Chapter 6
Genetic Manipulation Via Gene Transfer
Paul C. Edwards1 and Michael J. Passineau2
1
Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry, Indianapolis, IN, USA
2
Division of Cardiovascular Medicine, Allegheny Health Network, Pittsburgh, PA, USA
Chapter Contents
6.1 Introduction to Basic Biology of Gene
Transfer 75
6.2 Gene Transfer Methodologies 76
6.2.1 Specific Gene Delivery Approaches 77
6.2.1.1 Viral Vectors 77
6.2.1.2 Nanoparticles 77
6.2.1.3 Naked DNA Entry Through Membrane
Disruption 77
6.2.2 Gene Delivery Considerations 78
6.2.2.1 Endosomal Escape Mediated by Viral
Vectors and Nanoparticles 78
6.2.2.2 Cytoplasmic Trafficking of Genetic
Payload 78
6.2.2.3 Nuclear Import and Fate of Genetic
Payload 78
6.2.2.4 Matching Vector and Target 79
6.1  INTRODUCTION TO BASIC BIOLOGY
OF GENE TRANSFER
Gene transfer is a potentially powerful approach to stem cell
biology and tissue engineering since it is the one method
that allows manipulation of cells and tissues in their own
language. By employing the subcellular machinery under-
lying the central dogma of molecular biology, gene transfer
engages and alters the software of cellular behavior. While
drug or chemical-based therapeutics can certainly modify
cellular behavior, no method matches the potential of gene
transfer for precise manipulation of cellular programming.
It should be appreciated that manipulation of the RNAi pro-
cess shares with gene transfer its mechanistic proximity to
the translational machinery of the cell, but is not considered
gene transfer per se, as RNAi strategies can only affect gene
expression by an inhibitory pathway and cannot introduce a
therapeutic transgene to a cell or tissue.
At its most basic level, gene transfer involves the trans-
portation of nucleic acid (single or double-stranded) across
Stem Cell Biology and Tissue Engineering in Dental Sciences. http://dx.doi.org/10.1016/B978-0-12-397157-9.00008-4
© 2015 Elsevier Inc. All rights reserved.
6.3 Host Response to Gene Transfer 79
6.3.1 Classical Anti-Viral Response to
Viral-Mediated Gene Transfer 79
6.3.2 Intracellular Response to Gene Transfer 80
6.3.3 Implications for Human Gene Therapy 80
6.4 Examples of Successful Genetic Manipulation
Via In Vivo Gene Transfer 80
6.4.1 Pre-Clinical Ex Vivo and In Vivo Gene Transfer
Strategies 80
6.4.2 Future Potential Approaches to Gene Therapy
Currently at Earlier Stages of Development 81
6.4.3 Limitations of Pre-Clinical Studies 82
6.5 Pathway to Clinical Implementation of
Therapeutic Gene Transfer 82
6.6 Conclusion 83
References 83
the cell and nuclear membranes. The gene drug, or “vector,”
serves as the template for synthesis of mRNA molecules
encoding the transgene sequence, and thus the transgene-
encoding portion of the vector must survive the process and
somehow arrive within the host cell nucleus intact. This
transmembrane/­ transcytoplasmic voyage crosses multiple
barriers and engages multiple subcellular mechanisms, and
this diversity of challenges explains in part why gene therapy
has been so slow to demonstrate clinical fulfillment of its
enormous potential.
Transit of a gene transfer vector across the cell membrane
has historically been regarded as the most challenging leg of
the journey. The phospholipid bilayer of a cell is impervious
to charged molecules, and nucleic acid is strongly anionic.
Further, the highly regulated transmembrane transport em-
ployed by cells occurs by means of channels, pumps, and
other membrane complexes that regulate pores much too small
for polynucleic acid molecules to transit. Thus, an intact cell
membrane presents an absolute barrier to gene transfer. If a
cell membrane is impervious to the transit of polynucleic acid
75
76  PART | II  In Vitro Regulation of Cell Behaviour and Tissue Development
FIGURE 6.1  Artist’s representation of the steps of viral-mediated gene transfer to a target cell.
vectors, gene transfer must rely upon one of three mechanisms:
(1) receptor-mediated endocytosis (and to a much lesser ex-
tent pinocytosis); (2) disruption of the cell membrane; or (3)
complexation of the polynucleic acid vector with an amphipa-
thic molecule that can mask its charge and allow it to diffuse
through the cell membrane. Gene transfer strategies have been
devised utilizing each of these membrane transit mechanisms,
and the advantages and challenges of each respective approach
will be discussed in more detail in the following section.
Once the transgene-coding region of vector enters the
cytoplasm, it engages the host cell’s microtubule network
to move toward the nucleus. This process has been studied
and appreciated, but is generally given far less attention than
transit of the cell membrane. This is at least partially due
to the observation that inclusion of appropriate non-coding
e­ lements (e.g., enhancers) in the vector engages native cellular
proteins that facilitate nuclear transport along microtubules.
