapter I. Nucleic Acid Delivery Methods: A Review
This chapter is adapted from published work. Please see:
B.F. Canine, and A. Hatefi, Development of cationic recombinant polymers for gene therapy research,
Adv Drug Delivery Rev, 2010. (In press)
A. Hatefi and B.F. Canine, Perspectives in vector development for systemic cancer gene therapy. Gene
Ther Mol Biol 13 (2009) 15-19.
1.1. Gene Therapy
The concept of gene therapy has long been alluring to researchers because of the promise to
treat genetic based diseases at their origins through the transfer of genes into specific cells of a
patient. The technology of recombinant DNA was the first step in the realization of single gene
manipulation at the nucleic acid level. Precise control at the genetic level was a major stride
toward the realization of gene therapy as a therapeutic treatment.
The science of gene therapy has experienced great progress since the concept of gene therapy
was suggested. The first approved clinical trial for somatic gene therapy treatments was
performed in 1990. Thousands of patients have been involved in gene therapy clinical trials with
the greatest number of trials targeting cancer (64.6%) followed by vascular diseases (8.9%) and
monogenic diseases (8.1%).[1] The journey to this point has however revealed significant
problems, and solutions to these problems must be realized for gene therapy to reach its full
potential.
2
In the late 60’s the first gene therapy patients were treated in an unapproved clinical trial to
express arginase. Two girls with Arginase deficiency syndrome were treated with Shope
papilloma virus with little success.[2] Decades later, in the 1980’s β-thalassemia patients were
treated with bone marrow cells which were transfected with a a β-globin encoding plasmid. This
therapy was also not successful. Despite these early failures, gene therapy continued to be
pursued as a treatment option and two major scientific advances gave hope that gene therapy
would be viable. These were the sequencing of the human genome, the advent of recombinant
DNA technology, transfection reagents, and technologies which allowed for mammalian cell
transefction both in vivo and ex vivo. In 1990, an adenosine deaminase gene was transferred into
T-lymphocytes with a retroviral vector in attempt to treat patients with severe combined
immunodeficiency disease (SCID).[3]
While this trial showed promise, a more publicized gene therapy trial would bring doubt to
the safety and effectiveness of this new treatment. In 1999 at the University of Pennsylvania a
patient with ornithine transcarbamulase (OTC) received a gene encoding OTC in an adenovirus
vector. The patient, Jesse Gelsinger suffered a severe inflammatory response and died from
multi-organ failure believed to be due to immune system sensitization as a result of previous
adenovirus exposure. [4, 5] In 2000 a murine leukemia virus (MLV) vector carrying a γ-c chain
cytokine receptor was used to transduce hematopoietic stem cells ex vivo. These transduced cells
were then reinfused into patients with T and natural killer cell deficiencies. This trial was highly
successful in its intended treatment however two of the tree patients developed a leukemia like
disorder due to the randomness of the retrovirus genome integration which activated the LMO2
oncogene. [6, 7]
3
Hundreds of genes have been identified as targets for gene therapy, but as of yet, the FDA
has not approved gene therapy as a primary treatment for any disease. This is not due to the lack
of target genes, but due to the lack of a suitable way in which to deliver these genes both either
transiently or permanently. The following sections will address perhaps the most important
aspect of gene therapy—the delivery system. The current problems and future directions of the
existing technology will be discussed.
1.2 Gene Silencing
RNA inference (RNAi) is a highly conserved system found in eukaryotes that utilizes double
stranded RNA (dsRNA) to inhibit gene expression through degradation of mRNA in a sequence
specific fashion. It is a mechanism that has a role in controlling gene activity as well as
protecting the cells from foreign nucleic acids such as those introduced by pathogens.[8]
MicroRNA (miRNA) and short interfering RNA (siRNA) molecules are two types of small
RNAs that are the most well understood. siRNA results in gene knockdown in a sequence –
specific manner via degradation of complementary mRNA while miRNA results in translational
suppression in a less-sequence specific manner; it does not have to be a perfectly complementary
sequence to be degraded.[9] The pathway begins with the Dicer enzyme which recognizes long
double stranded RNA (dsRNA) and cleaves them into shorter fragments of ~20-25 base pairs.
These short pieces are then recognized by the RISC protein complex which unwinds the dsRNA .
One strand remains associated with the RISC complex and then binds it to the complementary
mRNA strand. This results in either translation arrest and/or cleavage of the mRNA. siRNA can
be utilized in this RNAi mechanism in three separate pathways: 1) introduction of synthetic
4
siRNA to the cytoplasm which leads to RISC complex loading, 2) introduction of longer siRNA
which is recognized by Dicer, or 3) introduction of a plasmid to express dsRNA which would be
made via complementary hairpin structures. [10]
The foremost concern of siRNA knockdown is off-target effects. This includes concerns of
non-specific binding to non-target sequences as well as induction of an immunogenic response.
Non-specific binding can be reduced by better genomic optimization and secondary structure
analysis. The induction of the immune response can also be reduced by examining secondary
structure or by utilizing a delivery vector which can also act as a targeting vehicle.
The first uses of siRNA in clinical trials were in treatment of macular degeneration and
respiratory syncytial virus (RSV). In both of these disease delivery vehicles were not required for
the siRNA as the eye and the lungs are easily accessible through direct injection or inhalation.
The first antisense oligonucleotide drug approved by the FDA was Vitravene in 1998. This was
to treat cytomegalovirus retinitis in AIDS patients. [11] Other antisense oligonucleotides have
been developed with limited success. The first siRNA therapeutic in clinical trials was Cand5 in
2004 which targets VEGF mRNA for age related macular degeneration (AMD) treatment and
showed visual improvement in Phase I trial patients. Another RNAi also for AMD, Sirna-027
also showed improvement in vision and was well tolerated. [12] Each of these therapies,
however required frequent, intraocular injections and as a result have not been pursued past
Phase II trials. In 2009, a therapy for AMD, bevasiranib, failed its Phase III trial due to a
nonspecific inflammatory responses and failure to result in more effective treatment than other
already approved therapies although the study identified no systemic or ocular safety issues.
Other trials examined the use of ALV-RSV01 in RSV infected lung transplant patients. Phase II
5
trials were completed in 2009 and showed a 40% reduction in viral load. Recruitment for Phase
2b trials are ongoing.[13] Systemic delivery of siRNA to solid melanoma tumors demonstrated
in Phase I trials that the siRNA inhibited expression of ribonucleotide reductase M2 (RRM2)
when delivered by a synthetic delivery vector that targets transferrin receptors. RMM2 is an
enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. [14] This
study showed that the tumors had reduced RRM2 levels several weeks after treatment. [15]
Other studies looking at treatment of other cancers, high cholesterol, and prevention of transplant
rejection are ongoing.
The understanding of the RNAi pathway has progressed rapidly in recent years and the
therapeutic targets pegged for siRNA therapy are numerous, however the efficient and targeted
delivery of siRNA still remains the largest challenge.
1.3 Nucleic Acid Delivery
Delivery and transfection of polynucleotides into specific cells of the body is the critical, and
to date, most elusive aspect of gene therapy. The main factor limiting successful gene therapy
seems to be the lack of a suitable delivery system to carry the nucleic acid therapeutics safely
and efficiently to the target tissue. [16] For systemically administered therapeutic nucleic acids
to successfully reach the target cells, a carrier (vector) should be designed to overcome cellular
barriers which are outlined in Figure 1 for both plasmid DNA and siRNA delivery.
6
Figure 1.1 Barriers to entry for nucleic acids, DNA and siRNA pathways
Schematic illustration of proposed pathway of gene delivery mediated by cationic vectors. The
positively charged vector interacts with nucleic acids to form condensed nano-size particles. The
particles must then be internalized. If the particles contain a targeting motif the targeting motif
binds to receptors over-expressed on the surface of target cells. This allows for internalization of
the complexes via receptor-mediated endocytosis. After internalization, endosomal escape is
necessary or the complexes will be degraded. Upon endosomal escape, a nuclear localization
signal, if present shuttles the pDNA towards the nucleus. If the cargo is pDNA upon entry to the
nucleus transcription of the gene would occur. If there is no nuclear localization signal is
present, then the cargo remains in the cytosol. siRNA would at this point enter the RNAi
pathway.
Accordingly, the carrier should be able to: a) condense DNA from a large micro-meter to a
smaller nano-meter scale suitable for endocytic uptake and protection from nuclease degradation,
b) be recognized by specific receptors on the target cells and internalize, c) promote the escape
of the gene from the endosomal compartment into the cytosol, and d) assist the translocation of
7
DNA from the cytosol to the nucleus for gene delivery or remain in the cytosol in the case of
siRNA delivery. [17] In addition, features of suitable vectors for clinical applications are low
toxicity/immongenicity while having high transfection efficiency, tissue specificity and being
cost effective to produce. Vectors carrying nucleic acids are commonly divided into viral and
non-viral categories. [4] A number of delivery vectors are used and are discussed below.
Unfortunately, all vectors have significant limitations, and research to find an improved delivery
vehicle continues.
1.3.1 Physical delivery
Physical methods of delivery are via technological means, meaning no exogenous carrier is
required and naked DNA is delivered.[18] These systems are often costly and complex as they
often require specialized equipment and are most widely used in in vitro studies, however as
technologies improve and costs are lowered these concerns may be addressed. In addition
physical methods generally have low throughput with transient expression.
1.3.1.1 Direct injection
Delivery of pure genetic material through direct tissue injection is a logical place to start with
gene delivery. While direct delivery of naked DNA can induce transgene expression when
delivered intramuscularly in vivo, degradation is an issue as DNase rapidly degrades and clears
the plasmid from tissue. [19, 20] Intramuscular injections localizes the DNA to the injection site
but cellular uptake must occur for transfection. This process is relatively inefficient as most of
the DNA is degraded and cleared by phagocytotic cells. [21] Multiple injections can increase the
level and duration of transgene expression, but the variability level is high. Additionally,
8
targeting specific cells only occurs in regard to spatial relationship to the injection and to cells
which are amenable to take up the DNA. For example, muscle cells are the only known non-
dividing cells that can be tranfected via this method thus limiting this avenue’s usefulness at
delivery of genetic material.
1.3.1.2 Gene Gun (Biolistic)
Originally developed as a technique to transform genes into plant cells, the gene gun has
recently been used to introduce genetic material into animal and human cells especially in
delivery of DNA vaccines. This technique is a bio-ballistic method for mechanical delivery of
DNA into cells both in vitro and in vivo. Cells are bombarded by micron sized or smaller
particles made of DNA coated with gold that are accelerated by pulses of compressed helium
gas. The advantage of this technique is that it works on cell types that are difficult to transfect
including neurons and other terminally differentiated cells. [22] The delivery is limited to surface
cells in the immediate area of treatment.
1.3.1.3 Electroporation
A commonly used in vitro technique, electroporation, uses an electric field to increase the
permeability of the cell membrane through the shifting of lipid molecules to form a nanometer-
sized pore that fills with water. [23] This technique has been used in primary neurons
successfully, however its in vivo use is severely limited. [24] The use of the electric field in
close proximity to cells limits its use to surface only applications and the cells are transfected in
a non-specific matter.
9
1.3.1.4 Magnetofection
Magnetofection uses nanoparticles made out of iron oxide that are coated with cationic
molecules. [25] These magnetic nanoparticles interact with the nucleic acids through electrostatic
interactions and colloidal aggregation. Particles are applied to cells followed by an external
magnetic field to draw the complexes into the cells. This increased concentration of the vector in
the cells results in more efficient transfection than if no magnetic field was used. The complexes
are taken in via endo or pinocytotic pathways and depending upon the formulation of the
nanoparticles, they can be released intracellularly via different mechanisms such as the proton
sponge effect or viral pathways. This technology has shown improved efficiency in a variety of
transfection applications. It has enhanced non-viral gene delivery as well as improved viral
transductions. The major downfall of this method is that complex formulations are needed as
magnetic nanoparticles alone are not sufficient to ensure high gene expression and depth of
penetration is limited. [26]
1.3.2 Viral Vectors
Another approach to gene delivery is through the use of viral vectors including retroviruses,
adenoviruses, adeno-assosciated viruses, or herpes simplex viruses. Viruses have evolved to
efficiently infect their host, overcome the cellular barriers, and transfer their genetic material into
the cell’s nucleus. There are five main classes of viral vectors which can be categorized into two
groups (Table 1) according to whether their genomes integrate into host cellular chromatin
10
(oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as
extrachromosomal episomes (AAVs, adenoviruses and herpes viruses).
Table 1. 1 Viruses Used in Gene Therapy
Viruses used for gene therapy can be divided into two categories 1) integrated meaning they result in
persistent gene transfer or 2) episomal meaning they result in transient gene transfer
1.3.2.1 Integrating
Integrating viruses used in gene therapy insert the therapeutic gene into the host genome for
expression. Commonly these are retroviruses or lentiviruses. These are desirable as vectors
because the insertion into the genome results in long term expression. They are also relatively
easy to produce and have cellular tropism.
1.3.2.2 Retrovirus & Lentivirus
Vector Immunogenic
Potential
Specificity Limitation Major Advantage
Integrated
Retrovirus Low Dividing
Cells only
Integration may induce
oncogenesis
Persistent gene transfer in
dividing cells
Lentivirus Low Broad Integration may induce
oncogenesis
Persistent gene transfer in
most cells
Episomal
AAV Low Broad Small packaging
capacity
Non-inflammatory and
non-pathogenic
Herpes Simplex
Virus
High High in
neurons
Transient expression in
some non-nueronal
cells
Large packaging capacity
Adenovirus High Broad (CAR
receptor)
Capsid may induce
inflammatory response
Efficient transduction of
most cells
11
Retroviruses are enveloped diploid single stranded positive sense RNA viruses that are
between 8-10 kilobases. The viral RNA is reverse transcribed into double stranded DNA which
is then integrated into the host chromosome after transport into the nucleus. Lentiviruses
specifically undergo an active nuclear import through the nuclear pore complex in non-dividing
cells. Advantages are low immunogenicity, large packaging capacity. Limitations include low
production yield, instability due to the envelope proteins, and random integration patterns that
pose a risk of insertional mutagenesis and potential oncogene activation. [27] Retroviruses
have been successful in treating the immune disorder SCID and continues to be used. However,
insertional mutagenesis has occurred in SCID-XI patients leading to leukemia due to activation
of oncogenes. [27-29]
1.3.2.3 Episomal (Non-Integrating)
Non-integrating viruses are used when transient gene expression is preferred and the
therapeutic DNA is retained as an independent extrachromosomal episome. [30] Examples of
episomal viruses include adenovirus and adeno-assosciated virus.
1.3.2.3.1 Adenovirus
One viral vector that has received considerable attention in cancer gene therapy is adenovirus
(Ad). Ad is a non-enveloped 36 kilo-base pair double stranded DNA virus. It is able to grow to
high-titer as a recombinant virus, has a large transgene capacity, and is able to transduce dividing
and non-dividing cells. [31-33] The basic elements of the trafficking pathway for adenovirus
include high affinity binding of the capsid to receptors on the cell surface, internalization by
12
endocytosis, lysis of the endosomal membrane resulting in escape to the cytosol, trafficking
along microtubules, binding to the nuclear envelope, and insertion of the viral genome through
the nuclear pore. [34] Adenoviruses have high affinity for the coxsackievirus-adenovirus
receptor (CAR) and use it to enter the cells. Although they are highly efficient in transducing
cells that over-express CAR on their surface, they are considered poor gene delivery systems in
cells that have low expression of CAR. [35] In addition, CAR is expressed on many normal cells
which undermines the ability of this vector to specifically reach target cancer cells when
administered systemically. Thus, adenovirus is not considered a universally efficient vehicle for
cancer gene therapy as the majority of cancer cells do not over-express CAR. [36]
Attachment of a targeting ligand to the viral capsid has been used as a means to make
adenovirus specifically bind cancer cells and internalize via receptor mediated endocytosis. One
example is attachment of the ligand, fibroblast growth factor 2 (FGF2), which has affinity for the
basic fibroblast growth factor receptor (FGFR). [37] This receptor is over-expressed in
subpopulations of lung, prostate and breast cancer. [38] While promising, the attachment of the
ligand to the virus capsid involves chemical conjugation during which a significant portion of
viruses could become inactive. As a result, obtaining high titers of active virus for delivery
becomes expensive. Alternatively, retargeted viruses can be genetically engineered through the
abrogation of CAR binding (e.g., Y477A mutation in adenoviral fiber protein) and insertion of a
receptor-specific binding peptide in the HI loop of the fiber protein. [39] In this approach, no
chemical conjugation step is involved. However, one potential problem with this approach is that
targeting peptides with considerable 3D structure could interfere with the proper packaging of
the viral capsid proteins and result in reduced transduction efficiency. Furthermore, such
13
alterations in receptor targeting could impact transduction efficiency of viruses due to the change
in trafficking routes and internalization pathways. [40]
Besides transduction of off-target cells a major limitation of Ad is the possibility of
preexisting immunity and the resulting vector immunogenicity upon administration. [41] Only
herpes simplex virus (HSV) and adenovirus (Ad) have been shown to be highly immunogenic. In
general, introduction of any non-self molecule, including viruses, into the body has the potential
to trigger an immune response. However, the level of immune response to the foreign entity is
dependent on the dose, the structure and any previous exposures. For example, a patient (Jesse
Gelsinger) who suffered from a partial deficiency of ornithine transcarbamylase (OTC) took part
in a gene therapy clinical trial conducted at the University of Pennsylvania in 1999. OTC is a
liver enzyme that is required for the safe removal of excessive nitrogen from amino acids and
proteins. Gelsinger received the highest dose of vector in the trial (3.8 × 1013
particles). After 4
hours of treatment Gelsinger developed a high fever and within four days of treatment he died
from multiorgan failure. A female patient who received a similar dose (3.6 × 1013
particles)
experienced no unexpected side effects. It has been speculated that previous exposure to a wild-
type virus infection might have sensitized Gelsinger’s immune system to the vector. [5] If a
lower dose of the adenovirus was administered, Gelsinger’s symptoms may not have been as
catastrophic. Therefore, drawing a firm conclusion that viral vectors are highly immunogenic
and deadly is premature.
1.3.2.3.2 Herpes Simplex Virus
14
Herpes Simplex Virus overcomes the deficiency of the CAR receptor by utilizing a
different receptor to enter cancer cells. The initial attachment of HSV involves the interaction of
viral envelope glycoproteins with the glycosaminoglycan moieties of cell surface heparin
sulfates. [42] However, like CAR, expression of heparin sulfates is not unique to cancer cells and
can be found routinely in normal cells. As a result, systemic administration of HSV could also be
problematic.
1.3.2.3.3 Adeno-Assosciated Virus
Adeno-Assosciated Virus (AAV) is a nonpathogenic, nonenveloped, 4.7 kb single stranded
DNA virus. AAV requires the presence of a helper virus, usually adenovirus or hepesvirus to
complete its lifecycle as it does not have sufficient proteins encoded in its genetic material to
complete its lifecycle independently. Thus far it has not been the source of any pathogenesis in
human disease and AAV vectors have resulted in long term gene expression in many different
cell types. [43-45] The capsid of the virus will determine which receptors the virus will bind to.
AAV binds to heparin sulfate proteoglycan (HSPG), sialic acid, fibroblast growth factor receptor
(FGFR) and platelet-derived growth factor receptor (PDGFR) among others. [46] Success with
AAV vectors has been demonstrated in retinal degeneration studies reported in 2008. [46-48]
The limitations of AAV include genome packaging size, transduction of off target cells, and poor
transduction of certain cell types as well as immunity concerns dealing with helper virus
concerns. [43, 47-50] Additionally, in 2006 it was reported that during AAV gene therapy
treatment for haemophillia resulted in an anti-viral immune response that destroyed and damaged
the transduced hepatocytes. [43] Other studies have shown some evidence that a majority of
15
people has been exposed to the most common AAV serotypes. This results in circulating
neutralizing antibodies and a conditioned immune response. [51]
1.3.2.4 Viral Delivery Vectors Summary
Attempts at gene therapy began over 30 years ago and the first attempts utilized viral vectors
to transfer genetic material to gene deficient cells. Viral vectors were, and continue to be, highly
efficient at gene transfer, however after more than three decades of research, critical issues still
surround their clinical use. These issues include maintenance of long-term efficacy and bio-
compatibility without inducing immunogenic or toxic responses.
While viruses have yielded a few successes, much recent work has gone towards
development of synthetic viral vectors. Such vectors would mimic the viral delivery pathways
used during infection.
1.4 Non Viral Vectors
Non-viral delivery approaches apply physical, chemical, or biological approaches to safely
and efficiently deliver nucleic acids to the target cells. Like all delivery methods, non-viral
vectors must protect the nucleic acid from degradation, circumvent the intracellular and
extracellular barriers to entry, and have minimal detrimental effects. Advantages for non-viral
vectors include greater safety and lower immunogenicity in comparison to viral vectors. This in
turn would allow for repeated delivery and more flexibility in systemic administration. Two
classes of non-viral vectors will be discussed, synthetic and recombinant biological vectors.
16
1.4.1 Non-viral Synthetic Vectors
To fulfill the deficiencies associated with viral vectors, synthetic non-viral vectors such as
cationic lipids and polymers have emerged as potential safer alternatives. There are two broad
categories of non-viral synthetic vectors 1) cationic lipids and 2) cationic polymers.
1.4.2 Cationic Lipids
While lipid based gene carriers (lipoplexes or liposomes) provide high transfection
efficiency, their large scale production, reproducibility and cytotoxicity remain a major concern.
[52] Two common cationic lipids are 1,2 dilexyloxy-3-trimethylammonium propane (DOTAP)
and N-[1-(2,3-diolexyloxypropyl ]-N,N,N trimethylammonium chloride (DOTMA)]. Lipofectin
2000™ which is a DOTMA/DOPE (dioleoylphosphatidylethanolamine) formulation is a
commercially available formulation. The cationic lipid interacts via electrostatic interactions
with the negatively charged DNA. The DNA is packaged inside the cationic liposome which
then targets the cells via cell surface proteoglycan receptors and DNA is released into the cell.
The transfection of the lipoplexes are affected by several factors including the structure of the
cationic lipid, lipid to DNA charge ratio, structure and proportion of the helper lipid in the
complex, the surface charge and complex size, the total dose of lipoplexes used, and the cell
type. [53] A highly positive surface charge is the major drawback of using cationic lipids for
gene delivery due to the non specific binding to cell membranes. This leads to significant
toxicity as well as serum protein binding which can lead to aggregation. [54] One method to
reduce the non-specific cytotoxicity of the lipid-based vectors is the attachment of targeting
peptides of PEG to their surface. This will reduce the surface positive charge, minimize non-
17
specific toxicity and enhance cellular uptake. [55, 56] Polyethylene glycol (PEG) is a common
surface modifier that is used to mask epitopes and stabilize surfaces and is generally considered a
safe polymer. However, repeated use of PEGylated liposomes has been implicated in eliciting
IgM/IgG response in rats and mice. [57-60] Another source of concern is the size heterogenicity
of lipoplexes which has an impact on the gene transfer reproducibility as size is directly tied to
the way in which particles enter a cell. In targeted lipoplexes, receptor mediated entry is through
the clathrin-coated pathway and optimal sizes are below 150 nm. At sizes above 150 nm,
cellular uptake is skewed towards other non-specific pathways. [61] While cationic lipids afford
relatively high gene transfer efficiency, reproducible large scale production methods and vector
related cytotoxicity remain as major points of concern. [52] Unfortunately, reproducible ligand
attachment is still a significant challenge, primarily due to the thermodynamically driven (as
opposed to genetically driven) limitations of the chemical synthetic methods. Reproducibility of
the ligand attachment process is critically important as it impacts ligand density on the
nanoparticle surface which in turn affects their binding affinity toward receptors. The importance
of ligand density and its impact on receptor binding and gene transfer efficiency has been
discussed in detail in reference [39].