Similarly, nuclear entry has received more modest atten-
tion, particularly when cellular targets are mitotic (e.g., stem
cells), as nuclear breakdown during mitosis greatly facilitates
nuclear entry. Assuming that most of the cell and tissue tar-
gets relevant to dental sciences are mitotic, this chapter will
focus mainly upon strategies to transport a gene transfer vec-
tor from the exterior of the cell to the cytoplasm. Figure 6.1
summarizes the major steps in exogenous gene transfer.
6.2  GENE TRANSFER METHODOLOGIES
As discussed above, gene transfer methodologies may
be classified according to the mechanism they employ to

FIGURE 6.2  Artist’s rendition of viral-mediated gene transfer (left), ultrasound-assisted gene transfer (center), and nanoparticle-mediated gene transfer
(right).
transit the cell membrane. Viral vectors utilize receptor-
mediated cell uptake and subsequent endosomal escape.
Naked DNA methodologies, primarily electroporation
and sonoporation, rely upon physical disruption of the cell
membrane, sometimes in combination with enhancement
of endocytosis. So-called “nanoparticle” techniques em-
body a continuum between the two, with such approaches
as simple transfection on one end and synthetic viruses on
the other. Figure 6.2 provides representative illustrations of
these various methodologies.
In dental and craniofacial applications of gene therapy, it
is important to note that ease of access to target tissues defines
the paradigm according to which gene delivery strategies are
devised. Many target tissues are mucosal, and deeper struc-
tures can usually be accessed easily and precisely by injection.
This facile accessibility is not the norm in other applications of
gene therapy, such as solid thoracic or visceral organs, where
vascular transport of the vector must be utilized. As a result,
some gene therapy innovations are relevant to dental and cra-
niofacial applications, and others (e.g., strategies to promote
extravasation) are not. Overall, the target tissues contemplated
in this chapter bear greatest similarity to those target tissues
where the greatest progress in clinical gene therapy has been
demonstrated (e.g., eye, muscle, skin), and these practical
considerations underscore the potential power of gene transfer
for dental and craniofacial therapeutics.
6.2.1  Specific Gene Delivery Approaches
6.2.1.1  Viral Vectors
Viral vectors have evolved specific receptor affinities that
govern their tropism for host tissues. These affinities mimic
Genetic Manipulation Via Gene Transfer Chapter | 6   77
classical ligand(virus)/receptor(host cell) interactions, with
the ligand composed of a protein moiety embedded in the to-
pology of the viral capsid (e.g., adenovirus, ­adeno-associated
virus (AAV)) or comprising a domain of a glycoprotein
embedded in the viral membrane (e.g., Herpes simplex or
lentivirus). Receptor binding stimulates endocytosis of the
receptor/virus complex by the host cell through the forma-
tion of an endosome budding from the cell membrane.
6.2.1.2 Nanoparticles
Nanoparticles are a broadly applied term for any complex
of naked DNA with an agent(s) acting as a carrier to en-
able the genetic payload to transit the cell membrane. It is
important to note that while classical lipofection methods
work efficiently in vitro, the techniques are extremely lim-
ited in their in vivo efficacy. For this reason, most nanopar-
ticle strategies involve a cellular entry process identical to
viral vectors, wherein there is a receptor/ligand interaction
resulting in endocytosis and endosomal encapsulation. The
DNA payload is compacted within a polymeric chemical
structure that often features multiple components. The va-
riety of nanoparticle formulations in the gene therapy lit-
erature is myriad, but polyethylene glycol (PEG) is very
commonly used as a component of nanoparticles due to its
apparent ability to extend the stability, and thus circulation
time, of nanoparticles in plasma.
6.2.1.3  Naked DNA Entry Through
Membrane Disruption
In contrast to viral vectors and most nanoparticles, naked
DNA methodologies rely on physical, rather than biological,
78  PART | II  In Vitro Regulation of Cell Behaviour and Tissue Development
entry to the cell. Pores are created in the cell membrane ei-
ther through the application of a brief electrical pulse (“elec-
troporation”) or the violent destruction of microbubbles
in an applied acoustic field, resulting in inertial cavitation
(“sonoporation”). Under optimal conditions, these pores are
large enough to permit the diffusion of naked DNA across
the cell membrane, while still small and transient enough to
avoid cellular toxicity from the reversal of transmembrane
ionic gradients and uncontrolled influx of water.
Electroporation and sonoporation are rarely used for
in vitro gene transfer due to the increased fragility of cells
in culture and the availability of reliable chemical transfec-
tion techniques (e.g., lipofection). For in vivo gene transfer,
both techniques have been widely studied in animal models,
with a number of clinical trials now underway to evaluate
the safety and efficacy of gene electrotransfer in superfi-
cial tissues such as prostate and skin [1]. Sonoporation has
not yet advanced to human clinical trials, although micro-
bubbles have found wide application in clinical practice as
ultrasound contrast agents and have been determined to be
safe, even at relatively large concentrations.
While electroporation and sonoporation are mechanisti-
cally similar, the instrumentation required to execute them
are fundamentally different, and these differences have im-
portant implications for application in dental and craniofa-
cial tissues. Electroporation requires current to arc between
two electrodes, and thus the tissue target must be positioned
between the electrodes. In superficial structures, this is usu-
ally accomplished by needle-like electrodes inserted into
the target tissue. In contrast, the sonoporation relies upon an
externally applied ultrasound pulse that propagates through
soft tissue, requiring only that an uninterrupted pathway
through soft tissue exists between body surface and the
target tissue. For this reason, sonoporation is considered to
be somewhat amenable to organ targeting through the en-
gineering of an emitter that creates an ultrasound “beam.”
Supporting this concept is the feasibility of formulating
antigen-targeted microbubbles intended to accumulate at a
disease target, such as a tumor.
6.2.2  Gene Delivery Considerations
6.2.2.1  Endosomal Escape Mediated by
Viral Vectors and Nanoparticles
If cellular entry occurs via receptor-mediated uptake, the
mechanism utilized by viral vectors and nanoparticles, the
membrane budding that occurs results in the formation of an
endosomal compartment containing the vector. Once encased
within an endosome, a gene transfer vector must effect escape
before the payload is degraded by nucleases present within
the endosome. Several theories have been advanced as to the
mechanism by which endosome escape is accomplished, via
naturally occurring viruses or by chemical agents incorpo-
rated into nanoparticles. The ­process remains incompletely
understood, but it is clear that the mechanism of endosomal
escape is vector-specific and is accomplished by a wide va-
riety of viral capsid protein interactions. In at least one case,
that of AAV, endosomal escape may not even occur until the
virus reaches the nucleus [2]. In the current state of the art,
protein-mediated endosomal disruption is not sufficiently un-
derstood to impart these properties to nanoparticles, and thus
endosomal escape in nanoparticle strategies is mediated by
chemical structures that buffer the low pH environment of
the endosome and lead to net water influx, leading to escape
through bursting of the endosome [3].
6.2.2.2  Cytoplasmic Trafficking
of Genetic Payload
Cytoplasmic trafficking occurs via vector-specific pathways,
but there is commonality in that the microtubule network
is the physical substrate. For example, adenoviral infec-
tion utilizes a predictable and rapid pathway involving viral
uncoating, endosomal escape, and m ­ icrotubule-associated
transport involving dynein [4,5]. AAV, in contrast, ­appears to
enter cells and traffic intracellularly via multiple ­pathways,
and viral uncoating is delayed until nuclear entry [6].
Nonviral vectors follow a pathways similar to a­ denovirus,
but are far less efficient at both endosomal escape and
­nuclear translocation [7,8].
6.2.2.3  Nuclear Import and Fate
of Genetic Payload
Nuclear import of exogenous DNA occurs efficiently in di-
viding cells since the nuclear envelope disassembles dur-
ing the M phase of cell division [9]. Mitotically active cells
comprise many, but not all, of the tissue targets relevant to
this volume. For non-dividing cells, nuclear entry is more
complex, as the DNA molecules utilized in gene therapy
applications are too large to pass the nuclear pore com-
plex and must enter the nucleus through facilitated trans-
port. Characterization of so-called nuclear shuttle proteins,
which bind exogenous DNA and subsequently interact with
nuclear transport mechanisms, has been an active area of
research [10,11]. Fortunately, the practical upshot of this
complex topic is that viral vectors containing commonly
utilized enhancer sequences, such as that from SV40, are
sufficient to drive nuclear import and robust expression in
non-dividing cells.
Depending on the vector system used, genetic payloads
assume one of two distinctly different nuclear fates: epi-
somal persistence or chromosomal integration. Episomal
persistence is a key determinant of the duration of trans-
gene expression, but is poorly understood, and may in-
volve subcompartments of the nucleus, at least in the case
of AAV [6]. Vectors such as lentiviruses, HSV, and others
rely upon the natural physiology of the parent virus to in-
tegrate the genetic payload into host cell chromosomes.

This ­integration has the appearance of being random, or at
least unpredictable, and presents a risk of “genotoxicity,” a
phenomenon in which the integration of the genetic payload
occurs proximal to an oncogene. Genotoxicity has been ex-
tensively observed and studied in preclinical models, and
has been conclusively linked to oncogenesis in human pa-
tients treated with gene therapy [12–14]. Interestingly, the
genotoxic risk associated with integration of a therapeu-
tic expression cassette is not matched by the expected in-
crease in duration of expression, as integrated transgenes
are silenced within approximately the same timeframe as
transgenes persisting as episomes. In the aggregate, these
safety concerns explain the relative preference for the non-
integrating vectors adenovirus and AAV in dental and cra-
niofacial applications of gene transfer.