1.4.3 Synthetic Cationic Polymers
The positive charges on the polymers interact with the negatively charged phosphate groups
on the DNA resulting in the formation of polyplexes. Two main categories of synthetic cationic
polymers have emerged; non-degradeable and degradeable polymers. The most common non-
degradeable polymer for DNA delivery is polyethyleinimine (PEI) which is water soluble. PEI
18
is available as a linear or branched polymer and in many different molecular weights, with the
most common being 25 kDa.
Figure 1.2 Structure of Linear Polyethyleneimine Monomer[62]
The positive charge of PEI comes from the amino group on each monomer. While they have
shown some efficacy in vivo, the major drawback to PEI is their toxicity profile and studies have
shown both immediate and delayed cytotoxicities. [52] The high number of positive charges
often leads to aggregation and interactions with serum proteins. To reduce these interactions
PEG and other polymers have been used to shield and stabilize the surface of particles. While
PEI has shown cellular toxicity, it is believed to be non-immunogenic and have low
immunogenic potential. [52] PEI also cannot be metabolized or eliminated by the body which
leads to tissue accumulation further exacerbating toxicity concerns especially in repeated dosing
schemes.
Efforts have been made to overcome these toxicity concerns by using degradeable polymers.
These include poly(L-lysine) (PLL), poly (α-[4-aminobutyl]-L-glycolic acid (PAGA) and poly
(4-hydroxy)-L-proline ester. These polymers contain hydrolysis sensitive bonds which allow
enzymatic or chemical decomposition. It has been shown that the lower the molecular weight
the lower the toxicity of the polymer. In the case of these polymers, the lower toxicity can be
19
attributed to the hydrolysis rate. Degradeable polymers, while low in toxicity, also have lower
transfection efficiencies due to rapid removal from circulation. [63, 64]
1.5 Biologically Inspired Motifs for Use in Nucleic Acid delivery
Biologically inspired product development is the process of designing products inspired by
processes and structures found in nature. Called biomimetics, this area of science aims to imitate
the materials seen in nature. Bio-inspired designs utilize blueprints that nature has been
perfecting for eons, making this a highly effective design strategy. Natural designs are also
highly efficient as survival is often based upon using the least amount of energy and fewest
resources possible. Nature is also highly adaptive due to both short-term and long-term
evolution. Many products have evolved from borrowing ideas from the natural world including
Velcro which was inspired by plant burrs, polymer ceramics modeled after seashells, and even
synthetic silk based upon naturally produced spider silk. Utilizing biologically inspired motifs
from nature for nucleic acid delivery is no different from the examples above. Viruses have
evolved to overcome the naturally occurring cellular barriers to entry, and often times, specific
peptides perform precise basic functions. As described previously, there are many intra and
extracellular barriers to entry (Figure 1) and the importance of each functional group in
overcoming these hurdles will be discussed.
1.5.1 Nucleic Acid Condensation
The first hurdle in nucleic acid delivery is condensation of the material into sizes suitable for
cellular uptake. Non-condensed pDNA, for example, is microns in size and this bulk is
20
prohibitive for cellular internalization. Condensation of pDNA is a vital first step and the
formation of stable DNA-vehicle complexes is a requirement for efficient gene delivery as it
protects the nucleic acid cargo from endonuclease degradation. Physical stability refers to the
stable encapsulation due to electrostatic interactions between the vector and plasmid DNA.
Generally non-viral delivery systems condense DNA through electrostatic charge interactions.
This means that the surface charge of the complexes relies heavily on the cation/anion ratio. A
net positive charge is desirable in terms of binding activity and results in higher cellular uptake
in vitro. In vivo, however a net positive charge makes binding to negatively charged molecules
more likely. This includes the highly abundant serum albumin proteins and leads to aggregation
and loss of biological activity of complexes.
DNA condensation in nature is often facilitated by cationic peptides rich in arginine,
histidine, and lysine, all amino acids which contain basic groups. In chromosomal DNA, a
handful of histone peptides work in concert to result in strong condensation. For the purposes of
this work only a few of the most studied DNA condensing motifs will be discussed. Table 1.2
shows some examples of these peptide motifs, their origins, and their function. The adenoviral
core peptide, mu (µ), is a cationic peptide rich in arginine and shows superiority to the VP1
peptide from polyomavirus and the protein protamine in both charge neutralization and
condensation. [65] Mu-DNA complexes were between 80-100 nm and showed size stability
amenable to stable particle formation. [66] Additionally, µ was used in vivo to enhance
transfection of cholesterol cationic liposomes into differentiated and undifferentiated neuronal
cells. [67] Despite advantages in DNA neutralization and condensation, µ peptide did not
enhance endosomal disruption or nuclear localization, both major limitations.
21
Histones, as mentioned previously are naturally occurring proteins of a highly cationic nature
and are involved in DNA packaging in cells. Histones also have an inherent nuclear localization
signal to facilitate their nuclear import. [68] This dual functionality peptide has a string of
lysine/arginine which both condenses DNA and facilitates nuclear translocation. Full length
histones have not been efficient gene delivery agents and required complexation with polymers
or liposomes to mediate efficient gene delivery. [69]
Motif Function Origin
µ peptide[70] DNA neutralization/condensation Adenoviral core peptide
VPI[65] DNA neutralization /condensation Polyomavirus
Protamine[70] DNA neutralization/ condensation Spermatogenesis
SPKK
repeats[71]
DNA neutralization/ condensation Histone H1
Histone [72] DNA neutralization/condensation and nuclear
localization
Chromosomal packaging in
cells
Table 1. 2 Naturally Occurring Biological Motifs for Nucleic Acid Condensation
1.5.2 Targeting and Cellular Uptake
Negatively charged, naked nucleic acids do not associate with the cell surface.
Neutralization of the negative charge by cations, has been shown to allow accumulation on the
cell surface and triggers the accumulation of heparin sulfate proteoglycans (HSPGs) which are
involved in cellular uptake. This is not cell specific and does not discriminate between cell type.
In order to perform targeted gene delivery to the specific cells of interest, and spare normal cells
22
from treatment, different biologically active targeting motifs have been linked to the delivery
vector. For instance folate and transferrin have been covalently linked to both cationic polymers
and liposomes. As more biomarkers are discovered for specific disease states the number of
targeting ligands will also expand.
1.5.3 Membrane Lysis/Endosomal Escape
Uptake via endocytosis results in the particles being encapsulatd inside endsosomes. The
acidity of the endosomal compartment will gradually drop to pH 5.5 as the ATPase proton pump
is activated. These late state endosommes will fuse with lysosymes and pH will drop to 5.0. The
acidification will activate lysosomal enzymes and lead to degradation of contents. Endosomal
entrapment is one of the major reasons for low efficiency gene transfer in non-viral delivery
systems. There have been several biomimetic strategies utilized to disrupt endsosomal
compartments. The first is disruption via osmotic pressure through the use of lysosomotropic
agents. Perhaps the most well known of these is chloroquine, a lysosomotrophic agent which
works by buffering the acidic pH of the endosome resulting in swelling and bursting of the
endosomal compartment. In vivo this would not be a feasible due to the non-specific cytotoxicity
of the drug. Cationic polymers also have lysosomotropic effects to do the tertiary amine group
which protonates when the proton (H+) influx acidifies the environment. As the tertiary amines
absorb the protons, an excessive influx of H+ increases the osmotic pressure and results in
osmostic rupture of the endosome. This is known as the proton sponge effect. [73]
Borrowing again from nature, endosomes may also be disrupted with fusogenic peptides also
commonly referred to as amphiphillic peptides. The influenza virus utilizes this strategy. The
23
hemagglutinin-A2 (HA2) peptide is a random coil at physiological pH and converts to an α-helix
when the pH becomes acidic. The α-helix fuses with the membrane and leads to pore formation
in the endosomal membrane. The 20 amino acids of HA2 (GLFGAIAGFIENGWEGMIDG)
shows the presence of glutamate residues at regular intervals which will protonate under acidic
conditions. Several variations of the HA2 peptide, discussed below, have been made to have
different pH trigger levels to modulate their endosomal disruption behavior. GALA is 30 amino
acids long with Glu-Ala-Leu-Ala repeats (WEAALAEALAEALAEHLAEALAEALEALAA)
and forms the alpha-helix confirmation at pH 5.0 due to increases in hydrophobicidy on one side
of the helical strucure which enhances interaction with the lipid bilayer. At physiological pH the
charge repulsion between Glu residues destabilizes the helical structure and prevents membrane
interaction. [74] GALA does not participate in DNA condensation or aid in nuclear localization,
but has been shown to successfully deliver siRNA in a bifunctional R8/GALA. [75]
To overcome GALA’s inability to condense nucleic acids a variant, KALA, was designed
(WEAKLAKALAKALAKHLAKALAKALKACEA). [76] The lysine-alanine-leucine-alanine
units result in both a hydrophobic and hydrophilic region while also having lysines to bind DNA
and glutamates to trigger pH dependent protonation. The KALA peptide was used to deliver
DNA in a gal-PLL system and showed expression than when delivery was done even without
chloroquine treatment. [77] It was also used to deliver VEGF siRNA and when compard to PEI
and PLL had the lowest IC50. Sub 200 nm sizes resulted in 90% VEGF inhibition. [78]
In 1998, another cationic derivative of the N-termainal HA2 peptide was reported. [79]
H5WYG is designed to trigger conformational changes at the slightly acid pH of 6.8. The
glutamate residues were substituted with histidine residues which protonate at pH 6.0. This
24
results in endosomal membrane permeabilization before reaching lower pH levels of late
endosomes or early lysosomes.
Motif Function Origin
Mellitin[80] Membrane lysis Bee Venom
HA2[81] Membrane disruption Influenza
H5WYG Membrane disruption Derivative of HA2
GALA Membrane disruption Derivative of HA2
KALA Membrane disruption Derivative of HA2
Table 1. 3 Naturally Occurring Biological Motifs for Endosomal Escape
1.5.4 Nuclear Localization and Import
Many non-viral gene delivery systems have been designed to overcome the barrier of the cell
membrane to result in gene delivery to the cytoplasm. This delivery to the cytoplasm, however
does not correlate with efficient cell transfection [82]. The specifics of the molecular
mechanisms involved are not fully understood, but it is believed that several factors including a
crowded cellular environment as well as the presence of cytoplasmic nucleases prevent the naked
DNA from reaching the nucleus. [83] Upon endosomal release, the nucleic acid/vector complex
must translocate to the site of action and in the case of plasmid DNA, the site of action is the
nucleus. The passive diffusion coefficient of DNA is low and inversely related to its size due to
the cystoskeletal matrix and numerous organelles and vesicles. While translocation mechanisms
25
have not been fully explained, several studies have implicated microtubules as the means through
which complexes are actively transported towards the peri-nuclear compartment through a motor
driven mechanism that utilizes a nuclear localization signal to direct transport. [84]
Nuclear import is another hurdle separate from nuclear translocation and there are believed to
be three scenarios through which particles enter the nucleus. The first is passive diffusion
through nuclear pores if particles are less than 10 nm in size.[85, 86] The second is active
transport through nuclear pore complexes (NPC) which are located in the nuclear envelope. This
has been shown to occur in particles less than 40 nm in size that also carry recognized
signals.[87] The third is that particles accumulate in the perinuclear area and enter the cell
during mitosis when the nuclear envelope dissolves. For larger particles this is thought to be the
most likely scenario. For the smaller particles, it is thought that the nuclear localization signal
(NLS) facilitates binding of the protein carrier to members of the importin-α family of proteins,
also referred to as karyoperins, Translocation through the nuclear pore then occurs in a GTP-
dependent manner. [88] The complex then falls apart inside the nucleus and releases the protein
in the presence of RanGTP. This process is depicted in Figure 2. [88][89]
26
Figure 1.3 Nuclear Import of Protein with NLS
Proteins containing a NLS are thought to interact with importin proteins which then facilitate
binding to the nuclear pore complex. Translocation is then facilitated in a GTP dependent
manner.
Another less accepted mechanism for PEI/DNA complexes has been hypothesized that after
osmotic disruption of endosomes the complexes are coated with a phospholipid membrane which
facilitates nuclear membrane fusion and contributes to nuclear import of the particles. [90]
Nuclear localization signals are short peptide sequences that are recognized by cellular
machinery and trigger the active nuclear translocation. Many NLS’s have been identified, but
the SV40 large T-Antigen is the classic example. [91] The simian virus 40 large tumor antigen
nuclear localization sequence (SV40 large T-Antigen NLS) is made up of primarily basic amino
acids and has the sequence PKKKRKV. [91] This sequence binds to the importin machinery in a
highly specific manner, as single amino acid changes have shown impaired nuclear import. [92]
27
Several groups have used the SV40 NLS and chemically conjugated it to either cationic
polymers with condensed DNA or directly to linear DNA. No improvement in transfection
efficiency was shown in these cases in a variety of cell lines. [93] It is speculated that the basic
residues in the NLS are electrostatically interacting with the DNA and reducing accessibility of
the NLS to the importin binding sites. PEG spaces between the DNA and the NLS increased the
transfection efficiency proportionally to the spacer length. [94]
Another question that is often raised concerns the number of NLS peptides needed to mediate
nuclear import. A study in 1999 by Zanta et al. showed that a single NLS was sufficient to
mediate nuclear entry. [95] A linear piece of DNA was conjugated to a single SV40 NLS
peptide and was used in conjunction with PEI (25 kDa) or Transfectam™. DNA that was
conjugated to the NLS was 10 to 1000 fold more efficient than DNA without NLS or mutated
NLS. Nuclear import of DNA-NLS/PEI complexes however, was not shown and the highest
efficiency was obtained in rapidly dividing cells while non-dividing cells had only a small
increase. If the sole reason for nuclear import was the NLS, one would expect no difference
between the dividing and non-dividing cells. Also of concern was the use of PEI to condense the
DNA. PEI does not condense DNA at a 1:1 ratio and it has been shown that PEI stays
complexed inside the cytoplasm, and as a result multiple NLS would be present in every
complex, making the single NLS statement invalid. [96]
Another common NLS is from the Rev protein which is found in human immunodeficiency
virus (HIV) where it regulates the transport of structural mRNAs from the cytoplasm to the
nucleus. [97] Rev peptide is 15 amino acids (RQARRNRRNRRRRWR) and is highly arginine
rich. The role of this protein was elucidated in 1990 by showing the accumulation of a luciferase
28
tagged peptide in the nucleus. [98] This was further proved by linking the Rev peptide to the
S413 peptide, a cell penetrating sequence from dermaseptin. [99] S413 alone did not enter the
nucleus but the linked Rev-S413 did show nuclear entry. [100]
The M9 peptide has the amino acid sequence GNYNNQSSNFGPMKGGNFGGRS
SGPYGGGGQYFAKPRNQGGY and is derived from the heterogeneous nuclear ribonuclear
protein (hnRNP) A1. hnRNP A1 has the ability to shuttle between the nucleus and the
cytoplasm due to its role as a pre-mRNA building protein. [101] The portion of hnRNP A1 that
was identified as the nuclear localization signal was been designated M9. [102] Unlike classic
NLS signals, which are highly rich in basic amino acids, the M9 NLS uses transportin for nuclear
import which is independent of the importin pathway discussed previously. [103] M9 was
conjugated to a 13 amino acid cationic peptide (ScT) and used to delivery a β-gal reporter
plasmid. This resulted in >75% transfection in bovine endothelial cells in vitro. Lipofectamine
was also used to disrupt endosomes in these studies, indicating M9 is not a viable endosomal
disruption peptide. [104, 105]
Transport to and across the nuclear membrane is a major barrier to non-viral gene delivery
and NLS peptides may be the solution to overcome this hurdle. As sequencing and molecular
studies become more proficient and new viral mechanisms are elucidated, more sophisticated
NLS peptides may be identified and utilized in delivery system strategies.
1.6 Recombinant Biopolymers for Gene Delivery
Many biological motifs have been utilized to deliver nucleic acids into mammalian cells.
None of the motifs discussed, or discovered to date have the ability to circumvent all cellular
29
hurdles in targeted gene therapy. Recombinant DNA technology gives researchers a tool which
allows combination of these motifs into a backbone. These fusion proteins have been inspired by
viruses and the multi-domain nature of these vectors allows for discrete functionality and
effective nucleic acid transport. It was suggested by Tecle that one potential solution would be
to combine positively charged peptides with peptides having other functions.[106] One concern
with using peptides to complex DNA is the production of homogenous particles and Tecle
showed more controlled condensation of plasmid DNA specifically with the fusion constructs.
[106]
In 1997 Overell, used recombinant DNA technology to create a protein vector made of
GAL4 (a DNA binding domain of a transcription factor in yeast) concatenated with a
binding/internalization domain.[107] The vector was complexed with a pDNA reporter and the
complexes did transfect cells, but failed to adequately condense DNA. PLL was added to further
condense the DNA and chloroquine was used to improve transfection efficiency. The inability
of this construct to condense pDNA in addition to inefficient endosomal escape, and the lack of a
nuclear localization signal are major weakenesses of this multifunctional non-viral vector. In
1998, Wel’s group used the same DNA binding domain, GAL4, in tandem with the translocation
domain of Pseudomonas exotoxin A for endosome disruption and the epidermal growth factor
(EGF) for a targeting motif. [108] Again pDNA was not condensed, but the DNA did target cells
over-expressing EGF. PLL was used to supplement condensation and chloroquine again
improved endosome disruption. No specific membrane lysis assays were performed so the
reason for suboptimal endosome disruption is not known. [108] Further work by this group
looked at another fusion protein named GD5. [109] GD5 has a C-terminal ErbB2 antibody
30
fragment to bind to tumors that express ErbB2 in addition to an N-terminal Gal4 binding domain
and a diphtheria toxin translocation domain between the two. [109] Again PLL was used to fully
condense the DNA and chloroquine enhanced endosomal escape. When creating these multi-
functional gene delivery systems it is of utmost importance that each domain retains its
individual specific activity while being on the same scaffold as the other domains. As these
early fusion vectors showed little promise, the development of these vectors was not pursued as
rigorously as other non-viral vectors. Great technological strides have been made in the years
since these early vectors. Even the advent of kits and standardized protocols for both protein
expression and gene manipulation has led to an increase in interest in these bio-inspired vectors.
In addition, the safety risks associated with viruses and concerns about chemicals in synthetic
polymers have made the biological expression of fusion proteins an appealing alternative. As the
inability to explain or predict transfection efficiency of non-viral vectors results partly from
insufficient understanding of the intracellular processes, development of a new class of
biomaterials is required to provide the possibility of performing reliable structure/activity
relationship studies. Such studies would help the scientists to better understand the rate limiting
steps to each specific vector, devise new approaches to overcome the deficiencies and develop
non-viral vectors that could potentially be more efficient than viruses. One class of biomaterials
that allows precise correlation of structure with function is recombinant polymers (biopolymers).
The advent of recombinant DNA technology allowed the design and development of
recombinant polymers for use in drug/gene delivery offering several advantages over more
conventional methods. Synthetic methods of polymer production utilize conventional
thermodynamically-driven chemical synthesis techniques which result in heterogeneous products
31
manifested by molecular weight distributions. If biological motifs (e.g., targeting peptides,
nuclear localization signals, etc.) are to be incorporated in the polymer structure, conjugation
must be followed by purification steps, which could add significantly to the costs. In contrast,
amino acid based polymers are synthesized using genetic engineering techniques in biological
systems (e.g., E. coli) resulting in homogeneous biopolymers with specific compositions where
functions can be dictated via amino acid sequence (programmability). [110] This allows multiple
functionalities to be incorporated onto a single biopolymer backbone by merely changing the
gene encoding the amino acid instructions. This concept is demonstrated in the following
chapters where a single chain biopolymer can perform several distinct tasks sequentially. In
terms of safety, the endotoxins (structural component in the bacteria cell wall) can be simply
removed during the washing steps of the biopolymer purification process by affinity
chromatography. Given the fact that there is no need for the removal of toxic solvents or un-
reacted monomers, such biopolymers could be just as cost-effective as synthetic polymers, if not
more so.
When compared to viral vectors, biopolymers can be made at significantly lower cost and
with fewer safety concerns. For example, virus production must be performed within Biosafety
Level 2 and 3 (BSL2/3) facilities, whereas recombinant biopolymers can be produced in large
amounts in BSL1 laboratories. Furthermore, biopolymers are produced in E .coli which is
among the most efficient and cost-effective methods of protein production. [111, 112] This is in
contrast to the methods of virus production which require painstaking and time-consuming
processes to reach high titers (>108 pfu/ml) suitable for clinical applications.
32
An alternative to biological synthesis of biopolymers is synthetic peptide production.
This approach generally relies on organic chemistry solid phase synthesis. Solid phase
synthesis is often limited by reaction yields and thus restrains the length of the peptide that can
be made. Longer peptides can be made by chemical linkage of these shorter fragments but this
is usually a prohibitively expensive process when large quantities are needed. [113] In the past,
technological hurdles have restricted the supply of readily available recombinant polymers. As
these technological obstacles are overcome, recombinant polymers will fill a void in the gene
delivery arena. This review highlights the evolution that recombinant polymers have undergone
thus far, ranging from simple bi-functional to the more complex multi-functional biopolymers.
For purposes of this article the word “biopolymer” refers only to recombinant amino acid based
polymers in order to differentiate from synthetic polymers (e.g., PEI) and natural polymers
(e.g., chitosan).
Aris et al. (2000) were among the first to report the genetic engineering of a gene delivery
system, namely 24 9AL, composed of a cationic lysine oligomer (K10) fused to a β-galactosidase-
derived protein displaying arginine-glycine-aspartic acid (RGD) cell attachment peptide. [114]
The role of K10 was to condense plasmid DNA (pDNA) and the RGD was for binding to the αVβ3
integrins on the cell surfaces. [115] It was also speculated, but not shown in this paper, that the
β-galactosidase could act as a DNA protector as well as a nuclear targeting motif.
The ability of the gene carrier to also mediate gene expression was examined by complexing
249AL with pDNA encoding luciferase reporter gene and transfecting CaCo2 cells. While the
total luciferase gene expression was reported, the percentage of transfected cells was not
measured. Because the 249AL was designed to target, the percent transfected cell results in terms
33
of the total gene expression could provide a better understanding of the efficiency of the gene
delivery system. The transfection efficiency of 249AL was compared with lipofectamine and
shown to be significantly less efficient. This was expected as 249AL is not well-equipped to
escape from the endosomal compartments. One point worth emphasizing is that comparing gene
transfer efficiency of targeted vectors such as 249AL with non-targeted ones (e.g.,
lipofectamine) may not be appropriate as they internalize via entirely different pathways. As a
result, the efficiency of non-targeted vectors should be discussed in the context of each cell type
rather than generalization to all mammalian cells. This importance is discussed in more detailin
reference [116]. Nonetheless, this study was among the early reports on the use of genetically
engineering techniques to make a fusion vector with gene delivery application.
In a similar approach, Furgeson’s group (2008) reported the development of a recombinant
elastin-based cationic diblock biopolymer for gene delivery. [117] This biopolymer consisted of
a cationic oligomer block (VGK8G) fused to a thermoresponsive elastinlike polymer (ELP) with
60 repeats of (VPGXG) where X is V, A, or G in a 5:2:3 ratio. They utilized a recursive
directional ligation method to synthesize the gene, which is a pseudo biosynthetic route achieved
in bacterial cell culture. [117, 118] ELPs are biocompatible and undergo a rapid reversible phase
transition at a temperature which is a function of the type of guest residue, the ionic state, and the
molecular weight among other factors. [119, 120] This system was specifically designed for use
in hyperthermic gene therapy. Hyperthermic gene therapy combines traditional local
hyperthermia after delivery of the biopolymer/pDNA to activate the biopolymer. The
biopolymer [K8-ELP(1-60)] was genetically engineered in E. coli and characterized. It was
shown that it can not only condense pDNA encoding green fluorescent protein (pEGFP) into
34
nanosize particles, but also responds to heat and goes through thermal transition. The results of
the MCF-7 cell transfection studies demonstrated the ability of the system to mediate gene
expression. Unfortunately, the transfection of the cells was only visualized and no total gene
expression or percent transfected cells were reported. Because the transfection studies were
performed in the presence of chloroquine, it can be deduced that the gene delivery system was
not able to escape from the endosomal compartments efficiently. This was expected as [K8-
ELP(1-60)] was not equipped with any endosomolytic motif to facilitate its escape from
endosomes. Before proceeding further in clinical administration as a gene therapy vector, the
efficiency, targeting ability, and the thermal transition point for this type of recombinant cationic
polymer need to be optimized.
In an attempt to overcome the endosomal barrier and also provide targetability, Ghandehari’s
group (2006) reported the structure of the first recombinant cationic biopolymer with tandem
repeating units composed of lysine (K) and histidine (H) residues fused to fibroblast growth
factor 2 (FGF2). [121] The biopolymer with the general structure of (KHKHKHKHKK)6-FGF2
or in short dKH-FGF2, contains 36 lysine residues (K) in the dKH segment to condense pDNA,
and 24 histidine residues (H) to promote endosomal escape via the proton sponge effect. [122]
Addition of FGF2 to the biopolymer was expected to give affinity towards FGFR expressing
cells such as T47D (breast cancer) and NIH3T3 (fibroblasts). As a starting point, the lysine
residues in the dKH tail (i.e., KHKHKHKHKK) were arranged as dispersed, while keeping the
lysine to histidine ratio constant at 60:40. The results demonstrated that the biopolymer was able
to condense DNA into nanosize particles. [121] It was also shown that the FGF2 motif in dKH-
FGF2 was functional and could induce significant cell proliferation, whereas the dKH segment
35
alone did not show any cell proliferative activity. While the result of the transfection efficiency
studies showed targeted gene transfer via the FGF receptor, the biopolymer efficiency was lower,
in comparison to other delivery systems, and requires further development.
Gopal, in 2007 created two fusion vectors Mu-Mu and Tat-Mu to transfect cells with the
reporter β-gal. [123] The condensation of the DNA was shown using an ethidium bromide
exclusion assay. The vectors were able to transfect cells, but endosomal escape was again an
issue. [123] They then reported the construction of a 3 domain vector containing 1) a cell
penetrating TAT domain, 2) nuclear localization from three repeating units of SV40 and 3) the
DNA condensing Mu domain. DNA condensation was again achieved, but transfection
efficiency was enhanced via the addition of chloroquine.
1.7 Conclusions
The development of nucleic acid delivery systems continues to be an area of interest in the
biological sciences. Biopolymer mediated delivery offers several advantages over alternative
methods. When compared to viral vectors, biopolymers can be made at significantly lower cost
and with fewer safety concerns. Batch to batch variation is less than that of synthetic polymers
and customizability, dictated by amino acid sequence is more controllable. Additionally
biopolymers can be composed of multiple functional domains to perform specific functions to
circumvent both extra and intracellular barriers to entry. The ability to interchange domains
allows for flexibility in targeting specific diseases, and also in what type of cargo is delivered.
The recombinant biopolymers that will be presented show potential as nucleic acid delivery
vehicles in both gene therapy and gene silencing.
36
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[107] R.W. Paul, K.E. Weisser, A. Loomis, D.L. Sloane, D. LaFoe, E.M. Atkinson, R.W.
Overell, Gene transfer using a novel fusion protein, GAL4/invasin. Hum Gene Ther 8(10)
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[108] J. Fominaya, C. Uherek, W. Wels, A chimeric fusion protein containing transforming
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[109] C. Uherek, J. Fominaya, W. Wels, A modular DNA carrier protein based on the structure
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[110] D.W. Urry, Physical chemistry of biological free energy transduction as demonstrated by
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[111] Y. Li, Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli.
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[117] T.H. Chen, Y. Bae, D.Y. Furgeson, Intelligent biosynthetic nanobiomaterials (IBNs) for
hyperthermic gene delivery. Pharm Res 25(3) (2008) 683-691.
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[118] D.E. Meyer, A. Chilkoti, Genetically encoded synthesis of protein-based polymers with
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of the molecular weight on the inverse temperature transition of a model genetically
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[121] A. Hatefi, Z. Megeed, H. Ghandehari, Recombinant polymer-protein fusion: a promising
approach towards efficient and targeted gene delivery. J Gene Med 8(4) (2006) 468-476.
[122] J.P. Behr, The proton sponge: A trick to enter cells the viruses did not exploit. Chimia 51
(1997) 34-36.
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47
2. Chapter II. Lysine-Histidine Recombinant Cationic Biopolymers
The results of the studies in this chapter are published. Please see:
B.F. Canine, Y. Wang, A. Hatefi, Evaluation of the effect of vector architecture on DNA condensation
and gene transfer efficiency. J Control Release 129 (2008) 117-123.
2.1 Background
In an attempt to overcome the endosomal barrier and also provide targetability, Hatefi and
Ghandehari (2006) reported the structure of the first recombinant cationic biopolymer with
tandem repeating units composed of lysine (K) and histidine (H) residues fused to fibroblast
growth factor 2 (FGF2) [121]. The biopolymer with the general structure of
(KHKHKHKHKK)6-FGF2 or in short dKH-FGF2, contains 36 lysine residues (K) in the dKH
segment to condense pDNA, and 24 histidine residues (H) to promote endosomal escape via the
proton sponge effect [122]. Addition of FGF2 to the biopolymer was expected to give affinity
towards FGFR expressing cells such as T47D (breast cancer) and NIH3T3 (fibroblasts). As a
starting point, the lysine residues in the dKH tail (i.e., KHKHKHKHKK) were arranged as
dispersed, while keeping the lysine to histidine ratio constant at 60:40. The results demonstrated
that the biopolymer was able to condense DNA into nanosize particles [121]. It was also shown
that the FGF2 motif in dKH-FGF2 was functional and could induce significant cell proliferation,
whereas the dKH segment alone did not provide any cell proliferative activity. While the result
of the transfection efficiency studies showed targeted gene transfer via FGFR, the biopolymer
48
efficiency was suboptimal and required further development. This previous work by Hatefi set
the stage for the studies presented below.
2.2 Evaluation of the Effect of Vector Architecture on DNA Condensation
and Gene Transfer Efficiency
2.2.1 Abstract
The objective of this study was to evaluate the effect of vector architecture on DNA
condensation, particle stability, and gene transfer efficiency. Two recombinant non-viral vectors
with the same amino acid compositions but different architectures, composed of lysine-histidine
(KH) repeating units fused to fibroblast growth factor, were genetically engineered. In one vector
lysine residues were dispersed (KHKHKHKHKK)6-FGF2, whereas in the other they were in
clusters (KKKHHHHKKK)6-FGF2. Organization of lysine residues in this manner was inspired
by the sequence of DNA condensing motifs that exist in nature (e.g., histones) where lysine
residues are organized in clusters. These two constructs were compared in terms of DNA
condensation and gene transfer efficiency. It was observed that the construct with KH units in
clusters was able to condense pDNA into more stable particles with sizes < 150 nm making them
suitable for cellular uptake via receptor mediated endocytosis. This in turn resulted in five times
higher transfection efficiency for the cKH-FGF2. This study demonstrates that in targeted non-
viral gene transfer, the vector architecture plays as significant a role as its amino acid sequence.
Thus, in the design of the non-viral vectors (synthetic or recombinant) this factor should be
considered of paramount importance.
49
2.2.2 Introduction
A major limiting factor to gene therapy is the lack of a suitable gene delivery system to carry
the therapeutic genes to the target tissues [1]. Advancements in gene therapy in general depend
on the development of novel gene delivery systems (vectors) with high transfection efficiency at
the target site and low toxicity.
Based on current understanding of the barriers to systemic gene transfer [2] and [3], serum
nucleases, endosomal entrapment, and the nuclear membrane play significant roles in limiting
the number of intact genes that reach the cell nucleus for successful transcription. Viruses have
evolved to efficiently overcome these barriers; however, safety and toxicity issues have limited
their use for systemic gene delivery [4].
In contrast, non-viral vectors can be utilized to deliver therapeutic genes to target cells
without significant toxicity; however, they suffer from low transfection efficiency, thus
conferring the need for the design and development of new vectors that are both efficient and
safe. For a vector to be maximally effective, it should protect the DNA from serum
endonucleases, disrupt the endosome membrane promoting escape of the DNA into cytosol, and
facilitate translocation of the genetic material towards the cell nucleus. Positively charged motifs
such as adenovirus µ peptide (arginine rich), histones (lysine rich), poly l-lysines, and others
have been found to be efficient in condensing plasmid DNA (pDNA) and protecting it from
degradation by nucleases. [5-7] After endocytic uptake, pDNA is sequestered in endosomes and
trafficked through the cytoplasm for eventual fusion with lysosomal vesicles. To avoid
degradation by lysosomal enzymes pDNA must escape from the endosomes prior to lysosomal
fusion. [8] It has been shown that histidine residues can be utilized in the vector structure to
50
disrupt endosome membranes thereby promoting endosomal escape. [9- 12] This endosomolytic
property is thought to arise from the “proton-sponge” effect. [13-14]. Although the science of
targeted non-viral gene transfer has come a long way, inefficiency still remains a significant
challenge due to the many hurdles that must be overcome. [15]
The focus of this research is to engineer well-defined non-viral vectors using genetic
engineering techniques and evaluating their potential for targeted gene transfer. Recombinant
DNA technology has empowered us to exert full control over vector structure at the molecular
level and fine tune its physicochemical properties for specific gene delivery needs.
The biosynthesis and characterization of a prototype recombinant vector with the structure
(KHKHKHKHKK)6-FGF2, namely dKH-FGF2, which contained 36 lysine residues (K) in the
dKH segment to condense pDNA, and 24 histidine residues (H) to promote endosomal escape
has been reported previously. [16] At the C-terminus of the dKH segment, FGF2 represents basic
fibroblast growth factor, a ligand for the basic fibroblast growth factor receptor (FGFR). This
receptor is known to be over-expressed in subpopulations of lung, prostate, and breast cancer,
thus conferring the potential for targeted gene delivery via receptor-mediated endocytosis. [17]
As a starting point, the arrangement of lysine residues in the dKH tail (i.e., KHKHKHKHKK)
was designed as dispersed, while keeping the lysine to histidine ratio constant at 6:4. This ratio
was chosen based on previous studies reported by Midoux and Monsigny. [18] Although dKH-
FGF2 was able to condense pDNA into nano-size particles (average 231 nm) and transfer genes
into target cancer and non-cancer cells [16], the percentage of the transfected cells in the absence
of serum was five times higher than in the presence of serum. This prompted us to further
characterize dKH-FGF2 and modify its structure to an extent that the transfection efficiency
51
increased in the presence of serum. It was hypothesized that by changing the arrangement of KH
residues in the KHKHKHKHKK repeating units and organizing the lysine residues in clusters,
the pDNA condensation efficiency will be improved resulting in more compact and stable
nanocarriers with higher transfection efficiency. This hypothesis was inspired by motifs that
exist in nature (e.g., histones and adenovirus µ peptide) that have lysine and arginine residues
arranged in clusters and have been shown to be highly efficient in DNA condensation. [19-21].
To test the hypothesis, an analogue of dKH-FGF2 was designed: (KKKHHHHKKK)6-FGF,
namely cKH-FGF2.
In this article, the word “architecture” literally refers to the manner in which the components
of the vector are organized and integrated.
2.2.3 Materials and Methods
2.2.3.1 Cloning and Epression of cKH-FGF2
The gene encoding cKH was designed, expression optimized, and synthesized (Blue Heron
Biotechnology Inc., Bothell, WA) with N-terminal NdeI and C-terminal EcoRI restriction sites.
The synthesized gene was double digested with NdeI and EcoRI (New England Biolabs, Ipswich,
MA) restriction enzymes and cloned into a pET21b-FGF2 expression vector which contained the
FGF2 gene in between EcoRI and HindIII. The parent pET21b vector was purchased from EMD
Biosciences (Gibbstown, NJ). The successful cloning of the cKH gene in pET21b-FGF2 vector
was verified by DNA sequencing and back translation into the corresponding amino acid
sequence.
52
The pET21b-cKH-FGF2 vector was transformed into E. coli BL21 (DE3) (Novagen, San
Diego, CA), grown using Barnstead-Labline MAX Q 4000 shaking incubator, and expressed by
the addition of IPTG to a final concentration of 0.4 mM at 30 °C. The cells were harvested,
lysed, and centrifuged for 40 minutes at 30,000 g (4 °C) to pellet the insoluble fraction. The
soluble fraction was removed and loaded onto a Ni-NTA column (Amersham Biosciences,
Piscataway, NJ) for purification. The column was washed with 50 volumes of wash buffer
(50 mM phosphate buffer, 10 mM Tris, 1 M NaCl, 20 mM immidazole) and eluted with 300 mM
immidazole. The purity and expression of the vector were confirmed by SDS-PAGE and western
blot analysis, respectively. The vector was dialyzed versus Dulbecco's phosphate buffered saline
(DPBS) and stored at − 80 °C after addition of 20 mM Tris buffer (pH = 7.4), 250 mM NaCl,
50 mM KCl, 2 mM β-mercaptoethanol and 20% glycerin.
The exact molecular weight and amino acid content of the purified cKH-FGF2 was
determined by mass spectroscopy and amino acid content analysis (Commonwealth
Biotechnologies Inc., Richmond, VA).
2.2.3.2 Particle Size and Charge Analysis
The mean hydrodynamic sizes and surface charges of vector/pDNA complexes were
determined using a Malvern zeta/particle sizer and software (Malvern Instruments, UK). Before
complexation, the vector solution was dialyzed versus 10 mM phosphate buffer and 5 mM NaCl
for 30 minutes. Various amounts of vector were added to 2 µg of pDNA (pEGFP) to form
complexes at N/P ratios of 0.5, 1, 2, 4, and 6. After 30 min of incubation time, the size and zeta
potential of the complexes were measured and reported as mean ± SEM (n = 3).
53
The particles size measurements are performed using Dynamic Light Scattering (DLS). The
particles are illuminated with a laser and the intensity of scattered light is collected. Due to
Brownian motion, particles continue moving resulting in fluctuating intensity of the scattered
light. A digital correlator measures the degree of similarity of scattered light intensity at different
times and generates a correlation curve that reflects the decay rate. Based on the Stokes–Einstein
equation, larger particles move more slowly and therefore the correlation decay rate is slower for
larger particles. The correlation function is then used to generate the size distribution of the
particles.
2.2.3.3 Mitogenic Assay
The details of the mitogenic assay for dKH-FGF2 have been reported previously. [16]
Briefly, NIH 3T3 cells were grown in F12/DMEM (1:1 ratio) with 10% fetal calf serum (FCS).
Cells were washed with a serum-free medium (SFM) and 5 × 103 cells were seeded in a 96-well
dish in 150 µl of SFM. A serial dilution of cKH-FGF2 and native FGF2 (Promega, Madison WI,
USA) was prepared across the plate ranging from 0 to 50 ng/ml. The control well with 0 ng/ml
concentration received PBS. Cells were incubated for 44 hr and after the incubation time, WST-1
(Roche Applied Science, IN, USA) reagent was added and after 4 hr the absorbance was
measured at 440 nm. The data is reported as Mean ± SD, n = 6.
2.2.3.4 Cell Toxicity Assay
Cell toxicity assays were performed in DMEM/F12 (90%) supplemented with FCS (10%) as
described previously for dKH-FGF2. [16] NIH3T3 cells (5 × 103/well) were seeded in a 96-well
54
dish in 150 µl of SFM and incubated overnight. A serial dilution of cKH-FGF2 or cKH-
FGF2/pEGFP complexes was prepared across the plate (equivalent of 0 to 80 µg/ml cKH-FGF2).
The control well with 0 µg/ml concentration received PBS. Cells were incubated with test groups
for 4 hr, washed, and incubated with fresh media overnight. The next day, WST-1 reagent
(Roche, Indianapolis, IN) was added, incubated for 4 hr, and the absorbance was measured at
440 nm. The measured absorbance for test groups is expressed as percent of the control (defined
as 1). The control cells were treated with PBS. The data is reported as Mean ± SD, n = 6.
2.2.3.5 Cell Culture and Transfection
NIH 3T3 cells (mouse embryo fibroblast) and T-47D cells (human breast cancer) were
propagated as suggested by the American Type Culture Collection (VA, USA). Cells were
seeded in 12-well tissue culture plates (in triplicate) at 5 × 104 cells per well in 1 ml growth
media with 10% heat-inactivated serum (Invitrogen, Carlsbad, CA). Cells were approximately
70–80% confluent at the time of transfection. pEGFP under the control of CMV promoter
(Clontech, CA) or pRLCMV-luc (Promega, Madison, WI) at a concentration of 3 µg/50 µl was
mixed with vector (N/P = 1) in 50 µl of 10 mM phosphate buffer and 5 mM NaCl and incubated
for 30 min at room temperature for complex formation. The complexes were then added to the
growth media supplemented with 10% heat-inactivated serum. This was added to the cells which
were then incubated at 37 °C in humidified 5% CO2 atmosphere. After 4 hours, the growth
media was removed and replaced with fresh growth media (DMEM 90% and serum 10%). The
GFP expression was visualized using a confocal microscope whereas luciferase activity was
measured by using Promega's luciferase assay kit and protocol. Using a previously reported
55
method [16], the percentage of transfected cells was calculated and reported as mean ± standard
deviation (n = 9) for this experiment. When used, 100 µM chloroquine (Sigma, St. Louis, MO)
was added to the culture media at the time of cell transfection. Bafilomycin A1 (Sigma, St.
Louis, MO) was added to the cell culture media at the time of transfection at a concentration of
100 nM. The data is reported as Mean ± SD, n = 3. Lipofectamine 2000 (Invitrogen, Carlsbad,
CA) was used as positive control.
2.2.3.6 Inhibition Assay by FGF2
This method has previously been reported for dKH-FGF2 [16]. In brief, NIH 3T3 cells were
seeded in 12-well tissue culture plates at 5 × 104 cells per well in 1 ml SFM. Cells were
approximately 70–80% confluent at the time of transfection. pEGFP (3 µg/50 µl) was mixed
with cKH-FGF2 vector at N/P ratio of 1:1 and incubated for 30 min at room temperature for
complex formation. In one set of wells, FGF2 (1000 ng/ml) was added followed by addition of
complexes. In the second set, SFM was added followed by addition of complexes (control). The
cells were incubated at 37 °C in humidified 5% CO2 atmosphere. After 4 hr, the growth media
was removed and replaced with fresh growth media with serum. The GFP expression was
quantified using a previously reported method. [15] The data is reported as Mean ± SD, n = 9.
2.2.4 Results
2.2.4.1 cKH-FGF2 Cloning, Expression, and Purification
Using the cloning strategy shown in Figure 2.1a, the gene encoding cKH-FGF2 was cloned
into a pET21b expression vector to make pET21b-cKH-FGF2 and sequenced to verify its fidelity
56
to the original design. The results of the DNA sequencing revealed that both sense and antisense
strands were free of any mutations and corresponded to the amino acid sequence shown in
Figure 2.1b.
Figure 2. 1 Cloning Strategy and Amino Acid Sequence for cKH-FGF21
(a) An overview of the cloning strategy used to make a pET21b expression vector containing the
gene encoding cKH-FGF2. The FGF2 gene was cloned into pET21b vector in between EcoRI
and HindIII restriction sites to make pET21b-FGF2 vector. The synthesized cKH gene was
cloned into pET21b-FGF2 vector in between NdeI and EcoRI restriction sites to make pET21b-
cKH-FGF2 expression vector. (b) The corresponding amino acid sequence of the cloned cKH-
FGF2 gene. The cKH sequence is shown in bold and the amino acid sequence of FGF2 is
underlined.
The expression of the vector was confirmed by western blot analysis using anti-His antibody
(Abcam, Cambridge, MA) and the purity of the vector was determined to be > 98% by SDS-
PAGE analysis (Figure 2.2). The expressed vector was further characterized by mass
spectroscopy and amino acid content analysis to determine the exact molecular weight and
1 Amino acid and DNA sequences can be found in Appendix A
57
amino acid composition. The observed molecular weight was determined to be 27,421 Da which
was in close agreement with the expected value (i.e., 27,486 Da) (data not shown). The observed
amino acid content of the purified vector was also in close agreement with the expected amino
acid composition (data not shown).
Figure 2. 2 SDS and Western blot of cKH-FGF2
(a) SDS-PAGE analysis of purified cKH-FGF2 with purity higher than 98%. (b) The western
blot analysis of the expressed cKH-FGF2 using monoclonal anti-histag antibody. The expected
molecular weight of cKH-FGF2 is 27486 Da.
2.2.4.2 Particle Size, Charge, and Stability Analysis
The ability of both vectors to condense model plasmid DNA (pEGFP) was examined in the
presence of 10 mM phosphate buffer and 5 mM NaCl. Various amounts of cKH-FGF2 and dKH-
FGF2 were complexed with 2 µg of pEGFP to form complexes. The best results were obtained at
58
N/P ratio of 1 with a resulting average particle size of 115 ± 15 nm for cKH-FGF2 and 205 ± 16
for dKH-FGF2 (Figure 2.3a).
Figure 2. 3 Particle Size and Stability Analysis of cKH-FGF2 and dKH-FGF2
(a) Particle size analysis of cKH-FGF2/pDNA and dKH-FGF2/pDNA complexes at various N/P
ratios in the presence of 10 mM phosphate buffer and 5 mM NaCl. (b) Stability of cKH-
FGF2/pDNA and dKH-FGF2/pDNA complexes in the presence of 10 mM phosphate buffer and
150 mM NaCl are demonstrated. Complexes were formed at N/P ratio of 1. For particles formed
with cKH-FGF2, the size slightly increased from 115 nm ± 15 to 156 ± 13, but remained stable
over 55 min. For particles formed with dKH-FGF2, the size increased steadily from 205 ± 16 nm
to 668 ± 24 nm.
59
The stability of particles under conditions close to physiologic ionic strength (i.e., 10 mM
phosphate buffer and 150 mM NaCl) was monitored over 55 min. The results showed that cKH-
FGF2 was able to form stable particles which only slightly increased in particle size
(115 nm → 156 nm) whereas dKH-FGF2 was not able to produce stable particles under the
stated conditions (205 nm → 668 nm) (Figure 2.3b).
The surface charge of the complexes at N/P ratio of 1 was also determined to be + 5 ± 3 for
dKH-FGF2 and + 7 ± 4 for cKH-FGF2.