6.2.2.4  Matching Vector and Target
It is now understood that ex vivo gene transfer to stem cells
and in vivo or in situ gene transfer to tissues or organs are
fundamentally distinct processes and require different, often
non-overlapping, methodologies. The epithelial origin of
most therapeutically relevant dental and craniofacial struc-
tures makes them natural targets of many viral vectors, and
indeed most prior literature reporting in vivo gene transfer
to this region has relied upon viral vector technologies. In
particular, adenovirus and adeno-associated virus account
for the majority of preclinical gene transfer studies in dental
science, and recently have advanced to clinical trials [15].
In contrast, the vectors of choice for gene transfer to
stem cells are usually retroviruses, specifically lentivirus
(although newer non-viral methods such as transposons and
zinc finger nucleases hold promise for permanent modifi-
cation of stem cells). One of the most important distinc-
tions between in vivo gene transfer to tissues and in vitro
gene transfer to stem cells explains much of the rationale
for lentiviruses. The therapeutic promise of stem cells often
assumes their expansion once delivered to the recipient’s
body. Cell division dilutes the effect of gene transfer with
non-integrating viruses like Ad and AAV, because the ge-
netic payload delivered by these viruses persists episomally
and is usually not replicated during mitosis. As described
above, the defining feature of retroviruses is their ability to
integrate a genetic payload into the host cell chromosomes,
usually resulting in the transgene being propagated across
subsequent generations. Thus, for therapeutic strategies
predicated upon in situ expansion of stem cells, retroviruses
offer this critical feature.
Finally, the issue of vector affinity for a target tissue rela-
tive to non-target tissues must often be considered. For ex-
ample, in a complex structure such as the root of a tooth,
many cell types with distinctly different characteristics are
present. Microsurgical injection of a gene transfer vector
to the root of a tooth will inevitably expose the vector to a
­variety of cell types, only one of which is likely to be the
Genetic Manipulation Via Gene Transfer Chapter | 6   79
target. One can imagine that expression of a therapeutic
transgene in a periodontal ligament fibroblast might have
a helpful effect, but expression of the same transgene in a
dental pulp cell could create undesired effects. While cel-
lular specificity may be needed in some applications, this is
an area of research that has not advanced beyond proof-of-
principle. In this respect, tropism modified viral vectors such
as alternate AAV serotypes or chimeric adenoviruses have
shown the most promise for cell-type-targeted gene delivery.
6.3  HOST RESPONSE TO GENE TRANSFER
Delivery of foreign DNA to a mammalian cell does not oc-
cur naturally and the few scenarios in which this phenom-
enon occurs, such as viral infection, are harmful to the host
organism. Accordingly, mammals have evolved multiple
layers of defense against the exogenous delivery of foreign
genes. The specific host response activated depends on the
mode of delivery, with two distinct pathways predominat-
ing. In the case of viral-mediated gene transfer, an activation
of cell-mediated immunity can occur, through the classi-
cal anti-viral immune response initiated by MHC Class II
presentation of viral antigens. In the case of non-viral gene
transfer, and in addition to the anti-viral cellular response in
the case of viral-mediated gene transfer, a more complex and
less understood intracellular immune response can occur.
6.3.1  Classical Anti-Viral Response
to Viral-Mediated Gene Transfer
Gene therapy appropriates a portion of the natural infection
cycle of viruses and aborts the cycle at the replication stage.
As a result, the cell binding, endocytosis, viral uncoat-
ing, and endosomal escape processes proceed in a manner
identical to a wild-type infection. Viral capsid fragments
are retained in the endosome, which in turn fuses with the
Golgi apparatus and allows these antigens to be presented
on MHC Class II receptors. Anti-viral immune response is
thus generated in a manner identical to wild-type infection,
although both humoral and cell-mediated responses to gene
therapy are usually more modest (depending on the vector
dose) due to the absence of the uncontrolled and sustained
viremia that normally accompanies wild-type infection.
The human immune system has a remarkable capacity
for immunological memory, with the result that repeat infec-
tions with the same virus are met with an increasingly reso-
lute and rapid response. As an example, pre-existing human
immunity to AAV [16] has been shown to confound human
gene therapy clinical trials [17]. While repeated administra-
tion of viral gene therapy vectors has not been conclusively
studied in human populations, it is assumed that repeated
administration of the same viral vector within the time in-
terval needed to sustain therapeutic transgene ­expression
(at least every few years) will be clinically infeasible. It is
80  PART | II  In Vitro Regulation of Cell Behaviour and Tissue Development
not yet clear to what extent transient immunosuppression
or viral sero-/pseudo-typing will circumvent this imposing
hurdle to lifelong gene therapy treatments.