2.2.4.3 Mitogenic and Toxicity Assays
Using a WST-1 cell proliferation assay (Roche, Indianapolis, IN), the bioactivity of the FGF2
segment of cKH-FGF2 was evaluated and compared with native FGF2 in NIH 3T3 fibroblasts
known to express the FGFR. The significant mitogenic activity as well as non toxicity of dKH-
FGF2 have been reported previously. [16] The FGF2 motif present in cKH-FGF2 was shown to
be active in terms of inducing cell proliferation in fibroblasts when they were exposed to
concentrations of vector that mimicked physiological FGF2 levels. The results of the mitogenic
assay showed that the mitogenic activity of cKH-FGF2 was as significant as native FGF2 up to
1 ng/ml; and overall, cKH-FGF2 induced significant cell growth in comparison to PBS control
(Figure 2.4a).
60
Figure 2. 4 Cell Proliferation and Toxicity Assays for cKH-FGF2 and FGF2 Only
(a) WST-1 cell proliferation assay for NIH 3T3 cells treated with cKH-FGF2 and native FGF2.
Cells were treated with various concentrations ranging from 0 (control) to 50 ng/ml and the
absorbance of soluble formazan was measured at 440 nm ( t-test, two-tailed, p < 0.05). (b)
WST-1 cell toxicity assay for NIH 3T3 cells treated with various concentrations of cKH-FGF2 or
cKH-FGF2/pEGFP (equivalent of 0 to 80 µg/ml vector). In the tested range, no toxicity was
observed with the vector (t-test, two-tailed, p < 0.05).
The toxicity of the cKH-FGF2 or cKH-FGF2/pEGFP in NIH3T3 cells was also evaluated by
exposing the cells to super-physiological concentrations of the vector ranging from 100 to
61
80,000 ng/ml. The results demonstrated that the vector does not have any significant effect on
cell viability regardless of the dose (Figure 2.4b).
2.2.4.4 In Vitro Cell Ttransfection by Vector/pEGFP Complexes
To evaluate transfection efficiency in terms of percent transfected cells, the pEGFP plasmid
was condensed with each vector and functioned as a reporter to monitor the percentage of
transfected cells in NIH 3T3 and T-47D cells in the presence of serum. Both vectors were
complexed with pEGFP at N/P ratio of 1 and used to transfect NIH3T3 and T47D cells. At N/P
ratio of 1, the percent transfected cells with cKH-FGF2 in NIH3T3 and T47D was 41 ± 4 and
28 ± 5, respectively (mean ± SD, n = 9). The percentages of transfected cells with dKH-FGF2 in
NIH3T3 and T47D were 9 ± 3 and 7 ± 2, respectively (Figure 2.5).
62
Figure 2. 5 Percentage of Transfected Cells and Fluorescent Microscopy Images
(a) Representative confocal images of NIH3T3 (left) and T47D (right) cells transfected with
cKH-FGF2/pEGFP complexes. The green dots are the cells expressing green fluorescent protein
(GFP). (b) Percentage of cells transfected with cKH-FGF2/pEGFP (closed bar) and dKH-
FGF2/pEGFP (open bar). Cells were transfected with vectors in DMEM supplemented with
serum. The percent transfected cells with cKH-FGF2 in NIH3T3 and T47D was 41 ± 4 and
28 ± 5, respectively (mean ± SD, n = 9). The percentages of transfected cells with dKH-FGF2 in
NIH3T3 and T47D were 9 ± 3 and 7 ± 2, respectively.
2.2.4.5 Inhibition Assay
To evaluate whether specific uptake occurred through FGFR, transfection experiments were
conducted on NIH 3T3 cells in the presence of 1000 ng/ml free FGF2 (n = 3). The results of
inhibition assay demonstrated that the presence of FGF2 in the media saturated the FGF2
receptors and significantly inhibited the transfection efficiency of the cKH-FGF2 vector. The
63
transfection efficiency reduced from 49 ±8% to 6 ± 3% (Figure 2.6). This is similar to what we
observed for dKH-FGF2. [16]
Figure 2. 6 Inhibition Assay
Inhibition assay was performed to demonstrate the transfection of cells via FGF2 receptor-
mediated endocytosis. (a) Confocal microscopy image of NIH 3T3 cells transfected with cKH-
FGF2/pEGFP in serum free media (SFM); (b) confocal microscopy image of NIH 3T3 cells
transfected with cKH-FGF2/pEGFP in SFM with addition of 1000 ng/ml FGF2.
2.2.4.6 Influence of Histidine on Endosomal Escape and Transfection Efficiency
Using plasmid DNA encoding luciferase (pRLCMV-luc), the levels of gene expression by
cKH-FGF2 and dKH-FGF2 in NIH3T3 cells were also quantitated. It was observed that cKH-
FGF2 was able to transfect NIH3T3 cells approximately five times more than dKH-FGF2.
Luciferase activity was determined to be 95 ± 5 and 16 ± 4 RLU/µg protein for cKH-FGF2 and
dKH-FGF2, respectively (Figure 2.7).
64
Figure 2. 7 Luciferase Activity to Compare Transfection Efficiency
Comparison of the transfection efficiency between cKH-FGF2 and dKH-FGF2 in NIH3T3 cells.
Luciferase activity was measured as described in Materials and Methods and determined to be
95 ± 5 and 16 ± 4 for cKH-FGF2 and dKH-FGF2, respectively. Lipofectamine was used as
positive control.
The effect of histidine residues in promoting the endosomal escape was also evaluated by
complexing the vectors with pRLCMV-luc and transfecting NIH3T3 cells in the presence of
100 µM chloroquine and 100 nM bafilomycin A. In the presence of chloroquine, the transfection
efficiency of cKH-FGF2 increased from 95 ± 5 to 142 ± 9 and for dKH-FGF2 it increased from
16 ± 4 to 29 ± 6 RLU/µg protein (Figure 2.8). In the presence of bafilomycin A, the transfection
efficiency of cKH-FGF2 dropped from 95 ± 5 to 15 ± 4 RLU/µg protein, whereas the
transfection efficiency of dKH-FGF2 decreased from 16 ± 4 to 6 ± 2 RLU/µg protein.
65
Figure 2. 8 Effect of Chloroquine on Transfection Efficiency
Comparison of transfection efficiency of cKH-FGF2 and dKH-FGF2 in the absence (closed bar)
and in the presence of chloroquine (hatched bar) and bafilomycin A (open bar). NIH3T3 cells
were transfected with both vector/pDNA complexes and the luciferase activity was measured.
The results are shown as mean ± SD (n = 3) ( t-test, two-tailed, p < 0.05).
2.2.5 Discussion
Recombinant DNA technology has allowed the production of amino acid based polymers
containing repeating blocks of amino acids with defined compositions, sequences and lengths.
[22-24] In this study, the pDNA condensation and gene transfer efficiency of two constructs with
similar compositions but different backbone architectures, i.e., dKH-FGF2 and cKH-FGF2, were
examined. In the latter, the lysine and histidine residues are arranged in clusters whereas in the
former they are dispersed. The biosynthesis and characterization of dKH-FGF2 has been
reported previously. [16] The SDS-PAGE and western blot analysis results demonstrated
successful cloning, expression and purification of the cKH-FGF2 vector in E. coli expression
system. The molecular weight as well as amino acid content of the expressed vector was also
66
confirmed by mass spectroscopy and amino acid content analysis. Approximately 800 µg of
cKH-FGF2 with 98% purity was obtained from one liter of culture media.
The ability of the purified cKH-FGF2 vector to condense pDNA into stable nanosize
particles was studied in solutions with different ionic strengths. Various amounts of cKH-FGF2
and dKH-FGF2 were complexed with 2 µg of pEGFP (N/P ratios 0.5, 1, 2, 4, and 6) to form
complexes in the presence of 10 mM phosphate buffer and 5 mM NaCl. The best results were
obtained when N/P ratio was 1. At higher N/P ratios the average particle size dramatically
increased (> 300 nm) which could be due to the interaction between the hydrophobic residues in
FGF2 sequence resulting in the aggregation of particles. Such particles are too big to fit in
clathrin coated vesicles and cannot be endocytosed via receptors. [14] and [25] However, they
could sediment readily and be taken up by the cells via phagocytosis. Apropos, they were
eliminated from the pool of data and not reported in transfection studies. This study shows that
both vectors were able to condense pDNA into nanosize particles, but cKH-FGF2 was able to
interact more efficiently with pDNA producing smaller particles. To examine the particle
stability under conditions close to physiologic ionic strength, complexes were formed at N/P of
1, the concentration of NaCl was increased to physiologic levels (i.e., 150 mM), and the particle
size was monitored over 55 min. The results showed that cKH-FGF2 was able to form stable
particles with slight increase in particle size (115 nm → 156 nm), whereas dKH-FGF2 was not
able to produce stable particles under stated conditions (205 nm → 668 nm) (Figure 2.3b). This
difference in particle stability could be the result of more efficient interaction between cationic
residues in cKH-FGF2 with negatively charged phosphate groups in pDNA. Once the
electrostatic charges are neutralized, vector/pDNA complex collapses into a condensed particle
67
stabilized by hydrophobic pockets. These hydrophobic pockets are created at the points of
interaction between neutralized amine groups in lysine residues and phosphate groups in pDNA.
Such pockets could inhibit diffusion of water molecules into the core and minimize the
possibility of buffer and salt molecules to interfere with the electrostatic interactions; hence,
formation of more stable particles. More studies with other KH-FGF2 analogues could provide a
clearer picture to better understand the observed differences. Although the histidine residues are
positively charged at physiologic pH (pKa = 6.0) and participate in pDNA condensation, lysine
residues (pKa = 10.5) are the major players in this event. Therefore, in comparison to dKH, cKH
with blocks of K residues could have produced dominant hydrophobic pockets resulting in more
stabilized particles.
As expected, the surface charges of the complexes formed with both vectors were determined
to be close to zero. In targeted gene transfer, particle surface charge does not play as significant a
role as in non-targeted vectors because particles are internalized via receptors.
Before utilizing the cKH-FGF2 vector for in vitro cell transfection, it was further
characterized in terms of mitogenicity and toxicity in NIH3T3 (fibroblast) cells by using WST-1
cell proliferation and toxicity assays. The mitogenicity assay was performed to confirm the
activity of the FGF2 motif in cKH-FGF2, whereas the cell toxicity assay was carried out to
evaluate the toxicity of the vector in normal fibroblast cells known to over-express FGF2
receptors. [26] The results showed that in comparison to PBS control, cKH-FGF2 induced
significant cell growth (Figure 2.4a). This indicates that presence of C-terminal histag or N-
terminal cKH did not have any significant effect on the ability of FGF2 to bind to FGFR. We
have previously reported similar observations with dKH-FGF2 and have also demonstrated that
68
the lysine-histidine repeating units by themselves do not have mitogenic activity. [16] Therefore,
the observed mitogenicity is likely due to the presence of FGF2 segment in cKH-FGF2.
Although the treatment of cells with peptide based vectors could have altered the cellular
signaling pathways, these changes did not have any deleterious effect on cell growth/viability
(Figure 2.4b). This could be due to the susceptibility of KH repeating units to serine proteases
inside endosomes degrading them into smaller fragments with less toxicity. This concept has
been demonstrated by other groups elsewhere. [27]
So far, it was demonstrated that the vector is able to condense pDNA efficiently, target
FGFR and internalize, while showing no detectable toxicity. The next logical step is to evaluate
the ability of cKH-FGF2 vector in terms of gene transfer efficiency in cells over-expressing
FGFR such as NIH3T3 (fibroblasts) and T47D (breast cancer) cells. [28] To compare the
transfection efficiency of cKH-FGF2 with dKH-FGF2, they were complexed with pEGFP at N/P
ratio of 1 and used to transfect both NIH3T3 and T47D cells in the presence of heat-inactivated
serum. In both cell lines, the highest number of transfected cells was observed with cKH-FGF2
in comparison to dKH-FGF2 (Figure 2.5). These results were expected because the size of the
particles that were formed with cKH-FGF2 at N/P of 1 were not only stable (Figure 2.3) but in
the optimum range for receptor mediated endocytosis (< 200 nm). [25] We used two different
cell lines (cancer and non-cancer) to examine whether the cell type had any effect on the
observed differences between the two vectors. The vector cKH-FGF2 was more efficient in gene
transfer than dKH-FGF2 in both cell lines. In addition, higher numbers of NIH3T3 cells were
transfected in comparison to T47D. This could be due to the presence of higher levels of FGFR
on the NIH3T3 cell membranes. Here, all the variables were kept constant except the vector
69
architecture (i.e., cKH versus dKH) which influenced the particle size and stability. Thus, it was
deduced that the observed difference in transfection efficiency between the two vectors was
directly influenced by the manner the KH repeating units were arranged. In subsequent studies
we used NIH3T3 cells in testing the hypotheses, because NIH3T3 cells responded to the
treatments with higher sensitivity (Figure 2.5).
To investigate whether the targeted cKH-FGF2 vector delivered the pEGFP to the NIH3T3
cells via FGFR, an inhibition assay was performed and cells were transfected in SFM in the
presence and absence of native FGF2. The results demonstrated that the presence of FGF2 in the
media saturated the FGF2 receptors and significantly inhibited the transfection efficiency of the
vector (Figure 2.6). This observation reveals that internalization of complexes was facilitated by
FGFR, although some non-specific uptake was also observed.
The levels of gene expression were also measured quantitatively by transfecting the NIH3T3
cells with vector/pRLCMV-luc complexes (Figure 2.7). It was observed that cKH-FGF2 was
able to transfect cells approximately five times more than dKH-FGF2. This could be the result of
higher numbers of cKH-FGF2/pDNA complexes that were internalized by the cells which
correlates directly to the particle size. In this set of experiments, due to its high efficiency,
Lipofectamine 2000® was used as positive control to validate the transfection efficiency process.
Polymers such as poly l-Lysine are not suitable positive controls because of their inability to
escape from endosomes and very low transfection efficiency. The transfection efficiency of dKH
has been reported previously. [16] It is noteworthy that commercially available gene transferring
agents (e.g., lipofectamine, DOSPER, Poly Lysine, PEI) are not targeted vectors and internalize
mostly via phagocytosis or pinocytosis, while the targeted vectors in this study internalize via
70
receptor mediated endocytosis. Therefore, the efficiency of targeted vectors is controlled by the
number of receptors available on the surface of the target cells. This concept where the
internalization of targeting motifs is dependent upon the availability of the receptors on the
surface of cells has been demonstrated elsewhere. [29] Thus, cKH-FGF2 could be highly
efficient in transfecting cells that over-express FGFR but very poor in transfecting cells that
express low levels of FGFR. Hence, lipofectamine (non-targeted) was used merely as a positive
control not as a point of reference to be compared with targeted vectors.
One question that still needs to be answered is whether histidine residues in the vector
backbone had any effect on endosomal escape and transfection efficiency. Histidine residues can
effectively increase the delivery of pDNA into the cytosol via membrane destabilization of acidic
endocytotic vesicles containing vector/pDNA complexes following the protonation of the
imidazole groups. This was assessed by transfecting NIH3T3 cells in the absence and presence
of bafilomycin A1 and chloroquine. Chloroquine is a buffering agent known to disrupt the
endosomal membrane by increasing the pH of the endosome environment and facilitating escape
of the cargo into cytosol. [18] In contrast, bafilomycin A is an inhibitor of vacuolar ATPase
endosomal proton pump which significantly reduces the escape of the cargo into cytosol. [30-
[31]
When NIH3T3 cells were transfected in the presence of chloroquine an approximately 50%
increase in transfection efficiency was observed with both vectors (Figure 2.8). This indicates
that some particles remained trapped inside a subpopulation of endosomes and could not escape
into cytoplasm without the help of chloroquine. It was also observed that the transfection
efficiency of dKH-FGF2 was not as high as cKH-FGF2 even in the presence of chloroquine
71
suggesting that fewer numbers of particles were internalized in the first place. The fact that
chloroquine had a significant positive effect on transfection efficiency implies that addition of
more histidine residues to the vector structure could enhance the endosome disrupting efficiency
of the vectors. This can be examined by carefully designing and testing other KH-FGF2
analogues with different architecture containing more or less histidine content.
In the presence of bafilomycin, the transfection efficiency of both vectors dropped
significantly as a result of the entrapment of vector/pDNA complexes inside endosomes and
inability to escape into cytosol. Therefore, it is conceivable that the observed difference between
the transfection efficiency of the two vectors was due to the internalization of higher number of
cKH-FGF2/pDNA complexes. Consequently, this observation can directly be attributed to the
vector architecture highlighting the considerable effect of vector architecture on transfection
efficiency.
2.2.6 Conclusion
This study demonstrates that besides the amino acid composition, the vector architecture
could also play a significant role in targeted gene transfer. This factor becomes more important
when using random copolymerization to synthesize polymeric non-viral vectors. Drawing a
decisive conclusion at this point seems premature, but the data suggests that more information
can be obtained by generating other KH analogues (e.g., KKHHKKHHKK or KKKHHKKKHH)
followed by conducting comprehensive physicochemical and biological characterization.
Recombinant DNA technology has made it possible to design limitless number of constructs
with minute differences in amino acid sequence (up to single amino acid) in order to study the
72
effect of vector structure on transfection efficiency at all levels. By manipulating the vector
structure at the molecular level, it can be correlated with DNA condensation and transfection
efficiency potentially leading to the creation of vectors with high transfection efficiency and low
toxicity. It is expected that the next generations of recombinant non-viral vectors would rival
viruses in terms of efficiency without compromising safety.
2.2.7 Acknowledgement
This work was funded in part by the American Cancer Society Institutional Research Grant
(IRG-77-003-26).
2.2.8 References
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[7] M. Yamagata, T. Kawano, K. Shiba, T. Mori, Y. Katayama, T. Niidome, Structural
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[9] Q. Leng, J. Kahn, J. Zhu, P. Scaria, J. Mixson, Needle-like morphology of H2K4b
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[10] Q. Leng, L. Goldgeier, J. Zhu, P. Cambell, N. Ambulos, A.J. Mixson, Histidine-lysine
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[11] D. Putnam, A.N. Zelikin, V.A. Izumrudov, R. Langer, Polyhistidine-PEG: DNA
nanocomposites for gene delivery, Biomaterials 24 (24) (2003) 4425–4433.
[12] K. Shigeta, S. Kawakami, Y. Higuchi, T. Okuda, H. Yagi, F. Yamashita, M. Hashida,
Novel histidine-conjugated galactosylated cationic liposomes for efficient hepa- tocyte-
selective gene transfer in human hepatoma HepG2 cells, J. Control. Release 118 (2) (2007)
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[13] J.P. Behr, The proton sponge: A trick to enter cells the viruses did not exploit, Chimica 51
(1997) 34–36.
[14] D. Putnam, C.A. Gentry, D.W. Pack, R. Langer, Polymer-based gene delivery with low
cytotoxicity by a unique balance of side-chain termini, Proc. Natl. Acad. Sci. U. S. A. 98 (3)
(2001) 1200–1205.
[15] M. Haider, A. Hatefi, H. Ghandehari, Recombinant polymers for cancer gene therapy: a
minireview, J. Control. Release 109 (1–3) (2005) 108–119.
[16] A. Hatefi, Z. Megeed, H. Ghandehari, Recombinant polymer–protein fusion: A promising
approach towards efficient and targeted gene delivery, J. Gene Med. 8 (4) (2006) 468–476.
[17] V.D. Blanckaert, M. Hebbar, M.M. Louchez, M.O. Vilain, M.E. Schelling, J.P. Peyrat,
Basic fibroblast growth factor receptors and their prognostic value in human breast cancer,
Clin. Cancer Res. 4 (12) (1998) 2939–2947.
[18] P. Midoux, M. Monsigny, Efficient gene transfer by histidylated polylysine/pDNA
complexes, Bioconjug. Chem. 10 (3) (1999) 406–411.
[19] R. Rajagopalan, J. Xavier, N. Rangaraj, N.M. Rao, V. Gopal, Recombinant fusion proteins
TAT-Mu,Mu andMu-Mumediate efficient non-viral gene delivery, J. Gene Med. 9 (4)
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[20] J.R. Khadake, M.R. Rao, Condensation of DNA and chromatin by an SPKK- containing
octapeptide repeat motif present in the C-terminus of histone H1, Biochemistry 36 (5)
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[21] M. Tecle, M. Preuss, A.D. Miller, Kinetic study of DNA condensation by cationic peptides
used in nonviral gene therapy: Analogy of DNA condensation to protein folding,
Biochemistry 42 (35) (2003) 10343–10347.
[22] J. Cappello, J. Crissman,M. Dorman,M.Mikolajczak, G. Textor,M.Marquet, F. Ferrari,
Genetic engineering of structural protein polymers, Biotechnol. Prog. 6 (3) (1990) 198–202.
[23] W. Liu, M.R. Dreher, D.Y. Furgeson, K.V. Peixoto, H. Yuan, M.R. Zalutsky, A. Chilkoti,
Tumor accumulation, degradation and pharmacokinetics of elastin-like polypep-tides in
nude mice, J. Control. Release 116 (2) (2006) 170–178.
[24] D.W. Urry, C.M. Harris, L. CX, L. C-H, D.C. Gowda, T.M. Parker, S.Q. Peng, J. Xu,
Transductional protein-based polymers as new controlled-release vehicles, in: K. Park (Ed.),
Controlled drug delivery: Challenges and Strategies, American Chemical Society,
Washington, DC, 1997, pp. 405–436.
[25] J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Size-dependent internalization of particles
via the pathways of clathrin- and caveolae-mediated endocytosis, Biochem. J. 377 (Pt 1)
(2004) 159–169.
[26] B.A. Sosnowski, A.M. Gonzalez, L.A. Chandler, Y.J. Buechler, G.F. Pierce, A. Baird,
Targeting DNA to cells with basic fibroblast growth factor (FGF2), J. Biol. Chem. 271 (52)
(1996) 33647–33653.
[27] D.S. Manickam, D. Oupicky, Multiblock reducible copolypeptides containing histidine-rich
and nuclear localization sequences for gene delivery, Bioconjug. Chem. 17 (6) (2006) 1395–
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[28] L.A. Chandler, B.A. Sosnowski, L. Greenlees, S.L. Aukerman, A. Baird, G.F. Pierce,
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3. Chapter III. Arginine Histidine Recombinant Cationic
Biopolymers for Gene Delivery
The results of the studies in this chapter are published. Please see:
B.F. Canine, Y. Wang, and A. Hatefi, Biosynthesis and characterization of a novel genetically
engineered polymer for targeted gene transfer to cancer cells. J Control Release 138 (3) (2009)
188-196.
3.1 Background
For a gene carrier to successfully overcome intracellular barriers and reach the nucleus of
target cells, it must accurately mimic viral vectors. This includes protecting the DNA from
endonucleases by condensation, binding to the surface receptors on the target cells followed by
internalization, escape from endosomes into the cytosol, rapid shuttling of DNA toward the
nucleus via microtubules, entering the cell nucleus and mediating gene expression. However, no
single system had yet been designed to systematically overcome all hurdles. The comparison of
dKH-FGF2 and cKH-FGF2 vectors in Chapter 2 demonstrate that the vector architecture and
not just amino acid composition plays a significant role in gene transfer. This highlights an
important point that random copolymerization methods used to make synthetic polymers results
in a myriad of structures making structure activity relationships hard to elucidate. The
homogeneous nature of recombinant polymers makes them a more appropriate tool for
examining the contribution of architecture to gene delivery and its effect on efficiency.
77
Another important point that needs to be mentioned is that the production of dKH-FGF2 and
cKH-FGF2 peptides in soluble form in E. coli was problematic. For example, expression of ca.
800 µg of soluble cKH-FGF2 in 1 L of cell culture yielded ca. 100 µg of purified biopolymer
after purification process. While this level of biopolymer expression in E. coli can support the in
vitro structure/activity relationship studies, it is not sufficient for in vivo studies. To overcome
the large scale biopolymer production problem as well as improving escape from the endosomal
compartments, the next generation of biopolymers was designed which is discussed in the
following section. The design, production and characterization of the first arginine histidine
based vectors was published in 2009 in the Journal of Controlled Release.[7]
3.2 Biosynthesis and Characterization of a Novel Genetically Engineered
Polymer for Targeted Gene Transfer to Cancer Cells
3.2.1 Abstract
A novel multi-domain biopolymer was designed and genetically engineered with the purpose
to target and transfect cancer cells. The biopolymer contains at precise locations: 1) repeating
units of arginine and histidine to condense pDNA and lyse endosome membranes, 2) a HER2
targeting affibody to target cancer cells, 3) a pH responsive fusogenic peptide to destabilize
endosome membranes and enhance endosomolytic activity of histidine residues, and 4) a nuclear
localization signal to enhance translocation of pDNA towards the cell nucleus. The results
demonstrated that the biopolymer was able to condense pDNA into nanosize particles, protect
pDNA from serum endonucleases, target HER2 positive cancer cells but not HER2 negative
78
ones, efficiently disrupt endosomes, and effectively reach the cell nucleus of target cells to
mediate gene expression. To reduce potential toxicity and enhance biodegradability, the
biopolymer was designed to be susceptible to digestion by endogenous furin enzymes inside the
cells. The results revealed no significant biopolymer related toxicity as determined by impact on
cell viability.
3.2.2 Introduction
Currently, an efficient non-viral gene delivery vehicle (vector), which mimics each step of
viral entry and infection, has yet to be developed. As a result, non-viral gene transfer technology
remains stagnant due to the limitations of the existing vectors. Viral vectors are the current gene
delivery vehicle of choice because of their high transduction efficiency and success in preclinical
trials. However, immunogenicity and toxicity issues have arisen and clinical results have not
translated into a successful commercial product. Lipoplexes are a viral alternative without the
immunogenicity concerns; however, they have reproducibility and cytotoxicity issues. [1]
Cationic polyplexes have less biocompatibility concerns, but their transfection efficiencies
remain low. [2] For a non-viral vector to successfully overcome biological barriers, it must
mimic viral characteristics. Accurate mimicry of viral infection and ultimately full control over
gene transfer processes requires a greater understanding of the natural mechanisms involved at
the molecular scale and subsequent development of a class of biomaterials that would allow
precise correlation of one dimensional design (amino acid sequence) with three-dimensional
functionality.
79
Amino acid based polymers can be synthesized using genetic engineering techniques
resulting in biopolymers with precise compositions, molecular weights, sterotacticity and
specified functions. [3-4] Compared to other conventional methods, the principal advantages are
(i) monodisperse material, (ii) full control over polymer architecture at the molecular level, (iii)
precise covalent attachment of functional moieties (e.g., targeting motifs), and from a
manufacturing standpoint, (iv) elimination of the conjugation steps. Since most conventional
polymers are synthesized using free radical addition or similar methods, the resulting polymer is
heterodisperse. Heterogeneity in turn undermines conjugation or covalent coupling of other
moieties at precise locations which complicates characterization and controlled drug delivery. In
contrast, biopolymers can overcome these issues because of the fidelity associated with protein
expression. [5]
The overall objective of this research is to develop a gene delivery system that is
customizable, easy to engineer, efficient and non-toxic. As a first step towards achieving the
objective, a multifunctional biopolymer was designed and genetically engineered (Figure 3.1).
The architecture of this biopolymer is designed based on our current and past experience with
genetically engineered biomimetic vectors. [6-8] It features a unique domain with repeating units
of arginine-histidine (RH) with the general structure of (RRXRRXHHXHHX)n where X is any
amino acid except D and E and n is the number of repeating units. This domain is named the
DNA condensing and endosomolytic motif (DCE) where the ratio of R to H is kept constant at
50:50. Other domains include a fusogenic peptide (FP) for endosomal disruption [9], a C-
terminal HER2 targeting motif (TM) [10] and a M9 nuclear localization signal (NLS) to enhance
translocation of genetic material towards nucleus. [11] A cathepsin D enzyme substrate (CS) has
80
also been engineered in between TM and NLS to facilitate dissociation of the targeting motif
from the biopolymer inside late endosomes where cathepsin D is abundant. [12] To simplify, the
biopolymer will be referred to as FP–(DCE)n–NLS–CS–TM. Biopolymers with the following
structures were also genetically engineered (DCE)n–NLS–CS–TM (biopolymer without FP) and
FP–(DCE)n–CS–TM (biopolymer without NLS) and used as controls.
Figure 3. 1 Schematic of Multidomain Biopolymer2
Schematic of the designed multidomain biopolymeric gene carrier composed of fusogenic
peptide, DNA condensing and endosomolytic motif, nuclear localization signal and targeting
motif. The corresponding amino acid sequence for each domain is also shown.
It is our central hypothesis that a biopolymer with multiple functional domains can be
engineered to condense plasmid DNA (pDNA) into stable nanosize carriers, target model HER2
positive cancer cells, disrupt endosome membranes efficiently facilitating escape into the
cytosol, and ultimately reach the nucleus to mediate gene expression.
2 Amino acid and DNA Sequences are in Appendix A
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3.2.3 Materials and Methods
3.2.3.1 Cloning and Expression of the Biopolymer
The gene encoding FP–(DCE)3–NLS–CS–TM was designed, expression optimized, and
synthesized by Integrated DNA Technologies (San Diego, CA) with N-terminal NdeI and C-
terminal XhoI restriction sites. The synthesized gene was double digested with NdeI and XhoI
(New England Biolabs, Ipswich, MA) restriction enzymes and cloned into a pET21b expression
vector (EMD Biosciences, Gibbstown, NJ) to make pET21b:FP–(DCE)3–NLS–CS–TM. After
cloning, the fidelity of the gene to its original design was verified by DNA sequencing. The
expression vector was transformed into E. coli BL21(DE3) pLysS (Novagen, San Diego, CA)
and grown in a Barnstead-Labline MAX Q 4000 shaking incubator at 30 °C. Circlegrow media
(MB Biomedicals, Solon, OH) starter cultures were gown overnight and used to inoculate
500 mL media containing 50 µg/mL carbenicillin. At OD600 of 0.6, gene expression was induced
by the addition of IPTG to a final concentration of 0.4 mM at 30 °C for 4 hr. Cells were collected
at 5000 rpm for 10 min and frozen until further use. The pellet was suspended in lysis buffer
(50 mM NaH2PO4, 500 mM NaCl, 8 M urea, 12 mM 2-mercaptoethanol, 0.5% Triton X-100,
10 mM immidazole, pH 8.0) and centrifuged for 60 min at 30,000 ×g at 4 °C to pellet the
insoluble fraction. The soluble fraction was incubated with 0.5 mL of Ni–NTA resin (Qiagen)
equilibrated with lysis buffer. Incubation at room temperature with gentle mixing allowed for
complete binding. The resin was centriguged at 1000 × g for 5 min. Supernatant was discarded
and resin was loaded onto a 0.8 × 4 mL BioRad PolyPrep chromatography column. The column
was washed with 40 volumes of wash buffer (50 mM NaH2PO4, 1000 mM NaCl, 7 M urea,
12 mM 2-mercaptoethanol, 0.5% Triton X-100, 20 mM immidazole pH 8.0) and then eluted with
82
5 mL of elution buffer (50 mM NaH2PO4, 250 mM NaCl, 5 M urea, 12 mM 2-mercaptoethanol,
250 mM immidazole pH 8.0). The purity and expression of the vector were confirmed by SDS-
PAGE and western blot analysis using monoclonal mouse anti-6XHis antibody (Abcam,
Cambridge, MA). The purified biopolymer was stored at − 20 °C after adding glycerol to 40%
final concentration. Mass spectrometry was used to determine the exact molecular weight of the
purified biopolymer. Prior to use in further assays salts and buffer exchange was performed
using a G-25 sepharose size exclusion resin. FP–(DCE)3–CS–TM (without NLS) and (DCE)3–
NLS–CS–TM (without FP) were also purified as above.
3.2.3.2 Recognition of Cathepsin D Substrate by the Cathepsin D Enzyme
Cathepsin D enzyme (human liver) was purchased from Calbiochem (Gibbstown, NJ) and
dissolved as per manufacturer's protocol in 0.1 M Glycine–HCl, 0.5% Triton X-100 and 150 mM
NaCl pH 3.5 to a final concentration of 1 unit enzyme per 50 µL buffer. Purified biopolymer
(10 µg) was incubated with 1 unit of enzyme for 3 h at 37 °C. Enzymatic reaction was stopped
by addition of 2X Laemelli buffer (BioRad, Hercules, CA). Samples were boiled at 95 °C for
5 min and loaded onto 15% SDS-PAGE gel. Undigested biopolymer was used as control.
3.2.3.3 Susceptibility of Biopolymer to Proteolytic Activity of Furin
Furin protease was purchased from New England Biolabs (Ipswich, MA). Biopolymer
(10 µg) was prepared in 50 µL of buffer (100 mM sodium phosphate, 0.5% Triton X-100, 1 mM
CaCl2, 1 mM 2-mercaptoethanol) and pH was adjusted to 7.2, 6.0 and 5.5. One unit of furin
enzyme was added and incubated at room temperature for 3 h. Reaction was stopped by addition
83
of 2X Laemelli buffer (Sigma). Samples were boiled at 95 °C for 5 min and subjected to SDS-
PAGE gel.
3.2.3.4 Hemolysis Assay
Two milliliters of sheep red blood cells (Innovative Research, Novi, MI) were washed 2
times with Dulbeccos Phosphate Buffered Saline (Invitrogen, Carlsbad, CA). Cell numbers were
adjusted to 10 × 108 cells/mL in DPBS at pH 7.4 or 6.0. Various amounts (0.5, 1.0 or 20 µg) of
the biopolymer or biopolymer without fusogenic peptide were added to the cell suspension and
incubated at 37 °C for 1 hr in a shaking incubator set at 60 rpm. Cells were pelleted by
centrifugation and the absorbance of the supernatant was measured at 541 nm. Triton X-100
(1%) was used as a positive control while buffers only (pH 7.4 and 6.0) were the negative
controls. The percentage of hemolysis was reported relative to the positive Triton X-100 control
which was defined as 100%. Data is reported as mean ± s.d. n = 3. Statistical significance was
evaluated using t-tests (p < 0.05).
3.2.3.5 DNA Neutralization at Different pH
Gel mobility assays were performed to examine the neutralization of the pDNA negative
charges by the biopolymer. pEGFP (1 µg) was complexed with the biopolymer at various N:P
ratios (Nitrogen to Phosphate) at pH 7.4 and pH 5.5. N:P ratios were calculated based on the
number of arginine, histidine and lysine residues in the biopolymer sequence. After incubation at
room temperature for 15 min, the mobility of the pDNA was visualized on an agarose gel by
ethidium bromide after electrophoresis.
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3.2.3.6 Particle Size and Charge Analysis
Various amounts of biopolymer in 20 mM Tris–HCl buffer at pH 5.5 were added to 1 µg
pEGFP to form complexes at a range of N:P ratios in a total volume of 100 µL. For example, at
N:P ratio of 1, 1.5 µg of biopolymer was used to condense 1 µg of pEGFP. After incubation at
room temperature for 15 min, the mean hydronamic particle size and charge measurements were
performed using Dynamic Light Scattering (DLS) and Laser Doppler Velociometry (LDV) on a
Malvern Nano ZS90 instrument running DTS software (Malvern Instruments, UK). Size and zeta
potential were measured and reported as mean ± SEM (n = 3). Each mean is the average of 15
measurements and n represents the number of separate batches prepared for the measurements.
3.2.3.7 Particle Stability in the Presence of Serum
One µg plasmid DNA (pEGFP) was complexed with the biopolymer at N:P ratio of 14 and
incubated for 15 min. After stable particles had formed, fetal bovine serum (Invitrogen, Carlsbad
CA) was added to a final concentration of 10% (v/v). Complexes were incubated for 30 min at
37 °C. Subsequently, 1% heparin (Sigma, St. Louis, MO) was added to decomplex pDNA from
biopolymer. Samples were then electrophoresed on a 1% agarose gel and visualized with
ethidium bromide.
3.2.3.8 Cell Culture and Transgene Expression
SK-OV-3 cells (human ovarian cancer) with high levels of HER2 expression and MDA-MB-
231 (human breast cancer) and PC-3 (human prostate cancer) cells with low levels of HER2
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expression were seeded in a 96-well plate at 2.0 × 104 cells per well. Cells were incubated
overnight at 37 °C until 80-90% confluent. Cells were transfected with biopolymer/pEGFP
complexes at various N:P ratios in the presence of McCoy media (Hyclone, Logan, UT)
supplemented with antibiotic, transferrin, selenium, ovalbumin, dexamethasone, and fibronectin.
Three hours after transfection the media was removed and replaced with fresh McCoy media
supplemented with 10% serum. If used, 100 µM chloroquine or 100 nM bafilomycin (Sigma,
Milwaukee, WI) were added 5 min prior to transfection. 10 µM nocodazole (Sigma, Milwaukee,
WI) was added 20 min prior to transfection when used. An epifluorescent microscope (Carl
Zeiss) was used to qualitatively visualize expression of the green fluorescent protein (GFP).
Total green fluorescence intensity and percent transfected cells were measured using a FACS
Calibur flowcytometer (BD Biosciences). The total fluorescence intensity of positive cells was
normalized against the total fluorescence intensity of untransfected cells (control) to account for
cellular auto-fluorescence. The total green fluorescence intensity is an indicator of overall GFP
expression levels. The data are presented as mean ± s.d, n = 3. Statistical significance was
evaluated using t-test (p < 0.05).
3.2.3.9 Inhibition Assay
The details of this method has been reported previously. [6-7] In brief, SK-OV-3 cells were
pre-treated with the competitive inhibitor (HER2 targeting motif) at various concentrations (2.5,
50, and 100 µg/ml) followed by transfection with biopolymer/pEGFP complexes at N:P ratio of
14 in serum free media. After 3 hr of incubation at 37 °C, media was removed and replaced with
media supplemented with 10% serum. Cells were collected 48 hr post transfection and total
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fluorescence intensity was measured using flow cytometry. Untreated SK-OV-3 cells (0 µg) were
transfected with biopolymer/pEGFP complexes at N:P ratio of 14 and used as control. The data
are presented as mean ± s.d, n = 3.
3.2.3.10 Toxicity Assay
SK-OV-3 cells were seeded in 96-well plates at 2 × 104 cells per well in McCoy media plus
10% serum. Cells were treated with various amounts of biopolymer/pEGFP complexes or
phosphate buffer saline (PBS) for 3 hr. The media was removed and replaced with fresh media
supplemented with 10% serum followed by overnight incubation at 37 °C humidified CO2
atmosphere. Twenty four hr after incubation with biopolymer/pEGFP complexes, WST-1 reagent
(Roche Applied Science, Indianapolis, IN) was added, incubated for 4 hr followed by measuring
absorbance at 440 nm. The measured absorbance for test groups is expressed as percent of the
control where the control is defined as %100 viable. The data are reported as mean ± s.d., n = 3.
The statistical significance was evaluated using a t-test (p < 0.05).
3.2.4 Results
3.2.4.1 Cloning, Expression, and Characterization of Biopolymer
Using the cloning strategy shown in Figure 3.2a, the biopolymer DNA sequence was cloned
into pET21b expression vector. The fidelity of the DNA sequence to its original design was
confirmed by DNA sequencing. The biopolymer was expressed in E. coli and purified with a
10 mg/L yield. SDS-PAGE and western blot (anti-6XHIS)analysis confirmed the expression and
high purity of the biopolymer (Figure 3.2b). The exact molecular weight of the biopolymer was
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determined to be 22,682 Da which is in close agreement with the theoretical value of 22,613 Da
(Figure 3.2c).
Figure 3. 2 Cloning, Expression and Characterization of the Purified Biopolymer
a) An overview of the cloning strategy used to clone FP–(DCE)3–NLS–CS–TM gene into
pET21b expression vector. b) SDS-PAGE (left panel) and western blot analysis (right panel) of
purified biopolymer. c) The MALDI-TOF spectra of the purified biopolymer. The observed
molecular weight was 22,682 Da.
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Furin and cathepsin D substrates were incorporated into the biopolymer architecture to
enable biopolymer digestion upon entry into the cell. The accessibility of the furin substrate in
the biopolymer structure to the enzyme was evaluated at different pH conditions (Figure 3.3a,
lanes 2, 3 and 4). The results demonstrate that biopolymer can be digested by furin at various pH
values with optimum digestion at pH 5.5.
Figure 3. 3 Digestion of Biopolymer by Proteases
a) Furin cleavage; PM: Protein Marker, lane 1: undigested biopolymer, lane 2: furin digestion at
pH 5.5, lane 3: furin digestion at pH 6, lane 4: furin Digestion at pH 7. b) Cathespin D cleavage;
PM: Protein Marker, lane 1: undigested biopolymer, lane 2: cathepsin D digestion, i — cathepsin
D fragment ( 30 kDa), ii — uncut biopolymer ( 22.6 kDa), iii — biopolymer fragment (
14.4 kDa) and cathepsin D fragment ( 15 kDa), iv — biopolymer fragment 8.4 kDa.
The results of biopolymer digestion by cathepsin D illustrates that the substrate is readily
available to the protease (Figure 3.3b, lane 2). Digestion by cathepsin D results in bands at
14.2 kDa (iii) and 8.4 (iv) kDa. The bands at 30 kDa (i) and 15 kDa (iii) are a result of autolysis
of the cathepsin D enzyme at the conditions used. [13]
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3.2.4.2 Effect of pH on DNA Neutralization
The gel mobility assays were performed at pH 7.4 and 5.5 to evaluate the effect of partially
charged histidine residues on DNA neutralization. At pH 5.5, 15 µg of biopolymer (N:P 10)
was sufficient to fully neutralize 1 µg of pEGFP. However, at pH 7.4, 37 µg of biopolymer
(N:P 25) was needed to fully neutralize the same amount of pEGFP (Figure 3.4a and b).
Figure 3. 4 DNA Neutralization at Two pH Values
a) Gel mobility assay at pH 5.5 at various N:P ratios. b) Gel mobility assay at pH 7.4 at various
N:P ratios.
3.2.4.3 Evaluation of Particle Size, Charge, and Stability
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Biopolymer/pEGFP complexes were formed at various N:P ratios ranging from 1 to 20 at pH
5.0 followed by the size and zeta potential measurements. At N:P ratios greater than 5.5,
nanoparticles with sizes below 100 nm were obtained (Figure 3.5a). For example, at N:P ratio of
15, the size of the nanoparticles were 78 ± 4 nm with a zeta potential of + 10 ± 1 mV.
Figure 3. 5 Size, Charge and Serum Stability of Bioplymer/pEGFP Complexes
a) Sizes and zeta potentials of complexes at various N:P ratios b) Lane 1: pEGFP only, Lane 2:
pEGFP incubated with serum, Lane 3: Bipolymer/pEGFP in the absence of serum, Lane 4:
Biopolymer/pEGFP incubated with Serum, Lane 5: released pEGFP from the vector/pEGFP
complexes after incubation with serum.
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The ability of biopolymer to shield the genetic material from serum endonucleases was
examined by incubating the biopolymer/pEGFP with serum. The results demonstrated that the
nanoparticles were stable in the presence of serum and the pEGFP was protected (Figure 3.5b,
lanes 4 and 5). Plain pEGFP was fully degraded by the serum nucleases (Figure 5b, lane 2).
3.2.4.4 Determination of Optimum N:P ratio for Cell Transfection
SK-OV-3 cells were transfected with biopolymer/pEGFP at various N:P ratios ranging from
5 through 15. The results revealed that at N:P ratio of 14 the highest level of transfection
efficiency was achieved (Figure 3.6a). At N:P ratio of 14, 21 ± 1.0% of cells were transfected
with total green fluorescence intensity of 724,000 ± 65,000. At N:P 15, the total green
fluorescence intensity and the percentage of transfected cells significantly decreased.
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Figure 3. 6 Transfection Efficiency and Cell Toxicity at Various NP Ratios
Determination of optimum N:P ratio for transfection efficiency and biopolymer related toxicity.
a) Transfection efficiency of biopolymer/pEGFP at various N:P ratios in SK-OV-3 cells. b)
WST-1 cell toxicity assay for SK-OV-3 cells treated with various concentrations of
biopolymer/pEGFP complexes equivalent to 0 to 110 µg/ml of biopolymer.
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A toxicity assay was also performed to examine the potential toxicity of biopolymer and its
effect on cell transfection. The results showed no significant toxicity in the SK-OV-3 cells at up
to 110 µg/mL concentrations (Figure 3.6b).
3.2.4.5 Evaluation of the Functionality of Targeting Motif
To show targetability for HER2, SK-OV-3 (high HER2 expression), MDA-MB231 and PC-3
(low HER2 expression) cells were transfected with biopolymer/pEGFP complexes at N:P ratio of
14. The results of flowcytometry showed 21 ± 2 percent cell transfection in SK-OV-3 versus
2 ± 0.5 and 0.1 ± 0.02 percent for MDA-MB-231 and PC-3, respectively (Figure 3.7a).
Lipofectamine in complex with pEGFP was able to transfect all three cell lines non-selectively
with relatively high efficiency.
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Figure 3. 7 Evaluation of the Targeting Motif
a) Epifluorescent images and corresponding percent transfected cells by biopolymer/pEGFP and
lipofectamine. Percent transfection was determined by flow cytometry. b) Inhibition assay: SK-
OV-3 cells were pre-treated with various amounts of targeting peptide (competitive inhibitor)
followed by transfection with biopolymer/pDNA complexes.
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Receptor mediated endocytosis of nanoparticles via HER2 was examined by using an
inhibition assay. The results of this assay showed that as the concentration of the targeting motif
in the solution (competitive inhibitor) increased the levels of gene expression shown by total
fluorescence intensity significantly decreased (Figure 3.7b).
3.2.4.6 Functionality of the Fusogenic Peptide
pH dependent membrane disrupting activity of the fusogenic peptide was examined by
hemolytic assay. The results of this assay revealed that the biopolymer was significantly lytic at
pH 6.0 and only at high concentration (20 µg) (Figure 3.8a). At pH 6.0 and low concentrations
the biopolymer did not show significant hemolytic activity. At pH 7.4 and 20 µg concentration,
slight hemolytic activity was observed. Biopolymer without fusogenic peptide [biopolymer (−)
FP] did not show any significant hemolytic activity at any concentration at pH 6.
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Figure 3. 8 Evaluation of the Functionality of the Fusogenic Peptide
a) The hemolytic activity of biopolymer and biopolymer (−) FP were evaluated at different pH
(i.e., 7.4 and 6.0) and concentrations. Triton X-100 was used as positive control and DPBS
buffer as the negative control. b) SK-OV-3 cell transfection with biopolymer, biopolymer plus
chloroquine, biopolymer plus bafilomycin, and biopolymer without fusogenic peptide
[biopolymer (−) FP].
The ability of the biopolymer to efficiently disrupt endosome membranes was evaluated by
transfecting SK-OV-3 cells with the biopolymer/pEGFP complexes in the presence and absence
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of chloroquine and bafilomycin at N:P 14. Significant reduction in total green fluorescent protein
expression was observed in cells treated with bafilomycin (Figure 3.8b). However, no
significant difference in total green fluorescent protein expression was observed in cells
transfected in the presence or absence of chloroquine. In comparison to biopolymer, a significant
reduction in green fluorescence intensity was observed when cells were transfected with
biopolymer without fusogenic peptide.