6.3.2  Intracellular Response
to Gene Transfer
The intracellular response to foreign genetic material is a
very ancient, genetically encoded innate signaling system.
The relevance and role of this multi-component immune
cascade to gene therapy is not entirely clear, due both to its
complexity and the practical difficulty of separately evalu-
ating anti-viral intracellular responses from those that are
directed purely against foreign genetic material. With the
increasing applicability of non-viral gene transfer technolo-
gies such as electroporation or sonoporation, this area is
now somewhat simpler to study and is gaining in its rel-
evance to clinical translation.
A number of recent reviews [18–21] have summarized
current thinking about the multiple innate immune signal-
ing pathways that likely mediate the intracellular response to
gene transfer. Recently, a landmark study has used microarray
technology to probe the transcriptional changes that occur in
muscle as a result of gene electrotransfer, using electropora-
tion without gene transfer as a control [22]. The relatively
clean experimental design of this study allowed the authors to
parse out the intracellular response to a naked DNA plasmid
in isolation from the gene transfer methodology and with-
out the confounding effects of viral proteins. It remains to
be determined whether intracellular immune responses can
be minimized or eliminated by purging all non-human se-
quences from the vector backbone and expression cassette.
6.3.3  Implications for Human
Gene Therapy
As gene transfer vectors have steadily improved over the
past two decades, human gene therapy has become a clini-
cal reality [23]. As the gene therapy field has progressed
from proof-of-principle into the serious consideration of
clinical practicality, host response has emerged as probably
the most important obstacle to wider impact. Notably, the
most successful and visible gene therapy clinical trials to
date have involved intervention in immunologically privi-
leged tissues, such as the retina [24], brain [25], and the
immune system itself [14]. Clinical trials involving gene
transfer to immunologically active organs, such as the liver
[17], have demonstrated the challenge that the host immune
response continues to pose to this promising field.
This pivot to consideration of clinical practicality is
cause for celebration as it brings gene therapy into a new
chapter wherein research can focus on challenges, most
­notably host response, that heretofore were postponed pend-
ing demonstration of clinical efficacy. At the forefront of
these challenges is how to match therapeutically ­meaningful
duration of transgene expression to the progress that has
been made in magnitude of gene expression. In many, if
not most, applications of gene therapy, transgene expression
spanning years or even decades is needed, and this require-
ment exceeds the capability of any available clinical grade
vector. Inevitably, many gene therapy applications will re-
quire episodic re-administration of gene transfer.
Considerations of host response to gene transfer are am-
plified when considering a paradigm that involves periodic
re-administration. In particular, repeated priming and am-
plification of cell-mediated immunity presents a potentially
dire complication, as the end result is not simply mitigation
of therapeutic effects, but destruction of the target tissue it-
self. As clinical application of gene therapy moves forward,
strategies aimed at suppression or evasion of cell-mediated
host immune response will have to be considered. Less clear
is the impact of repeated administration on intracellular pro-
gramming and the native structure and function of the target
tissue. As discussed above, the intracellular response to gene
transfer is only beginning to be investigated and it is rea-
sonable to hope that vector improvements made in consider-
ation of intracellular response might minimize these effects.
Overall, serial re-administration of gene transfer represents
the next chapter in gene therapy technological development.
6.4  EXAMPLES OF SUCCESSFUL GENETIC
MANIPULATION VIA IN VIVO GENE
TRANSFER
While the early focus on potential applications for gene trans-
fer among researchers and clinicians with a primary interest
in the dental field and head and neck area has largely focused
on promoting repair of dentoalveolar bone defects (e.g., sec-
ondary to trauma, tumor resection, or congenital deformities)
[26] and periodontal regeneration [27,28] as detailed in depth
in later chapters in this book, the potential applicability of
gene transfer is much broader than these two areas alone.
6.4.1 Pre-Clinical Ex Vivo and In Vivo
Gene Transfer Strategies
Animal and cell culture-based studies have clearly dem-
onstrated the feasibility of gene transfer, both ex vivo and
in vivo. These studies have encompassed a wide range of
potential applications, from increasing the success rate
of osseointegration by stimulating the local environment
into which the implants are placed (e.g., bone marrow-
derived mesenchymal stem cells induced ex vivo with
hypoxia-inducible factor-1a-expressing lentiviral vector
[29] to promote bone growth around the implant surgical
site), to articular cartilage repair (e.g., recombinant adeno-­
associated virus delivery of SOX9 to a rabbit knee joint os-
teochondral defect model to enhance the repair of cartilage

lesions [30]), wound ­repair (e.g., delivery of bone marrow-
derived mesenchymal stem cells transfected ex vivo with
both human vascular endothelial growth factor 165 and
human beta-defensin 3 to enhance wound repair in a rat
combined radiation-wound injury model [31]), pulp cap-
ping/dentin regeneration (e.g., primary cultured human
dental pulp cells infected with recombinant human bone
morphogenetic protein-7-­expressing adenoviral vector with
the goal of promoting the differentiation of human pulp
cells into odontoblast-like cells in order to promote dentin
mineralization [32]), the prevention and management of
­radiation-induced salivary hypofunction (e.g., administration
of an adenoviral vector overexpressing human keratinocyte
growth factor to murine submandibular glands by retrograde
ductal instillation in order to reduce radiation damage to the
salivary glands [33]) and the management of immune-
mediated conditions (e.g., administration of adeno-associated
viral vectors expressing interleukin-27 to suppress disease
severity in a murine model of Sjøgren’s syndrome [34]).