3.2.4.7 Effect of Nuclear Localization Signal on Gene Transfer
The effect of the NLS on gene transfer efficiency was examined by transfecting SK-OV-3
cells with biopolymer and biopolymer without NLS. Comparison of the transfection efficiencies
in these two constructs showed a marked decrease in total fluorescence intensity and percent cell
transfection in the NLS deficient biopolymer (Figure 3.9).
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Figure 3. 9 Evaluation of the Functionality of the Nuclear Localization Signal
Biopolymer and biopolymer without NLS [(FP–(DCE)3–CS–TM] were complexed with pEGFP
at N:P 14 and used to transfect SK-OV-3 cells. Cells pretreated with nocodazole were also
transfected with biopolymer/pEGFP complexes at N:P 14.
The effect of the microtubules network on gene transfer efficiency was evaluated by
transfecting SK-OV-3 cells in the presence of nocodazole (microtubule depolymerizer).
Treatment with nocodazole showed a significant decrease in transfection efficiency (Figure 3.9).
At the concentration used (10 µM), nocodazole did not show any significant cell toxicity which
could impact transfection efficiency. This concentration is in agreement with the literature. [8
and 14]
3.2.5 Discussion
In order to facilitate correlation of vector structure with function and help identify the rate
limiting steps to gene transfer by the targeted vector in this study, a chimeric biopolymer was
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designed. It is composed of multiple functional domains with potential to mimic some of the
major viral characteristics including pDNA condensation, cell targeting, endosome disruption,
and gene expression mediation.
The biopolymer was genetically engineered to contain at precise locations four major
domains (Figure 3.1). The first is a DNA condensing and endosomolytic (DCE) motif comprised
of three repeating units of RRX1RRX2HHX3HHX4, where X can be any amino acid except
aspartic acid (D) or glutamic acid (E) as these negatively charged residues could interfere with
DNA condensation. The role of the DCE is to primarily condense pDNA, and secondarily to
disrupt endosomes via the proton sponge effect. [15] Histidine residues will become protonated
in the low pH environment of the endosome resulting in swelling and eventual bursting of
endosomes releasing the contents into the cytosol. In the DCE motif (RRX1RRX2HHX3HHX4)
the arginine and histidine residues were arranged in clusters based on previous work by our
group which found that a clustered arrangement in lysine-histidine biopolymers resulted in more
stable DNA condensation as compared to a dispersed architecture. [6] To maximize the proton
sponge effect without compromising the DNA condensing ability, the ratio of R:H in DCE
domain is adjusted to 50:50 ratio.
Residue X1 was designed to be valine (V) in order to generate RVRR sequences along the
DCE unit. This sequence is a substrate for the protease furin which is ubiquitously present in the
cell, and specifically abundant inside endosomes. [16] The furin enzyme recognizes the (R-X-
R/K-R↓) sequence and cleaves at the ↓ site. [17] This site was engineered in the biopolymer
structure to enhance the biodegradability of the DCE unit which could potentially be toxic. Large
blocks of cationic amino acids can be toxic to cells as they could bind to various organelles and
100
enzymes inside the cells and interfere with their functions. Degradation of the biopolymer into
smaller pieces could reduce the cytotoxicity due to reduction in molecular weight and increase
intracellular release of the genetic cargo. [18-19]
Residues X2 and X4 are designed to be serine (S) and threonine (T). T and S were selected to
increase the solubility of the biopolymer and yield of production while maintaining the balance
of R to H (50:50) with no negative impact on DNA condensation. In addition, the terminal
hydroxyl groups in S and T could potentially be involved in hydrogen bonding with the DNA
backbone enhancing the complex stability.
Residue X3 in the first and second repeating unit is R and in the third repeating unit is H. This
is to not only maintain the R to H balance, but incorporate an intrinsic histidine tag into the
biopolymer sequence which facilitates its purification via Ni-NTA chromatography.
The second functional domain in the biopolymer structure is a targeting motif that is an
affibody with high specificity for biorecognition by model cancer cells over-expressing HER2.
[10] This is to enhance the targetability and internalization of the biopolymer/pDNA complexes
via receptor mediated endocytosis. The third functional domain in the biopolymer sequence is an
endosme destabilizing motif, namely 5HWYG, which is a synthetic derivative of the influenza
virus fusogenic peptide. While influenza virus fusogenic peptide changes conformation at pH 5
to fuse with endosomal membranes, 5HWYG destabilizes endosomal membranes at pH 6.8. [9]
This fusogenic peptide was designed in the biopolymer structure to cooperatively enhance the
endosomolytic activity of the histidine residues. The fourth major domain is a M9 nuclear
localization signal (M9-NLS) derived from the heterogeneous nuclear ribonucleoprotein (hnRNP
A1). [11] This NLS was deliberately selected because it does not contain clusters of basic amino
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acid residues minimizing its interaction with pDNA and involvement in the DNA condensation
process. Finally, a minor domain encoding cathepsin D substrate (CS) was engineered in
between the NLS and the HER2 targeting motif (TM) to facilitate dissociation of the targeting
motif from the biopolymer inside the endosomal compartment. This could enhance exposure of
NLS to the cell's importin machinery and facilitate the transport of pDNA across cytoplasm
towards nucleus. To correlate structure with function and identify the shortcomings of the
biopolymer, several experiments were performed and the functionality of each domain was
examined.
The biopolymer sequence was designed, cloned into pET21b expression vector, expressed in
E. coli and purified (Figure 3.2). The biopolymer was characterized in terms of susceptibility to
digestion by furin and cathepsin D enzymes. As mentioned above, three furin substrates (RVRR)
were engineered in the DCE structure to facilitate its degradation. It was expected that the furin
enzyme digest the biopolymer from these three sites resulting in multiple byproducts. However,
Figure 3.3a shows that only one site was accessible to the furin enzyme resulting in appearance
of one band at 15 kDa and one at below 10 kDa (unresolved). Because furin is a ubiquitous
enzyme inside the cells, the degradation of biopolymer at three different pH values was
evaluated. As expected maximum degradation of the biopolymer occurred at pH 5.5 which is
close to endosomal pH because RVRR is an optimum substrate for endosomal furin. [16] The
proteolytic cleavage results by cathepsin D showed that the substrate in the biopolymer structure
is accessible and has the potential to be digested by the enzyme (Figure 3.3b). As a result, it is
expected that the NLS become exposed and play a significant role in gene transfection.
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In order to identify the minimum required quantity of biopolymer needed to effectively
neutralize the negative charges in pEGFP and minimize possible toxicity, a DNA neutralization
study was performed. Various amounts of biopolymer were added to pEGFP at two different pH
values (i.e., 7.4 and 5.5) to neutralize the negative charges. At pH 7.4, histidine residues are
partially charged and do not make significant contribution to pDNA condensation. At pH 5.5
which is below the pKa of histidine (i.e., 6.0), histidine residues are protonated creating a
continuous block of positive charge in the DCE segment with significant contribution to pDNA
condensation. Thereby, at the lower pH, significantly less biopolymer is required to neutralize
and condense pDNA (Figure 3.4). Based on this observation, all the biopolymer/pDNA
complexes were formed at pH 5.5.
In the next step, the ability of the biopolymer to efficiently condense pDNA into nanosize
carriers suitable for receptor binding and cellular uptake was examined. Particle size studies
demonstrated that at N:P ratios greater than 5 particles less than 100 nm in size can be obtained
(Figure 3.5a). This size range (< 150 nm) is optimal for targeted vectors as they can easily fit
into clathrin-coated vesicles and internalize via receptor mediated endocytosis. [20-21] The
surface charge studies showed that the zeta potential of the nanoparticles remained slightly
positive even at high N:P ratios. This could be an indication that the affibody molecules are
exposed at the surface imparting slight positive charge. It is noteworthy that the pI value for the
affibody is 9.4 which make it positively charged at physiologic pH or lower. The exposure of the
affibody on the surface of the nanoparticles was further investigated using an inhibition assay
which will be discussed later.
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The stability of nanoparticles in the presence of serum proteins and protection from serum
nucleases is also of paramount importance. The serum stability was demonstrated by incubating
the biopolymer/DNA complexes with serum. It was observed that the serum proteins were not
able to dissociate the complexes and the biopolymer effectively protected the pDNA from serum
endonucleases (Figure 3.5b). Thus far, we have shown that the biopolymer is able to condense
pDNA into stable nanosize particles in a size range that is suitable for cellular uptake. To
examine the ability of these nanoparticles to enter the cells and mediate gene transfer, a cell
transfection study was performed.
The optimum ratio of biopolymer to pEGFP for maximum gene transfer efficiency was
determined by transfecting SK-OV-3 cells with biopolymer/pEGFP complexes at various N:P
ratios. The highest rate of gene transfection efficiency was observed at N:P 14 (Figure 3.6a) and
used in subsequent studies. To examine whether biopolymer related toxicity had any impact on
determination of gene transfection efficiency, a cell toxicity assay was performed. The results of
the cell toxicity assay indicated that the biopolymer did not have any significant effect on the
viability of SK-OV-3 cells in the range tested and as a result, did not alter the absolute values of
transfection efficiency (Figure 3.6b).
It was mentioned that an affibody was engineered in the biopolymer structure to target HER2
positive cells and facilitate internalization of nanocomplexes via receptor-mediated endocytosis.
To demonstrate the functionality of the targeting moiety in the biopolymer structure, methods
similar to those used for immunolipoplexes and targeted biopolymers were performed. [7, 22 and
23] The cell targetability was demonstrated by transfecting HER2 positive and HER2 negative
cell lines with biopolymer/pEGFP complexes. SK-OV-3 was selected because the ability of the
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HER2 affibody to target this cell line has previously been illustrated. [10] Well characterized
MDA-MB-231 breast cancer and PC-3 prostate cancer cell lines with low levels of HER2
expression were used as controls. [24] The results of this study show that the biopolymer/pEGFP
complexes could selectively internalize into HER2 positive cells but not HER2 negative cells
(Figure 3.7a). Internalization of nanoparticles into SK-OV-3 cells specifically via HER2-
mediated endocytosis was illustrated by performing an inhibition assay (Figure 3.7b). As result
of these studies, it was concluded that the affibody in the biopolymer structure was functional
and exposed on the surface of the nanoparticles.
Thus far, we have examined the functionality of DCE and TM segments in the biopolymer.
The next motif in the biopolymer structure that was characterized was the pH responsive
fusogenic peptide. The role of this motif is to assist in escape of cargo from the endosomes into
cytosol. Fusogenic peptide 5HWYG assumes an alpha helical structure at pH 6.8 which helps the
biopolymer to fuse with the endosome membranes and destabilize its integrity. The results of
hemolysis assay show that lysis occurred only in high concentrations (20 µg) and at low pH (6.0)
indicating the potential for endosomolytic activity (Figure 3.8a). Under these circumstances, the
possibility of cell damage by the biopolymer while in the circulating blood stream is minimal, as
circulation will dilute the biopolymer concentration and the pH will remain at physiological
levels until taken into endosomal compartments. Therefore, the position of the FP at the N-
terminus of the biopolymer with the specified sequence preserves its pH-responsive
functionality. The ability of the biopolymer to effectively disrupt endosome membranes resulting
in the escape of the genetic material into cytosol was examined by transfecting the SK-OV-3
cells in the presence and absence of chloroquine and bafilomycin. Chloroquine is a buffering
105
agent with the capacity to burst endosomes and release the trapped complexes (if any) into
cytosol. [25] Transfection in the presence chloroquine revealed no significant change in gene
expression in comparison to cells transfected in its absence (Figure 3.8b). This suggests that the
biopolymer/pEGFP nanocarriers that were internalized via receptor mediated endocytosis, could
effectively escape from the endosomes and did not remain trapped. Moreover, treatment with
bafilomycin showed that a low pH was necessary for endosomal escape as a significant decrease
in gene expression was observed when cells were treated with baflomycin at the time of
transfection. This is due to the fact that baflomycin is an inhibitor of the vacuolar ATPase
endosomal protein pump. [26] Inhibition of this pump reduced the release of the complexes into
the cytosol and subsequent gene transfection efficiency due to the loss of proton sponge effect as
well as conformational change of FP. Interestingly, the results of cell transfection with
biopolymer without fusogenic peptide revealed the significant impact of this motif on efficient
escape of nanocarriers from endosomes. Even though significant number of histidine residues
were present in the biopolymer sequence, it was not sufficient to disrupt endosomes efficiently
without the help of fusogenic peptide. This could be one important reason behind the fact that
viruses never evolved to utilize proton sponge effect as a means to lyse endosomes.
It is well understood that the escape of the pDNA from the endosomes into cytosol is not
sufficient to mediate gene expression as the target site for gene expression is the cell nucleus. To
provide the means of active transport towards nucleus for pDNA, a M9-NLS was engineered in
the biopolymer structure. The effect of M9-NLS in the biopolymer structure on gene transfer
efficiency was evaluated by transfecting SK-OV-3 cells with the biopolymer and biopolymer
without NLS. The results showed significant decrease in transfection efficiency when cells were
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transfected with biopolymer without NLS (Figure 3.9). This was expected as Gustin et al.
(2001), have shown that when the M9-NLS is fused to GFP, the distribution of the resulting
protein (GFP–M9NLS) was restricted to the nucleus. [27-28] Using the SWISS-MODEL
program, the tertiary structure of M9-NLS was predicted (Figure 3.10). Interestingly, the M9-
NLS appears to be an unstructured peptide with no apparent α-helical or β-sheet structure. Lack
of secondary structure and the fact that M9-NLS does not possess the ability to condense DNA
could perhaps make it easier for the cell's transportin machinery to recognize its binding site and
assist in gene transfer process to the nucleus and subsequent transfection. Although our
speculation on the mechanism of transport is probable, more in depth studies on this subject are
required to unravel the details of the intracellular trafficking processes involved. The effect of
microtubules on the transport of the nanoparticles was also examined by transfecting cells in the
presence of nocodazole. Nocodazole (microtubule depolymerizer) is a drug that is normally used
to demonstrate the effect microtubules on the trafficking of non-viral vectors towards nucleus.
[14] This drug has also been used to demonstrate the effect of NLS on microtubule-mediated
transport of the viruses towards nucleus. [29] Pre-treatment of cells with nocodazole causes
collapse of the shuttling system that interacts with the NLS sequence. [30] Consequently, it was
expected that a significant reduction in gene transfection efficiency in the absence of microtubule
network would be observed which was confirmed in SK-OV-3 transfection studies. The
observed results were another indication that the NLS in the biopolymer structure utilized
microtubules to facilitate translocation of the nanoparticles towards nucleus (Figure 3.9).
Although this shuttling system does not guarantee nuclear entry; it does provide an opportunity
107
based on proximity for the nanoparticles to enter the cells during the mitosis phase of the cell
cycle when the nuclear membrane dissolves.
Figure 3. 10 The 3D Structure of M9-NLS Predicted by SWISS-MODEL Program
3.2.6 Future Directions
We demonstrated that a biopolymer with well defined architecture can be engineered that is
customizable, easy to engineer, non-toxic, and able to perform multiple tasks. The biopolymer in
this study can condense pDNA into stable nanosize particles suitable for cellular uptake, target
cancer cells, effectively disrupt endosomes and enhance translocation of the genetic material
towards nucleus. However, the transfection efficiency may still have room for improvement.
This opens the door for more studies aimed at optimizing the biopolymer architecture in order to
achieve higher rates of gene transfer. One area that is of particular interest is the optimization of
ligand density on the surface of the nanoparticles which could significantly impact particle
internalization. [31] This can be studied by varying the number of repeating units (n) in the
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biopolymer structure to create biopolymers with different molecular weights. Furthermore, by
changing the X amino acid residues in the biopolymer structure, more basic or lipophilic
constructs can be obtained which could in turn impact gene transfer efficiency. Such constructs
can be used as a basis to generate a library of biopolymers with various gene transfer
capabilities. For example, substitution of the HER2 targeting motif with other targeting peptides
could help to target and transfect different subpopulation of cancer cells. A series of studies are
in progress to evaluate the in vivo gene transfer efficiency as well as potential immunogenicity of
this biopolymeric gene delivery system.
3.2.7 Acknowledgements
This work was supported by the startup funds from the Washington State University to Hatefi
and NIH biotechnology training fellowship (T-32 GM008336) to Canine.
3.2.8 References
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Chapter IV: Arginine Histidine Recombinant Cationic
Biopolymers for siRNA Delivery
A manuscript is in preparation to submit the results of these studies for publication:
B.F. Canine, Y. Wang, W. Ouyang, and A. Hatefi. Genetically Engineered Multifunctional
Polymer Designed for Site-Specific Nucleic Acid Delivery. (In preparation)
4.1 Background
To date there has not been a delivery system designed specifically for siRNA delivery. Most
of the available siRNA delivery systems either have developed from systems initially designed
for pDNA delivery or from siRNA that has been modified directly. These backbone
modifications allow siRNA to enter cells in a non specific manner. It was hypothesized that by
altering the functional domains on the biopolymer described in Chapter 3 that we could
extracellulary target cells through the targeting motif and intracellarly target cellular
compartments depending upon the payload, i.e. siRNA or pDNA. Recombinant cationic
biopolymers allow for the delivery vector to be customized depending on the therapeutic agent
being delivered.
4.2 A Genetically Engineered Multifunctional Polymer Designed for Site-
Specific Nucleic Acid Delivery
4.2.1 Abstract
One of the major limitations to effective siRNA delivery is the lack of a siRNA-specific
delivery system. Currently, the same delivery systems that are used for plasmid DNA (pDNA)
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delivery to the cell nucleus are used for siRNA delivery to the cytoplasm. To fill this gap, the
objective of this study was to design a biopolymer that can be programmed via its amino acid
sequence to deliver siRNA specifically to cytoplasm. By modifying the amino acid sequence, the
same biopolymer can also be programmed to deliver pDNA to the cell nucleus. For siRNA
delivery, a biopolymer was designed composed of three functional moieties: 1) a nucleic acid
binding motif, 2) a fusogenic peptide to facilitate escape of the cargo from endosomes into the
cytoplasm, and 3) a HER2 targeting peptide. For pDNA delivery, a nuclear localization signal
(NLS) was added to the biopolymer structure to facilitate active translocation of the genetic
material towards nucleus. The biopolymers were complexed with pEGFP and GFP-siRNA and
used to transfect SK-OV-3 (HER2+) cells. The intracellular trafficking of the nanoparticles was
also monitored in real-time and live cells. The results demonstrated that the biopolymer with
NLS is a suitable carrier for pDNA delivery but not siRNA delivery. Conversely, the biopolymer
without NLS was suitable for siRNA delivery to the cytoplasm but not pDNA to the cell nucleus.
To examine the potential use of the biopolymers for cancer therapy, BCL2-siRNA in
combination with pSR39 (mutant thymidine kinase gene) were used. The results demonstrated
synergistic activity in cancer cell killing when biopolymers delivered both therapeutic nucleic
acids to SK-OV-3 cells. The results of this study demonstrate the versatility and potential use of
biopolymers in programmed delivery of nucleic acids specifically to their site of action, a goal
that had not been achieved before.
4.2.2 Introduction
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One of the major limiting factors in successful targeted siRNA and plasmid DNA
(pDNA) delivery is the lack of suitable vectors that by design have the ability to target cells and
specifically deliver siRNA to the cytoplasm or pDNA to the cell nucleus. Unlike gene therapy
where the gene must be delivered to the nucleus of the cell to initiate transcription, the site of
action for siRNA is the cytoplasm. Therefore, for maximum efficacy, a delivery system must be
designed that is capable of differentiating between delivery to the cytoplasm and the cell nucleus.
So far, pDNA delivery systems such as cationic lipids and polymers (e.g., PEI) are also utilized
to form complexes with siRNA and carry them to the cytoplasm for mRNA knockdown. [1-5]
While relatively effective, liposomes and synthetic polymers allow the siRNA to enter any
cellular compartment non-discriminatingly and do not necessarily localize the cargo (i.e.,
siRNA) to the cytoplasm (site of action). This is due to the fact that such delivery systems (e.g.,
liposomes) were originally designed for pDNA delivery to the cell nucleus. [6-8]
We have recently introduced a new class of genetically engineered multifunctional vectors
for pDNA delivery. [9-10]. In one design, we have genetically engineered a prototype multi-
functional polymer for gene delivery to the cell nucleus. More details about the characterization
of the biopolymer including biodegradation, efficiency, non-toxicity and cell targeting can be
found in reference. [11] The described biopolymer, features a DNA condensing and
endosomolytic (DCE) domain consisted of repeating units of arginine-histidine (RH) with the
general structure of (RRXRRXHHXHHX)n, where X is any amino acid except D and E, and n is
the number of repeating units. Other domains include a pH-dependent fusogenic peptide for
destabilization of endosomal membrane, a high affinity HER2 targeting affibody for targeted
gene delivery, and a M9 nuclear localization signal (NLS) to enhance active translocation of
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genetic material toward the cell nucleus. In addition, the RRXRR in the DCE domain is
engineered to not only condense pDNA but be a substrate for the intracellular furin enzyme,
which will facilitate biodegradation of the cationic domain intracellularly, resulting in less
biopolymer related toxicity. [11] This biopolymer which is composed of a Fusogenic peptide, a
DNA condensing and endosomolytic motif, a Nuclear localization signal and a Targeting motif
is named FDNT.
The objective of this study was to design a programmed biopolymeric platform that can
selectively deliver siRNA to cytoplasm and pDNA to the cell nucleus. It was hypothesized that
by removing the nuclear localization signal sequence from the FDNT biopolymer structure, the
function of the biopolymer could be tailor-made for siRNA delivery. Because we have
previously demonstrated that FDNT can target SK-OV-3 (HER2+) ovarian cancer cells and
mediate gene expression, we used this cell line as the model. [11]. Schematics of the two
biopolymers are shown in Figure 4.1. While biopolymer FDT is designed for siRNA delivery
(lacks NLS), biopolymer FDNT contains a NLS and is suitable for pDNA delivery.
Figure 4. 1 Schematic of FDT and FDNT
Schematics of the multi-domain biopolymers designed for siRNA delivery (FDT) and pDNA
delivery (FDNT).3
3 Amino acid and DNA Sequences can be found in Appendix A
Nuclear Localization
Signal
DNA Condensing &
Endosomolytic MotifFusogenic Peptide
Targeting
Motif
DNA Condensing &
Endosomolytic MotifFusogenic Peptide
Targeting
Motif
FDNT
FDT
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4.2.3 Experimental Methods
4.2.3.1 Genetic Engineering of the Biopolymers
The details of the methods used to genetically engineer FDNT have been reported
previously.[11] FDT biopolymer was genetically engineered using the same methods as for
FDNT. In brief, the genes encoding the FDT and FDNT biopolymers were designed in our lab
and synthesized by Integrated DNA Technologies (San Diego, CA) with C-terminal NdeI and N-
terminal XhoI restriction sites. Synthesized genes were double digested using NdeI and XhoI
restriction enzymes (New England Biolabs, Ipswich, MA) and subcloned into a pET21b
expression vector (EMD Biosciences, Gibbstown, NJ). The biopolymer genes were expressed in
E. coli BL21(DE3) pLysS (Novagen, San Diego, CA) expression system and purified using Ni-
NTA affinity chromatography. [11] The expression of the biopolymers as well as purity was
determined by western blot analysis using anti-6X His (Abcam, Cambridge, MA) and SDS-
PAGE.
4.2.3.2 Biopolymer/pDNA complextion and particle size analysis
pEGFP or GFP-siRNA was complexed with FDT or FDNT in Bis-Tris buffer at pH 6.0. A
Malvern Nano ZS90 (Malvern Instruments, UK) was used to determine the particle sizes of the
particles. For example, for plasmid DNA delivery, 21 µg FDNT or 18 µg FDT was used to
complex with 1µg of pEGFP for an NP (N-atoms in biopolymer to P-atoms in plasmid DNA)
ratio of 14. This ratio was selected based on our previously published data. [11] For siRNA
delivery, 5 µg of biopolymer (FDNT or FDT) was used to complex with 1 µg of siRNA to
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prepare complexes at a weight ratio of 1:5. This optimum 1:5 ratio was chosen based on the
findings from our preliminary studies. After 15 min of incubation, the size of the complexes
were measured and reported as mean ± SEM, (n=3). Each mean is the average of 15
measurements and n represents the number of separate batches prepared for the measurements.
4.2.3.3 Preparation of Stable SK-OV-3 cell Line that Overexpress GFP
SK-OV-3 cells (human ovarian cancer) were seeded in T-75 flasks (BD-Biosciences, San
Jose, CA) and grown to 80% confluence in McCoy’s 5A media (Hyclone) supplemented with
10% fetal bovine serum (Hyclone, Logan UT). Using lipofectamine (Invitrogen, Carlsbad, CA),
2 x 106
cells were co-transfected with 1 µg pUB-GFP (under the control of ubiquitin promoter)
and 0.2 µg pBABE (plasmid containing a puromycin selectable marker) in serum free McCoy’s
5A media containing transferrin, selenium, ovalbumin, dexamethasone, and fibronectin
(McCoy’s-SFM). Transfected SKOV3 cells were treated with puromycin to kill the non-
transfected ones and select for GFP expressing SK-OV-3 cells (SKOV3/GFP). Stocks of
SKOV3/GFP cells were frozen and stored in liquid nitrogen for future use. The level of GFP
expression was determined using flowcytometry (FACSCalibur, BD Biosciences, San Jose, CA)
and visualized by epifluoresent microscopy (AxioObserver, Carl Zeiss, Jena, Germany).
Flowcytometry data are reported as mean ± s.d. and statistical significance was evaluated using t-
test.
4.2.3.4 Preparation of stable SK-OV-3 cell line that overexpress BCL2
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1x105 SK-OV-3 cells were seeded in 6 well plates. After 24 hours media was removed and
replaced with McCoy’s-SFM. Using lipofectamine, cells were transfected with 1 µg
pcDNA:BCL2 (Addgene, Cambridge, MA) under the control of a CMV promotor. Media was
removed after 4 hours and replaced with McCoy’s 5A media containing 10% FBS and neomycin
to select for SK-OV-3 cells overexpressing BCL2 (SKOV3/BCL2).
To verify overexpression of BCL2 in SKOV3/BCL2 cells, from each well cells were
collected and lysed on ice using extraction buffer composed of 25 mM HEPES, 5 mM MgCl2, 1
mM EDTA, 1 mM DTT, 0.1% Triton X-100, 0.1 mM PMSF, and protease inhibitor cocktail
(Calbiochem, Gibbstown, NJ). Cell lysate was centrifuged and supernatant was removed.
Equivalent of 50 µg protein was loaded onto SDS PAGE gel and electrophoresed. The
expression of BCL2 in SKOV3/BCL2 cells was then verified by western blot analysis using anti-
BCL2 antibody (BD Biochem).
4.2.3.5 Cell Transfections
SK-OV-3 cells were seeded in 96 well plates at 2.0 x 104
cells per well. Cells were incubated
overnight in 37°, 5% CO2 environment until 80-90% confluent. Plasmid DNA encoding green
fluorescent protein (pEGFP) was complexed with FDNT or FDT at N:P ratio of 14 to form
nanoparticles. This optimum N:P ratio for cell transfection was based on our previous published
data. [11] At this N:P ratio, 18 µg of FDT or 21 µg of FDNT was complexed with 1ug of pEGFP
for cell transfection. SK-OV-3 cells were then transfected in McCoy’s-SFM with the
biopolymer/pEGFP complexes. Four hr post transfection, media was replaced with McCoy’s 5A
supplemented with 10% FBS and antibiotics. Visualization of the green fluorescence was
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conducted using a Zeiss epifluoresence microscope. Total green fluoresence intensity was
measured 48 hr post transfection using a FACS Calibur flow cytomter. Flow cytometry data are
reported as mean ± s.d, n=3.
For GFP knockdown studies, SKOV3/GFP cells were seeded in 96 well plates at 2.0 x
104 cells per well. After 24 hr, unmodified GFP-siRNA (Ambion, Cat#AM4626) or scrambled-
siRNA (Ambion, Carlsbad, CA, Cat#AM4613) was complexed with the biopolymers (i.e., FDT
or FDNT) at biopolymer:siRNA weight ratio of 5:1 for optimum gene knockdown. Four hr post
transfection the media was replaced with McCoy’s 5A supplemented with 10% FBS and
antibiotics. The GFP expression level was evaluated 48 hr post transfection using flow
cytometry.
For BCL2 knockdown studies, SKOV3/BCL2 cells were used as described above.
Various amounts of unmodified BCL2-siRNA (Ambion, Carlsbad, CA Cat#AM16706,
GGAUUGUGGCCUUCUUUGAtt) or scrambled siRNA (Ambion, Carlsbad, CA Cat# AM4613,
UGUACUGCUUACGAUUCGGtt) was complexed with FDT and used to transfect cells. After 2
days cells were harvested, lysed and equivalent of 50 µg proteins was subjected to SDS PAGE.
The proteins were then transferred onto PVDF membrane and blotted with anti-BCL2 (1:500) or
anti GADPH (1:1000) (Calbiochem) antibodies.
4.2.3.6 Cancer Cell Killing by Combination Gene and siRNA Delivery
1x104
SK-OV-3 cells were seeded in 96 well plates. At the time of transfection, media was
removed and replaced with McCoy’s–SFM. One µg Plasmid DNA encoding mutant Herpes
Simplex Virus thymidine kinase gene, namely pSR39, was complexed with FDNT at the NP
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ratio of 14. [23] pSR39 was generously provided by Dr. Margaret Black in the Department of
Pharmaceutical Sciences at Washington State University. FDNT/pSR39 complexes were added
to each well. After 4 hr, media was removed and replaced with McCoy’s 5A containing 10%
FBS.
Subsequently (48 hr post transfection with pSR39), media was changed to McCoy’s-SFM to
prepare cells for siRNA delivery. FDT in complex with BCL2-siRNA or scrambled-siRNA were
added to transfect cells. Four hr post siRNA transfection, media was removed and replaced with
McCoy’s 5A containing 10% FBS and 50 µM ganciclovir (GCV). On alternating days media
containing 50 µM GCV was added to cells. Eight days following initial transfection with
FDNT/pSR39 complexes, cell survival was assessed using WST-1 reagent (Roche, Applied
Science, Indianapolis, IN). Ten µL of reagent was added per 100 µL media and plates were
incubated for 4 hr followed by measuring absorbance at 420 nm. The measured absorbance for
test groups is expressed as a percent of the control where the control is defined as 100% viable.
The data are reported as mean ± SEM, n=6 and statistical significance was evaluated using t-test.
4.2.3.7 Real-time live cell particle tracking
2x105 SK-OV-3 cells were seeded in 35 mM glass bottom MaTek plates (MaTek Corp,
Ashland, MA) in the presence of McCoy’s 5A plus 10% FBS. After 24 hr media was replaced
with McCoy’s 5A media without FBS to starve the cells. 24 hr later, biopolymers were used to
transfect SK-OV-3 cells with pDNA which was labeled with Cy3 (Mirus LabelIT kit, Mirus Bio,
Madison, WI) according to manufacturer’s instructions. Images were captured sequentially
every 10 min for 80 min using an epifluorescene microscope (Carl Zeiss Axiobserver Z1 with
121
40X objective). For z-stack images, depths of 7 µM were captured with images taken every 300
nM. Image compilation was performed using AxioObserver software (Zeiss, Jena, Germany)
and National Institutes of Health Image J (Bethesda, MD) software.
4.2.4 Results
4.2.4.1 Genetic Engineering of FDT and FDNT Biopolymers
Both FDNT and FDT biopolymers were genetically engineered, expressed in E. coli and
purified using nickel column affinity chromatography. The purity of FDNT (>98%) is reported
previously. [11] The FDT was also purified and its expression verified by western blot
analysisusing anti-6XHIS (Abcam, Cambridge, MA) (Figure 4.1).
Figure 4.1: FDT Identification
SDS-PAGE (right panel) and western blot analysis (left panel) of the purified FDT biopolymer.
The expected molecular weight for FDT is 18,210 daltons. Lane 1: lysate; Lane 2: flow through;
Lane 3: protein marker; Lane 4: purified FDT biopolymer.
20
10
1 2 3 4
20
10
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4.2.4.2 Biopolymer mediated pDNA delivery
To examine the ability of the biopolymers to delivery pDNA, where the site of action is the
cell nucleus, each biopolymer was complexed with pEGFP and used to transfect SK-OV-3 cells.
The sizes of the FDT/pEGFP nanoparticles were 78±4 nm, whereas the sizes of FDNT/pEGFP
nanoparticles were 66±5nm. SK-OV-3 cells that were transfected with FDNT biopolymer
(contains NLS) showed significantly more GFP expression in comparison to FDT biopolymer
which lacks the NLS domain (Figure 4.2).
123
Figure 4. 2 Evaluation of pEGFP Delivery to SK-OV-3 Cells Using FDNT or FDT
a) Epifluorescent images of transfected SK-OV-3 cells by FDNT/pEGFP (left panel) and
FDT/pEGFP (right panel). b) Quantitative analysis of the gene expression in cells transfected
with FDNT/pEGFP and FDT/pEGFP. The fluorescence intensity was measured using flow
cytometry
124
4.2.4.3 Intracellular Trafficking of the Nanoparticles in Live Cells
To better understand the reason behind the observed differences in transfection efficiencies
of the biopolymers, the intracellular trafficking of the biopolymer/pDNA complexes was studied
using dynamic imaging (real-time in live cells). Labeled pDNA was complexed with FDNT or
FDT and used to transfect cells. It was observed that the FDNT/pEGFP complexes are trafficked
towards the nuclear membrane actively and in less than 80 min (Figure 4.3a). In contrast,
FDT/pEGFP complexes remain fixed, are more localized in their movements, and rarely reach
the nuclear membrane area (Figure 4.3b).
125
Figure 4. 3 Real Time, Live Cell Imaging of Delivery to SK-OV-3 Cells
Real time live cell imaging of the FDNT/pDNA and FDT/pDNA nanoparticles over 80 min
using an epifluorescent microscope. a) FDNT/pDNA complexes were used to transfect SK-OV-3
cells and their trafficking was observed by an epifluorescent microscope. The nuclear membrane
is shown by a white crooked line, whereas the white arrows point at the red fluorescent
nanoparticles. b) The FDT/pDNA complexes were used to transfect SKOV3 cells. The arrows
point at the nanoparticles, whereas the crooked line indicates the edge of the nuclear membrane.
To verify that the nanoparticles were indeed internalized and not immobilized on the cell
surface, z-stack images were taken each 300 nm apart. Z-stack images of the SK-OV-3 cells that
were transfected with FDNT/pDNA and FDT/pDNA complexes are shown in Figure 4.4 a and
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Figure 4.4 b. Highest levels of red fluorescence were observed in midsection slices (z-stacks)
highlighting the internalization of the nanoparticles.
127
Figure 4. 4 Z-stack of SK-OV-3 cells Transfected with FDNT/pDNA or FDT/pDNA
a) Z-stack of FDNT/pDNA nanoparticles labeled with Cy3 (b) Z-stack of FDT/pDNA
nanoparticles. pDNA (pEGFP) was labeled with Cy3 dye and used to complex with
biopolymers. Z-stack images 7 to 12 (inside the cell) show the highest levels of nanoparticle
fluorescence intensity. Each numbered frame represents a vertical section.
128
4.2.4.4 Biopolymer Mediated siRNA Delivery
To examine the ability of the biopolymers to deliver siRNA, where the site of action is the
cytoplasm, FDT and FDNT biopolymers were complexed with GFP-siRNA and used to transfect
SKOV3/GFP cells. The sizes of the nanoparticles formed with FDNT/GFP-siRNA and
FDT/GFP-siRNA were 121 ± 7 and 140 ± 5 nm, respectively. As shown in Figures 4.5 a and
Figure 4.5 b, the SKOV3/GFP cells (GFP+) have significantly higher green fluorescent intensity
above the background level (GFP¯ ). SKOV3/GFP cells treated with GFP-siRNA alone showed
no decrease in GFP expression. While SKOV3/GFP cells that were transfected with FDT/GFP-
siRNA showed significant GFP knockdown (*t-test, p<0.05), no significant GFP knockdown
with FDNT/GFP-siRNA was observed. Scrambled siRNA sequences also had no effect on
measured GFP levels.
Based on the results from the abovementioned studies, the potential use of the biopolymer
FDT in BCL2 (pro-survival protein) knockdown by delivering BCL2-siRNA was also examined.
It was observed that FDT has the ability to ablate the expression of BCL2 in SKOV3/BCL2 cell
line Figure 4.5 c. As a result, SK-OV-3 cells could become more susceptible to apoptosis that is
induced by drugs or therapeutic genes.
129
130
Figure 4. 5 Evaluation of GFP or BCL2 Knockdown Using FDNT or FDT Complexed
With siRNA for GFP or BCL2
a) SK-OV-3/GFP cells were transfected with GFP-siRNA alone, FDNT/GFP-siRNA and
FDT/GFP-siRNA to knock down GFP expression. 1:5 ratio refers to 1 ug of GFP-siRNA to 5 ug
of biopolymer. Scrambled siRNA was used as a control. The level of GFP expression was
measured using flowcytometry. b) Epifluorescent images of the untreated GFP¯ and GFP
+ cells
as well as SK-OV-3/GFP cells treated with biopolymers in complex with GFP-siRNA. C)
Knockdown of BCL2 protein expression in SKOV3/BCL2 cancer cells that overexpress BCL2.
BCL2-siRNA was complexed with FDT to knockdown the BCL2 expression. i) Knockdown was
detected by westernblot analysis using anti-BCL2 antibody. Lane 1: BCL2 protein (positive
control); Lane 2: FDT/BCL2-siRNA (1:1 ratio); Lane 3: FDT/BCL2-siRNA (1:5 ratio); Lane 4:
scrambled-siRNA (1:5 ratio); ii) GAPDH control.
4.2.4.5 Use of Biopolymers for Combination Gene and siRNA Therapy
The ability of one biopolymeric platform to deliver different therapeutic nucleic acids and
result in significant cell killing was evaluated by trasnfecting SK-OV-3 cancer cells with BCL2-
siRNA and pSR39 gene. The results demonstrated that FDNT/pSR39 complexes in combination
with prodrug ganciclovir (GCV) are able to kill SK-OV-3 cells significantly (*t-test, p<0.05)
(Figure 4.6). In comparison to cells treated with FDNT/pSR39 complexes plus GCV, maximum
killing efficiency was observed when FDNT/pSR39 plus GCV was combined with FDT/BCL2-
siRNA (*t-test, p<0.05). No other control group resulted in statistically significant cell killing in
comparison to the untreated cells.
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Figure 4. 6 Cell Killing Efficiency
Evaluation of cell killing efficiency by biopolymers in complex with therapeutic nucleic acids.
Cells were transfected with two test groups (FDNT/pSR39 plus GCV; and FDNT/pSR39 plus
GCV + FDT/BCL2-siRNA) and six control groups. The cell survival was assessed at day 7 with
WST-1 cell toxicity assay.
4.2.5 Discussion
In the past decade, since the discovery of RNA interference (RNAi), significant amounts of
money have been invested toward the therapeutic application of gene silencing in humans. [12]
Concurrently, significant efforts have been made in therapeutic application of genes to cure
various diseases. Despite early successes, however, the widespread use of RNAi and gene
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therapeutics for disease prevention and treatment has not been achieved. Future success requires
the development of nucleic acid delivery systems that are safe, efficient, targeted and cost-
effective. One of the major impediments to the effective siRNA and pDNA delivery is the
inability to efficiently localize such molecules to their site of action. While various gene delivery
systems have been designed for nucleic acid delivery, there has been no report on the design and
development of a vector that is tailor-made specifically for either siRNA or pDNA. To address
this issue, development of a class of biomaterials is required that will allow precise correlation of
structure with function. One class of biomaterials that provides the possibility of performing such
precise structure/activity relationship studies is genetically engineered polymers. Using
recombinant techniques, novel polymers can be synthesized with precise compositions,
molecular weights, sterotacticity and specified functions. Compared to synthetic methods of
polymer production, the principal advantages are (i) monodisperse material, (ii) full control over
polymer architecture at the molecular level, (iii) precise covalent attachment of functional
moieties (e.g. targeting motifs), (iv) programmability via amino acid sequence, and from a
manufacturing standpoint, (v) elimination of the conjugation steps. [9] In 1997, Dan Urry was
among the first to discuss the possibility of developing biopolymers that can be programmed via
their amino acid sequences to perform self-guided functions. [13] Here, we demonstrate that
multifunctional biopolymers can be designed where amino acid sequence could dictate not only
their function but localization site.
To achieve the objective, two multifunctional biopolymers were designed one with NLS and
the other without (Figure 4.1). Using genetic engineering techniques, they were cloned and
produced in E. coli expression system and isolated to high purity using nickel column
133
chromatography. Both purified biopolymers were used to complex with pEGFP to form particles
with sizes less than 150 nm which would make them suitable for receptor-mediated endocytosis.
[14] Our previous experience with targeted gene delivery shows that nanoparticles with sizes less
than 100 nm produce maximum efficiency. [11,15,16] These nanoparticles were then used to
transfect SK-OV-3 ovarian cancer cells which overexpress HER2 on their surfaces. [17] It was
observed that the cells that were transfected with vector FDNT had significantly higher gene
expression in comparison to the cells that were transfected with FDT (Figure 4.2). This
observation was somewhat expected as the site of action for plasmid DNA is the cell nucleus and
vector FDNT has all the major necessary tools to facilitate its localization in the nucleus. To
make a direct observation of the trafficking of the nanoparticles, we utilized real-time live cell
imaging technique. This technique has previously been described in detail by Hanes group. [18-
19] Vectors FDNT and FDT were complexed with Cy3 labeled pEGFP and used to transfect
cells. The trafficking of the nanoparticles was observed in a single SK-OV-3 cell using an
epifluorescent microscope for a period of 80 min. It was observed that the FDNT/pDNA
nanoparticles were actively shuttled towards nuclear membrane while FDT/pDNA remained
stagnant inside the cytoplasm (Figure 4.3). This observation reaffirmed our previous findings
that the presence of NLS in the biopolymer structure is necessary for microtubule mediated
translocation of genetic materials toward nucleus[124]. [11] To examine whether the labeled
nanoparticles could internalize and not merely attach to the outer membrane, z-stack images
were obtained (Figure 4). Observation of bright red signals in the midst sections illustrated that
the nanoparticles were indeed inside the cells.
134
To further examine the localization site of FDNT versus FDT, we changed the payload to
siRNA where the site of action is cytoplasmic. From the cell transfection studies with pEGFP
and imaging studies we understood that FDT/pDNA nanoparticles were inefficient in reaching
the cell nucleus and localized in the cytoplasm. Therefore, we hypothesized that FDT is a
suitable vehicle for siRNA delivery. To test the hypothesis, we used GFP-siRNA as a model
gene silencer to demonstrate the localization of FDT in the cytoplasm. FDNT biopolymer was
used as control to demonstrate that presence of NLS in the structure is detrimental to the
localization of siRNA in the cytoplasm. Unmodified GFP-siRNA was purchased from Ambion
and used as a gene silencer to evaluate the localization of the biopolymers in cytoplasm. It is of
paramount importance to mention that for true evaluation of the efficiency of siRNA delivery
systems, unmodified siRNA needs to be used. Several companies such as Dharmacon (Lafayette,
CO) have modified the siRNA structure by attaching lipids or other molecules to enhance its
internalization into the cells. Such modified siRNAs are not suitable for the evaluation of the
efficiency of the delivery systems as they themselves can enter the cells without the help of a
carrier. In this study, we purchased unmodified siRNA to ensure that siRNA by itself cannot
enter the cells. For the evaluation of the gene expression knockdown, we also engineered a SK-
OV-3 cell line that stably expresses GFP (SKOV3/GFP).
Both FDT and FDNT biopolymers were complexed with GFP-siRNA and used to
knockdown GFP expression in SKOV3/GFP cells. Significant GFP expression knockdown
indicated that vector FDT is the more suitable carrier for siRNA delivery (Figure 4.5 a and
Figure 4.5 b). To examine the potential use of FDT in cancer therapy, we delivered BCL2-
siRNA to SK-OV-3 cancer cells to knockdown one of their most prominent pro-survival
135
signaling pathway. When under stress (e.g., chemotherapy or gene therapy), cancer cells have the
capacity to up-regulate production of BCL2 protein which make them more resistant to
apoptosis. [20] Under normal conditions, the basal level of BCL2 production in SK-OV-3 cells is
low and hard to detect. Therefore, we engineered a SK-OV-3 cell line that overexpresses BCL2
(SKOV3/BCL2) which was used to evaluate the efficiency of the FDT biopolymer to
knockdown BCL2 expression. Both biopolymers were complexed with BCL2-siRNA and used
to transfect SKOV3/BCL2 cells. The results showed that only FDT was able to ablate the
expression of BCL2, whereas FDNT was not effective (Figure 4.5 c). This finding not only was
in agreement with our GFP-siRNA observations, but also indicated the potential use of the FDT
vector in siRNA delivery for cancer therapy. To examine this potential, we combined BCL2-
siRNA delivery with thymidine kinase gene delivery.
Herpes simplex virus thymidine kinase (HSV-TK) is a widely used suicide gene that in
conjunction with prodrug ganciclovir (GCV) results in cell killing by selectively converting
nontoxic prodrugs to highly toxic metabolites. In the case of HSV-TK/GCV, the viral enzyme
phosphorylates a nucleoside analog resulting in analog incorporation during DNA replication,
halting the replication process. [21] HSV-TK/GCV also demonstrates a bystander effect meaning
that cells not directly transfected with the gene may also be effected due to the transfer of the
cytotoxic metabolites. [22] This offers an advantage in systems with less efficient transfection
levels as fewer cells need to be transfected to see a substantial effect. In this study, we used the
SR39 gene which is a mutant HSV-TK developed by Black’s group and more selective to GCV
than wild-type. [23] The rationale behind the choice of BCL2 knockdown in combination with
pSR39 is that expression of thymidine kinase gene in transfected cells in combination with GCV
136
will cause significant stress in those cells. As a result, transfected cells would up-regulate the
pro-survival BCL2 signaling pathway to resist apoptosis. Subsequent transfection of the cells
with BCL2-siRNA would inhibit such defense mechanism and render the cells more responsive
to suicide gene therapy. Published data by others also reiterates the potential use of this approach
in combination gene and siRNA cancer therapy. [24]
This approach in cancer therapy was evaluated using FDNT/pSR39 complexes plus GCV in
combination with FDT/BCL2-siRNA complexes. Using a cell toxicity assay, we evaluated the
cell killing effects of combination siRNA and suicide gene delivery (Figure 4.6). In comparison
to untreated cells, we observed statistically significant cell killing with FDNT/pSR39 plus GCV
(*t-test, p<0.05). As discussed above, cells that undergo stress may up-regulate BCL2 pathway
to resist apoptosis. Therefore, by knocking down the BCL2 expression, we expected to observe
significantly higher cell killing efficiency. In comparison to the cell killing efficiency of
FDNT/pSR39 plus GCV, we observed significantly higher cell death in cells that were treated
with FDT/BCL2-siRNA in combination with FDNT/pSR39 plus GCV (*t-test, p<0.05).