6.4.2  Future Potential Approaches to
Gene Therapy Currently at Earlier
Stages of Development
Numerous other prospective applications for in vivo gene
transfer of interest to the clinician who focuses on pathologic
processes involving the oral and maxillofacial region are at
earlier stages of development. While by no means intended
as an all-inclusive list, areas of active investigation include
the treatment of less common oral and maxillofacial neo-
plasms, genetically modifying the plasticity of oral cavity-
derived cells for regeneration of extra-oral tissues, interfering
with unfavorable aspects of tissue repair (such as scar forma-
tion) with the goal of promoting regeneration, reducing caries
activity both by targeting pathogenic bacteria and behaviors
associated with increased tooth decay, and treatment of vari-
ous genodermatoses. These are discussed in sequence.
l As the molecular mechanisms underlying the less com-
mon primary neoplasms involving the oral cavity (e.g.,
malignant tumors of minor salivary glands such as ad-
enoid cystic carcinoma) are better characterized, gene
therapy interventions to improve treatment of these neo-
plasms are likely.
l Cells from the oral cavity, especially human periodontal
ligament-derived cells, appear to be excellent candidates
for ex vivo gene modification, with the goal of enhanc-
ing expression of specific growth factors to stimulate the
regeneration of non-oral tissues along specific lineages
(e.g., promoting neuroregenerative and/or neurotrophic
differentiation in the adult brain [35]).
l Gene therapy also offers promise for neural repair of
traumatic injuries to the sensory nerves (e.g., iatrogenic
paresthesia following third molar surgery or mandibular
Genetic Manipulation Via Gene Transfer Chapter | 6   81
cystectomy), by shifting the balance from scar forma-
tion towards axonal regeneration [36].
l Development of dental caries encompasses both be-
havioral and microbial components. Gene therapy ap-
proaches could be employed to increase the production
of anti-cariogenic salivary constituents, potentially
reducing caries activity in high-risk individuals. Jiang
et al. [37] demonstrated reduced levels of streptococ-
cus mutans in rats following the retrograde transfer of
the gene encoding the chemokine CCL28 to rat parotid
acinar cells. On the behavioral side, methamphetamine
abuse, in addition to its devastating social consequences,
is associated with extensive loss of tooth structure [38]
and an increased propensity to engage in high-risk be-
haviors that predispose to contracting HIV infection and
other sexually transmitted diseases [39]. Chen and Chen
[40] propose using recombinant AAV-mediated gene
transfer to achieve expression of anti-methamphetamine
antibodies, the goal being to reduce central nervous sys-
tem bioavailability of methamphetamine.
l The genodermatoses, many of which have both oral cav-
ity and extraoral head and neck manifestations, comprise
a heterogeneous group of skin disorders, many resulting
from single gene mutations. Hence these represent po-
tentially ideal candidates for gene replacement therapy.
Of the more common genodermatoses affecting the head
and neck area, a number are associated with a higher in-
cidence of developing benign or malignant neoplasms.
These include Gardner syndrome, Peutz-Jeghers syn-
drome, xeroderma pigmentosum, and nevoid basal cell
carcinoma syndrome (NBCCS). The latter results from
mutations in the patched-1 gene, a down-regulator of
the sonic hedgehog (Shh)-gli1 pathway, resulting in a
very high incidence of developmental cysts of the jaws
(specifically odontogenic keratocysts, also referred to
as keratocystic odontogenic tumors). NBCCS is also
associated with a predisposition to developing central
nervous system tumors (e.g., medulloblastoma) and
the development of hundreds to thousands of basal cell
carcinomas of the skin. The use of small molecular in-
hibitors of the sonic hedgehog pathway has received at-
tention as a possible therapy for both late stage basal
cell carcinomas and odontogenic keratocysts of the jaws
[41]. The potential for reconstitution of patched-1 ac-
tivity through gene replacement therapy may offer an
additional therapeutic approach for patients with both
NBCCS and late stage basal cell carcinomas. There are
a number of other genodermatoses, such as the different
variants of inherited epidermolysis bullosa, which are
characterized by a varying propensity to form extensive
skin blisters, and consequently may be associated with
significant morbidity. The potential for gene therapy ap-
proaches in the management of these conditions is dis-
cussed later in this chapter.