The significance of these studies is that one biopolymeric platform can be tailor-made with
minimum costs to deliver different types of nucleic acids to their site of action for maximum
therapeutic outcome. These proof-of-concept studies justify the use of animals in the next steps
to evaluate the potential application of such biopolymers to establish preliminary efficacy and
toxicity in vivo.
4.2.6 Conclusions
137
One of the most important features of using biopolymers for gene delivery is the enormous
possibility for recombinant engineering that facilitates accurate structure/activity relationship
studies. As a result, the rate limiting steps to non-viral gene transfer can be studied with higher
degree of accuracy and precision. These biopolymers allow full control over their architecture at
the molecular level; hence, customizable and programmable via their amino acid sequences. The
results of this study demonstrate the versatility and potential use of biopolymers in programmed
delivery of nucleic acids specifically to their site of action, a goal that had not been achieved
before.
4.2.7 References
[1] S.H. Lee, S.H. Kim, T.G. Park, Intracellular siRNA delivery system using polyelectrolyte
complex micelles prepared from VEGF siRNA-PEG conjugate and cationic fusogenic
peptide. Biochem Biophys Res Commun 357(2) (2007) 511-516.
[2] O.B. Garbuzenko, M. Saad, S. Betigeri, M. Zhang, A.A. Vetcher, V.A. Soldatenkov,
D.C. Reimer, V.P. Pozharov, T. Minko, Intratracheal versus intravenous liposomal
delivery of siRNA, antisense oligonucleotides and anticancer drug. Pharm Res 26(2)
(2009) 382-394.
[3] A. Inoue, S.Y. Sawata, K. Taira, Molecular design and delivery of siRNA. J Drug Target
14(7) (2006) 448-455.
[4] B.R. Meade, S.F. Dowdy, The road to therapeutic RNA interference (RNAi): Tackling
the 800 pound siRNA delivery gorilla. Discov Med 8(43) (2009) 253-256.
[5] Y. Wang, M. Saad, R.I. Pakunlu, J.J. Khandare, O.B. Garbuzenko, A.A. Vetcher, V.A.
Soldatenkov, V.P. Pozharov, T. Minko, Nonviral nanoscale-based delivery of antisense
oligonucleotides targeted to hypoxia-inducible factor 1 alpha enhances the efficacy of
chemotherapy in drug-resistant tumor. Clin Cancer Res 14(11) (2008) 3607-3616.
[6] H. Akita, H. Harashima, Nonviral gene delivery. Contrib Nephrol 159 (2008) 13-29
[7] X. Gao, K.S. Kim, D. Liu, Nonviral gene delivery: what we know and what is next. Aaps
J 9(1) (2007) E92-104.
138
[8] C. Louise, Nonviral vectors. Methods Mol Biol 333 (2006) 201-226
[9] B.F. Canine, A. Hatefi, Development of recombinant cationic polymers for gene therapy
research. Adv Drug Deliv Rev (2010 (In press)).
[10] H.O. McCarthy, Y. Wang, S.S. Mangipudi, A. Hatefi, Advances with the use of bio-
inspired vectors towards creation of artificial viruses. Expert Opin Drug Deliv 7(4)
(2010) 1-16.
[11] B.F. Canine, Y. Wang, A. Hatefi, Biosynthesis and characterization of a novel genetically
engineered polymer for targeted gene transfer to cancer cells. J Control Release 138(3)
(2009) 188-196.
[12] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in
siRNA delivery. Nat Rev Drug Discov 8(2) (2009) 129-138.
[13] D.W. Urry, Physical chemistry of biological free energy transduction as demonstrated by
elastic protein-based polymers. J Phys Chem B 101(51) (1997) 11007-11028.
[14] J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Size-dependent internalization of
particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J
377(Pt 1) (2004) 159-169.
[15] S.S. Mangipudi, B.F. Canine, Y. Wang, A. Hatefi, Development of a genetically
engineered biomimetic vector for targeted gene transfer to breast cancer cells. Mol Pharm
6(4) (2009) 1100-1109.
[16] Y. Wang, S.S. Mangipudi, B.F. Canine, A. Hatefi, A designer biomimetic vector with a
chimeric architecture for targeted gene transfer. J Control Release 137 (2009) 46-53.
[17] D.W. Rusnak, K.J. Alligood, R.J. Mullin, G.M. Spehar, C. Arenas-Elliott, A.M. Martin,
Y. Degenhardt, S.K. Rudolph, T.F. Haws, Jr., B.L. Hudson-Curtis, T.M. Gilmer,
Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2)
protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded
panel of human normal and tumour cell lines. Cell Prolif 40(4) (2007) 580-594.
[18] J. Suh, D. Wirtz, J. Hanes, Efficient active transport of gene nanocarriers to the cell
nucleus. Proc Natl Acad Sci U S A 100(7) (2003) 3878-3882.
[19] J. Suh, M. Dawson, J. Hanes, Real-time multiple-particle tracking: applications to drug
and gene delivery. Adv Drug Deliv Rev 57(1) (2005) 63-78.
[20] N. Sasi, M. Hwang, J. Jaboin, I. Csiki, B. Lu, Regulated cell death pathways: new twists
in modulation of BCL2 family function. Mol Cancer Ther 8(6) (2009) 1421-1429.
139
[21] S.D. Mahan, G.C. Ireton, C. Knoeber, B.L. Stoddard, M.E. Black, Random mutagenesis
and selection of Escherichia coli cytosine deaminase for cancer gene therapy. Protein Eng
Des Sel 17(8) (2004) 625-633.
[22] T.W. Nicholas, S.B. Read, F.J. Burrows, C.A. Kruse, Suicide gene therapy with Herpes
simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve
gap junctions and bystander effects. Histol Histopathol 18(2) (2003) 495-507.
[23] M.E. Black, M.S. Kokoris, P. Sabo, Herpes simplex virus-1 thymidine kinase mutants
created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell
killing. Cancer Res 61(7) (2001) 3022-3026.
[24] W. Hamel, L. Magnelli, V.P. Chiarugi, M.A. Israel, Herpes simplex virus thymidine
kinase/ganciclovir-mediated apoptotic death of bystander cells. Cancer Res 56(12) (1996)
2697-2702.
140
5. Chapter V. General Conclusions and Future Directions
5.1 General Conclusions
Nucleic acids have transformed biomedical research. They are used for diagnostic tools,
pathway elucidation, as well as in target validation applications. The discoveries that
exogenously delivered nucleic acids could result in protein expression (gene delivery) or mRNA
degradation (siRNA delivery) offered great promise as revolutionary new therapeutics for a
myriad of diseases. The translation of these research tools to therapeutically relevant nucleic-acid
based drugs however has not been as successful.
One of the major limiting factors in successful targeted siRNA and plasmid DNA (pDNA)
delivery is the lack of suitable vectors that by design, have the ability to deliver siRNA
specifically to the cytoplasm of the cell or pDNA to the nucleus.
In Chapter I, we provided a review of the current status of the pDNA and siRNA delivery
strategies as well as a short description of the barriers that need to be overcome by these delivery
vectors. Unfortunately all of the described vectors to date, have limitations. Viral vectors have
safety concerns and are limited by costly and difficult manufacturing processes. Cationic lipids
and polymers were introduced to overcome these concerns, however they are hampered by low
transfection efficiencies. As evidenced by the infrequeent FDA approval of nucleic acid
therapies a new strategy for delivery is needed. This provided the basis for the design and basis
of recombinant delivery systems. The overview given in Chapter 1, described the general
biomimetic strategy we used to design our recombinant biopolymers, and discussed other
recombinant systems that have been developed.
141
The overall objective of this research was to develop novel, recombinant vectors which are
targeted, biocompatible, and can perform several functional tasks to overcome intracellular
barriers. The target model for treatment was metastatic cancer cells that overexpress either
Fibroblast Growth Factor Receptor 2 (FGFR2) or Human Epidermal Growth Factor Receptor 2
(HER2). Using recombinant DNA and genetic engineering techniques mutifuncitonal vectors
were designed to either delivery pDNA or siRNA encoding reporter or therapeutic agents.
Chapter II discusses the design and development of a multifunctional vector utilizing a
lysine-histidine rich condensing core and a FGF2 targeting protein. This chapter demonstrates
the importance of vector architecture by comparing a dispersed and a condensed lysine-histidine
arrangement as well as demonstrating the ability of two functional groups to be incorporated
onto a single recombinant protein backbone. From this comparative study, it is clearly
demonstrated that not only the contents of the vector, but the arrangement of the contents, i.e.,
the architecture of the vector is important to consider in nucleic acid delivery vectors.
Manipulation at the molecular level, using recombinant DNA technology allows us to make
these comparisons and evaluate the effects of architectural changes on DNA condensation and
transfection efficiency.
Chapter III discusses the design, development, and characterization of a multifunctional
prototype vector utilizing an arginine-histidine rich core condensing region, a fusogenic peptide
for endosomal escape, a targeting motif for HER2 and a nuclear localization signal. Each domain
of the vector was characterized for functionality and it was demonstrated that the vector
containing multiple domains was able to successfully transfect specific cells with a reporter GFP
plasmid. In this in vitro proof of principle study, the vector was able to condense pDNA into
142
sizes suitable for cellular uptake, target SK-OV-3 cancer cells, mediate escape from endosomal
compartments, use microtubules to translocate to the nucleus and ultimately result in reporter
gene expression.
In Chapter IV we examined the customizeability of the RH based biopolymer to deliver
pDNA and siRNA. The ability to target specific compartments in the cell depending upon the
cargo i.e., pDNA or siRNA is important as the site of action for these molecules is vastly
different. These biopolymers were then evaluated for their ability to target intracellular
compartments as evidenced by microscopy studies as well as delivery of either pDNA or siRNA.
It was demonstrated that FDNT localizes to the nucleus and results in more GFP expression
when pEGFP was delivered. FDT localization, however, was cytoplasmic and demonstrated
knockdown of GFP when GFP-siRNA was delivered while FDNT did not. This emphasizes that
targeting of intracellular compartments is important for successful nucleic acid delivery.
Additionally a double treatment utilizing pDNA delivery (HSVTK-SR39 +GCV) and siRNA
delivery targeting BCL-2 was performed. The overexpression of BCL-2 has previously been
implicated on reduced efficacy of the suicide gene therapy utilizing the HSV-TK enzyme paired
with the prodrug, gancyclovir. The double treatment showed that by knocking down BCL-2 the
HSV-TK+GCV delivery resulted in higher cell killing than did the HSV-TK system.
Combination therapy is a common treatment strategy and the strategy demonstrated here could
be developed for gene therapy/siRNA delvery.
5.2 Future Directions
143
Delivery to target cells, continues to be the hurdle that hampers both gene and siRNA
delivery. Recombinant vectors have great promise as a potential solution to this problem;
however, they still have limitations. Before clinical application can be done several areas need
to be examined.
The first is to find an optimal formulation. This can be looked at in a variety of ways. .
While >20% transfection was achieved, a large number in non-viral delivery, this still does not
rival the transfection efficiency of viral delivery vehicles. Improvements in this could potentially
be achieved by substitution of various motifs.
The lab has already begun some of this work. The first study done was to replace the
H5WYG domain which is the endosomolytic domain with a KALA domain which is also
endosomolytic. This domain was chosen to alter as the solubility of the H5WYG domain was
limiting the ability of the purification of the entire protein and meant that perhaps altering the
ensosomolytic domain could result in increased expression levels. Initial studies show that the
KALA domain did not show improvement in transfection efficiency; however other domains
could be tried such as GALA.
Also of interest is to alter the ratio of arginine (R) to histidine (H) content in the DNA
condensation domain. The low pKA of the histidine resulted in much of the purification of the
FDNT protein being performed at low pH so that the amino group of the H would be charged
allowing for increased solubility. By reducing the number of H residues and increasing the
number of R residues it is hoped that the solubility issues will be resolved. The current
biopoloymer contains a ratio of 50:50 R to H. By increasing ratio to 60:40 it is hoped that the
solubility will improve without affecting the ability of the biopolymer to condense nucleic acids
144
into small nanosized particles. This alteration has been made and experiments are ongoing
examining this construct for future studies.
The M9 NLS was chosen due to its ability to target the nucleus without contributing to the
condensation of the DNA as it has no positively charged amino acids. Use of a more well
understood NLS such as SV40 may lead to improved nuclear localization but also might
decrease DNA unpackaging. To uncover the structure function relationships involved would
mean making a library of constructs that would need to be empirically tested for their respective
efficiencies.
Finally the targeting motif presents a unique challenge. The area of biomarker detection is
rapidly expanding with the advent of high throughput screening and any number of ligands could
be employed. As more peptide biomarkers are discovered, this motif could be interchanged and
used to target specific cellular receptors. The HER2 affibody is a part of a growing number of
similar molecules that target such things as EGFR, the Insulin Receptor, among an expanding of
list of targets developed by a Swedish Biotech company.[1] They are also developing an
additional targeting scaffold program utilizing the domains of serum albumin binding domains.
In addition to changing various motifs other challenges also still remain. At some point, in
vivo studies will need to be performed to look at the 1) in vivo efficacy, 2) immunogenicity, and
3) pharmacokinetics of these biopolymer nucleic acid complexes. Additionally these studies
should be done both intratumorally and systemically to fully characterize the system and its
potential applications. In vivo imaging studies would lead to further understanding of the
complex dynamics of this drug delivery system and would provide insight about the bottlenecks
in the system whether that be stability or tumor accumulation/targeting or elimination.
145
Other work that would be interesting would be to obtain crystal structures of the delivery
protein as a whole or in pieces. A preliminary crystal screen was done without production of
successful crystals but it would be of value to continue these efforts with each of the constructs
as well as with each functional domain as an individual entity.
In the polymer and lipid areas of drug delivery the use of PEG as a shielding agent to
increase circulation times and help with tumor accumulation is a common practice. Addition of
similar shielding entities could also be used with the cationic biopolymers. Use of ELP’s
(Elastin Like Polymers) which are repeating units of VPGVG are naturally occurring polymers.
These have also been used as thermoresonsive purification tags and may serve as both a
shielding motif as well as a purification aid for those biopolymer constructs which tend to bind to
intracellular entities.[2-6]
The recombinant biopolymers presented here have the unique ability to be designed
specifically for various applications depending on the domains incorporated in the biopolymer.
The interchangeability of the domains allows for great flexibility in not only the biopolymers
presented here but in future designs paving the way for many future projects in creation of a
delivery platform that is customizable depending upon the disease target and cargo being
delivered.
5.3 References
[1] Lofblom, J. et al. Affibody molecules: engineered proteins for therapeutic, diagnostic and
biotechnological applications. FEBS Lett 584, 2670-2680.
[2] Reguera, J., Urry, D.W., Parker, T.M., McPherson, D.T. & Rodriguez-Cabello, J.C.
Effect of NaCl on the exothermic and endothermic components of the inverse
146
temperature transition of a model elastin-like polymer. Biomacromolecules 8, 354-
358 (2007).
[3] Rincon, A.C. et al. Biocompatibility of elastin-like polymer poly(VPAVG)
microparticles: in vitro and in vivo studies. J Biomed Mater Res A 78, 343-351 (2006).
[4] Herrero-Vanrell, R. et al. Self-assembled particles of an elastin-like polymer as vehicles
for controlled drug release. J Control Release 102, 113-122 (2005).
[5] Reguera, J., Fahmi, A., Moriarty, P., Girotti, A. & Rodriguez-Cabello, J.C. Nanopore
formation by self-assembly of the model genetically engineered elastin-like polymer
[(VPGVG)2(VPGEG)(VPGVG)2]15. J Am Chem Soc 126, 13212-13213 (2004).
[6] Girotti, A. et al. Design and bioproduction of a recombinant multi(bio)functional elastin-
like protein polymer containing cell adhesion sequences for tissue engineering
purposes. J Mater Sci Mater Med 15, 479-484 (2004).
147
Appendix A: Amino Acid and DNA Sequences
Amino Acid and DNA Sequence of cKH-FGF2
Amino Acid Sequence
M K K K H H H H K K K G K K K H H H H K K K G K K K H H H H K K K G K K K H H H
H K K K G K K K H H H H K K K G K K K G K K K H H H H K K K G G E F M A A G S I T
T L P A L P E D G G S G A F P P G H F K D P K R L Y C K N G G F F L R I H P D G R V D
G V R E K S D P H I K L Q L Q A E E R G V V S I K G V C A N R Y L A M K D G R L L A
S K C V T D E C F F F E R L E S N N Y N T Y R S R K Y T S W Y V A L K R T G Q Y K L
G S K T G P G Q K A I L F L P M S A K S K L A A A L E H H H H H H Stop
DNA sequence:
5’atgaaaaaaaaacatcatcatcataaaaaaaaaggcaaaaaaaaacatcatcatcataaaaaaaaaggcaaaaaaaaacatcatcatcat
aaaaaaaaaggcaaaaaaaaacatcatcatcataaaaaaaaaggcaaaaaaaaacatcatcatcataaaaaaaaaggcaaaaaaaaagg
caaaaaaaaacatcatcatcataaaaaaaaaggcggcgaatttatggcggcgggcagcattaccaccctgccggcgctgccggaagatg
gcggcagcggcgcgtttccgccgggccattttaaagatccgaaacgcctgtattgcaaaaacggcggcttttttctgcgcattcatccggatg
gccgcgtggatggcgtgcgcgaaaaaagcgatccgcatattaaactgcagctgcaggcggaagaacgcggcgtggtgagcattaaagg
cgtgtgcgcgaaccgctatctggcgatgaaagatggccgcctgctggcgagcaaatgcgtgaccgatgaatgctttttttttgaacgcctgg
aaagcaacaactataacacctatcgcagccgcaaatataccagctggtatgtggcgctgaaacgcaccggccagtataaactgggcagca
aaaccggcccgggccagaaagcgattctgtttctgccgatgagcgcgaaaagcaaactggcggcggcgctggaacatcatcatcatcatc
atagcaccccg3’
148
Amino Acid and DNA sequence of FDNT
Amino Acid Sequence:
M G L F H A I A H F I H G G W H G L I H G W Y P G E G V P G E G V P G G G S R R V R
R S H H R H H T R R V R R S H H R H H T R R V R R S H H H H H H S S E L G G N Y N N
Q S S N F G P M K G G N F G G R S S G P Y G G G G Q Y F A K P R N Q G G Y V P G G G
F F L G G V P G E G V P G E G V P G G G K L V D N K F N K E M R N A Y W E I A L L P
N L N N Q Q K R A F I R S L Y D D P S Q S A N L L A E A K K L N D A Q A P K Stop
DNA Sequence:
5’catatgggcctgtttcatgccatcgcgcatttcattcacggcggttggcacggattgatccacggctggtacccgggcgagggggtaccg
ggagaaggcgtgccgggcggtggatcccgtcgtgtgcgtcgtagtcatcatcgtcatcacacgcgtcgtgtgcgccgttcccaccaccgtc
atcacacccgccgcgtacgccgttctcaccatcatcatcaccacagctcggagctcggaggcaattacaataaccagtcttccaactttggtc
ctatgaaaggcggcaactttggcggccgctctagtgggccatacggcggcggtggacaatactttgctaaaccacgtaatcagggaggat
atgtgccgggcgggggcttctttcttgggggagtccctggtgaaggcgtgccgggagaaggagtgccgggcgggggcaagcttgtaga
caataaatttaacaaggaaatgcggaacgcctattgggaaattgccctcttgccgaatctcaataaccagcagaaacgtgctttcatccgttca
ctgtacgatgacccttctcagtcggcgaacctgcttgcggaagccaaaaagctgaacgacgcgcaggcgcctaaataactcgag 3’
149
Amino Acid and DNA sequence of FDT
Amino Acid Sequence:
M G L F H A I A H F I H G G W H G L I H G W Y P G E G V P G E G V P G G G S R R V R
R S H H R H H T R R V R R S H H R H H T R R V R R S H H H H H H S S E L V P G G G F
F L G G V P G E G V P G E G V P G G G K L V D N K F N K E M R N A Y W E I A L L P N
L N N Q Q K R A F I R S L Y D D P S Q S A N L L A E A K K L N D A Q A P K Stop
DNA Sequence
5’catatgggcctgtttcatgccatcgcgcatttcattcacggcggttggcacggattgatccacggctggtacccgggcgagggggtaccg
ggagaaggcgtgccgggcggtggatcccgtcgtgtgcgtcgtagtcatcatcgtcatcacacgcgtcgtgtgcgccgttcccaccaccgtt
catcacacccgccgcgtacgccgttctcaccatcatcatcaccacagctcggagctgggcgggggcttctttcttgggggagtccctggtga
aggcgtgccgggagaaggagtgccgggcgggggcaagcttgtagacaataaatttaacaaggaaatgcggaacgcctattgggaaattg
ccctcttgccgaatctcaataaccagcagaaacgtgctttcatccgttcactgtacgatgacccttctcagtcggcgaacctgcttgcggaagc
caaaaagctgaacgacgcgcaggcgcctaaataactcgag 3’
150
Amino Acid and DNA sequence of Herpes Simplex Virus 1 thymidine kinase mutant SR39, 5
amino acids were mutated to achieve higher substrate (GCV) binding affinity.
Amino Acid Sequence:
MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKMPTLLR
VYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTT
QHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPP
ALTIFLDRHPIAFMLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDW
GQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWA
LDVLAKRLRSMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDL
ARTFAREMGEAN
SR39: 159~161: L I F I F L
168~169: A L F M
DNA sequence:
5’ATGGCTTCGTACCCCGGCCatcaaCACGCGTCTGCGTTCGACCAGGCTGCGCG
TTCTCGCGGCCATAGCAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAA
GAAGCCACGGAAGTCCGCCCGGAGCAGAAAATGCCCACGCTACTGCGGGTTT
ATATAGACGGTCCCCACGGGATGGGGAAAACCACCACCACGCAACTGCTGGT
GGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATGACTTACT
GGCGGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACAACA
CCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACA
AGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGTTCTGG
CTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCT
CACCATCTTCCTCGACCGCCATCCCATCGCCTTCATGCTGTGCTACCCGGCCG
CGCGGTACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGC
CCTCATCCCGCCGACCTTGCCCGGCACCAACATCGTGCTTGGGGCCCTTCCGG
AGGACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCT
GGACCTGGCTATGCTGGCTGCGATTCGCCGCGTTTACGGGCTACTTGCCAATA
CGGTGCGGTATCTGCAGTGCGGCGGGTCGTGGCGGGAGGACTGGGGACAGCT
TTCGGGGACGGCCGTGCCGCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGC
CCACGACCCCATATCGGGGACACGTTATTTACCCTGTTTCGGGCCCCCGAGTT
GCTGGCCCCCAACGGCGACCTGTATAACGTGTTTGCCTGGGCCTTGGACGTCT
TGGCCAAACGCCTCCGTTCCATGCACGTCTTTATCCTGGATTACGACCAATCG
CCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGGGATGGTCCAGA
CCCACGTCACCACCCCCGGCTCCATACCGACGATATGCGACCTGGCGCGCAC
GTTTGCCCGGGAGATGGGGGAGGCTAACTAA 3’