82  PART | II  In Vitro Regulation of Cell Behaviour and Tissue Development
6.4.3  Limitations of Pre-Clinical Studies
As noted above, although there is a growing body of litera-
ture investigating the potential benefits of gene transfer in
these specific areas, to date these studies have largely been
based on cell culture and/or animal models. There are clear,
and widely accepted, limitations to the translation of these
findings to human in vivo studies. This has been further ham-
pered by recent evidence that suggests that a significant mi-
nority of cell lines used in previously published studies were
not representative of the tissue from which they are claimed
to have been derived, as a result of both cross-­contamination
between cell lines and misidentification [42]. A list of the
several hundred cross-contaminated or misidentified cell
lines [43] identified to date includes several purported head
and neck-derived cell lines (e.g., adenoid cystic carcinoma,
oral squamous cell carcinoma, and papillary thyroid carci-
noma cell lines). A current impetus by both funding agen-
cies and journals to require verification of the actual cell
lines used in studies submitted for publication will obviate
this concern moving forward, but some degree of prudence
in interpreting previously published studies would seem
warranted. These concerns notwithstanding, the use of cell
line and animal studies remain a prerequisite to the develop-
ment of future in vivo human studies.
6.5  PATHWAY TO CLINICAL
IMPLEMENTATION OF THERAPEUTIC
GENE TRANSFER
Realistically, due to the large number of safety and regula-
tory issues involved in the use of in vivo, and perhaps to a
lesser extent ex vivo, gene transfer this approach is more
likely to find a role in the treatment of single gene defects
and multifactorial conditions associated with the greatest
potential for morbidity and mortality, i.e., malignant neo-
plasms such as oral squamous cell carcinoma, melanoma,
lymphoma, and adenoid cystic carcinoma. The treatment of
inherited conditions resulting from single gene defects in-
volves replacement of the single missing or defective gene
by means of gene therapy, whereas interventions for the lat-
ter conditions can range from approaches that interfere with
cell-cycle regulatory proteins involved in cancer cell pro-
liferation, to inhibition of angiogenesis, to enhancing anti-
tumor cell immune activation by delivering genes coding
for co-stimulatory proteins.
This is underscored by the available data on human gene
therapy. Of the 1800-plus ongoing, completed or approved
worldwide clinical trials involving gene therapy, 65% in-
volve therapy for cancer [44]. Of these, the majority are at
stage 1 phase (60%; evaluation of safety, determination of
a safe dosage range, and identification of side effects; typi-
cally involving 15-30 subjects), and stage 2 (35%; larger
study to determine potential effectiveness and to further
evaluate safety; typically involving fewer than 100 subjects)
[44,45]. Until recently, successful stage 3 trials (larger size
trials to confirm effectiveness, monitor for side effects,
and to compare commonly used alternative treatments
before being made widely available to the general popu-
lation) have been weighted towards the treatment of rare
single gene Mendelian-inherited diseases. In China, there
currently are two-stage four-gene therapy studies approved
for patients with advanced oral and maxillofacial squa-
mous cell carcinoma and advanced thyroid malignancies
involving adenoviral delivery of p53 protein (Gendicine,
Shenzhen SiBiono GeneTech Co., Ltd., China). For a com-
plete list of approved, ongoing or completed worldwide hu-
man gene therapy clinical trials, the reader is directed to
http://www.abedia.com/wiley/index.html, or for trials reg-
istered through the National Institutes of Health (NIH), the
NIH Genetic Modification Clinical Research Information
System (GeMCRIS®) at http://www.gemcris.od.nih.gov/
Contents/GC_CT_RPT_MAKER.asp.
Likewise, the preponderance of human clinical trials of
in vivo gene transfer specifically involving the oral and max-
illofacial complex constitute stage 1 and stage 2 trials for the
treatment of advanced oral and pharyngeal squamous cell
carcinoma and advanced thyroid carcinomas unresponsive to
conventional surgical excision and/or ablation with radioac-
tive iodine. For example, a phase 2 multicenter trial [46] dem-
onstrated the technical feasibility of perioperative delivery of
adenovirus-delivered p53 gene therapy for patients with head
and neck squamous cell carcinoma. P53, a pro-apoptosis
tumor suppressor gene, is mutated in up to 50% of patients
with head and neck squamous cell carcinoma. In this 12 site
multi-institutional study, spanning the period from March 1,
2003 to July 1, 2006, 13 individuals with newly diagnosed,
previously untreated surgically resectable squamous cell car-
cinoma of the oral cavity, oropharynx, larynx, and hypophar-
ynx with nodal disease but without distant metastases, were
given intraoperative and perioperative p53 gene therapy by
injection into both the primary surgical site and the neck dis-
section bed. This study highlighted some of the challenges
involved in patient recruitment for gene therapy studies (the
goal of accruing 30 patients per year was not met by a fac-
tor of 10) and the complex regulatory steps involved (of 12
institutions initially recruited to participate, only five cen-
ters were able to obtain both Institutional Review Board and
Institutional Biosafety Committee approval, and only three
of these ultimately enrolled patients into the protocol, with
one site taking 3 years from the time of initial regulatory
­application ­submission to receiving final approval). The au-
thors concluded that intraoperative p53 gene therapy, while
technically feasible, is “not logistically possibly when carried
out in a multi-institutional setting.”
Other approved phase 1 and 2 trials for treating ad-
vanced oral squamous cell carcinoma include gene therapy
with interferon-alpha cDNA, administration of combined

interferon-α/interleukin-12 cDNA, interleukin-2 cDNA,
interleukin-2-secreting semi-allogeneic human carcinoma
cell line transfected with autologous tumor DNA, HLA-B7/
beta 2-microglobulin (Allovectin-7) cDNA, tumor necrosis
factor cDNA, antisense epidermal growth factor receptor
DNA, granulocyte-macrophage colony stimulating fac-
tor cDNA, Herpes simplex virus thymidine kinase cDNA
administered with ganciclovir, recombinant Vesicular sto-
matitis virus expressing interferon beta, HPV 16 E7 cDNA
in HPV-16-associated head and neck cancer, attenuated
Vaccinia virus (GL-ONC1) with concurrent cisplatin and ra-
diotherapy, replication defective adenoviral vector express-
ing E. coli purine nucleoside phosphorylase in combination
with fludarabine monophosphate, and semi-allogeneic fi-
broblasts transfected with autologous tumor DNA. Phase 1
trials have also been approved for looking at the delivery of
sodium iodide symporter (NIS) using a modified measles
viral vector to treat advanced thyroid carcinomas.
While clearly in the majority, not all human clinical trials
involving in vivo gene transfer involve treatment of oral can-
cer. Other conditions of direct relevance to the head and neck
complex that have received regulatory approval include the
use of gene delivery for inherited connective tissue disorders,
release of endogenous pain killers for intractable pain, and
therapy for radiation induced xerostomia, as detailed below.
l Specifically, this includes phase 1 and 2 trials for patients
with the inherited blistering conditions recessive dystro-
phic epidermolysis bullosa and junctional epidermolysis
bullosa, conditions characterized by the development of
debilitating oral lesions that cause significant pain, scar-
ring, and, eventually, microstomia. These interventions
involve ex vivo therapy with autologous keratinocytes
transfected with retroviral vectors expressing the human
collagen 7A1 and laminin 5 beta 3 genes, respectively.
l A recently completed phase 1 trial utilizing intrader-
mal injection of a human preproenkephalin-expressing
­replication-defective modified herpes simplex viral vec-
tor in 10 patients with moderate to severe intractable pain,
despite treatment with more than 200 mg/day of morphine
[47,48] demonstrated the safety of this approach. This ap-
proach, if validated in phase 3 trials, could potentially be
of benefit for patients with facial pain syndromes such as
trigeminal neuralgia and atypical facial pain, or head and
neck cancer patients with intractable pain.
l To date, the most widely documented gene therapy
intervention in the head and neck complex involves a
phase 1 trial of an adenoviral vector encoding the hu-
man aquaporin-1 gene delivered to 11 patients with se-
vere xerostomia secondary to treatment for squamous
cell carcinomas involving the tongue base, tonsil, or
­hypopharynx [15]. In this proof-of-concept study, short-
term objective improvements in parotid gland salivary
flow rates between 60% and 540% over baseline were
Genetic Manipulation Via Gene Transfer Chapter | 6   83
noted in six patients, five of whom also reported subjec-
tive improvement in their degree of oral dryness.
l There have been scarce clinical trials involving gene
therapy in the field of orthopedics, focusing primarily
on therapies for rheumatoid arthritis, and osteoarthri-
tis, and one targeting aseptic prosthetic joint loosening
[49]. While there exists a substantial potential for this
approach in orthopedic tissue engineering and regenera-
tive medicine, there has been surprisingly little in the
way of human clinical studies in this area. This may be a
reflection of the complex regulatory process required for
study approval, the generally non-fatal nature of these
conditions, or the availability of mostly successful non-
gene based alternative therapies (e.g., osseointegrated
implants for tooth loss resulting from caries, periodontal
disease and trauma; bone morphogenetic protein; etc.).
6.6 CONCLUSION
As gene delivery approaches are further refined, acceler-
ating the transition from the laboratory setting to direct
clinical care, and as our knowledge of the underlying mech-
anisms involved in disease development continues to grow,
the potential for gene therapy to offer alternative and/or
complementary approaches to the treatment and manage-
ment of disease processes involving the craniofacial struc-
tures will continue to expand.
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