Review
Cells Tissues Organs 2006;183:169–179
DOI: 10.1159/000096508
Accepted after revision: August 22, 2006
Perspectives of Gene Therapy in
Stem Cell Tissue Engineering
Ulrich Reinhart Goessler a Katrin Riedel b Karl Hörmann a Frank Riedel a
a
Department of Otolaryngology, Head and Neck Surgery, Ruprecht-Karls University Heidelberg, Faculty of
Clinical Medicine, Mannheim, and b Department of Hand, Plastic and Reconstructive Surgery, Burn Center,
BG-Trauma Center, Plastic and Hand Surgery of the University of Heidelberg, Ludwigshafen, Germany
Key Words
Gene therapy Vectors Mesenchymal stem cells Cartilage Chondrocytes Tissue engineering Extracellular matrix Plastic and reconstructive surgery
Abstract
Tissue engineering is an interdisciplinary field that applies
the principles of engineering and life sciences toward the
development of biological substitutes that restore, maintain
or improve tissue function. It is hoped that forming tissue de
novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair.
Gene therapy is the science of the transfer of genetic material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Recently,
tissue engineering has been used in conjunction with gene
therapy as a hybrid approach. This combination of stem-cellbased tissue engineering with gene therapy has the potential to provide regenerative tissue cells within an environment of optimal regulatory protein expression and would
have many benefits in various areas such as the transplantation of skin, cartilage or bone. The aim of this review is to
outline tissue engineering and possible applications of gene
therapy in the field of biomedical engineering as well as basic principles of gene therapy, vectors and gene delivery.
Copyright © 2006 S. Karger AG, Basel
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Introduction
Historically, surgeons have sought and used different
procedures to augment the repair of various skeletal tissues. Recently, researchers have brought together the
knowledge of molecular and cellular biology, biochemistry, chemical engineering, genomics and material science
to provide clinicians with new modalities to aid this repair: tissue engineering and gene therapy.
Tissue engineering is an interdisciplinary field that
applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function [Langer and
Abbreviations used in this paper
bFGF
BMP
DNA
EGF
ESCs
IGF
IL
MSCs
NGF
PDGF
RNA
TGF
basic fibroblast growth factor
bone morphogenetic protein
deoxyribonucleic acid
epidermal growth factor
embryonic stem cells
insulin-like growth factor
interleukin
mesenchymal stem cells
nerve growth factor
platelet-derived growth factor
ribonucleic acid
transforming growth factor
Dr. Ulrich R. Gössler
Universitäts-HNO-Klinik Mannheim
Theodor-Kutzer-Ufer
DE–68135 Mannheim (Germany)
Tel. +49 621 383 1600, Fax +49 621 383 1972, E-Mail [email protected]
Vacanti, 1993]. It has become an area of intense research
in plastic surgery and other fields of medicine as the interplay between cells, growth factors and supporting matrices is investigated. It is hoped that forming tissue de
novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair, or tissue reconstruction that requires the
use of tissue from an alternative site with inevitable donor
site morbidity [Goessler et al., 2004].
Definition and Methodology of Tissue Engineering
in Plastic and Reconstructive Surgery
The most common need for cartilage and bone in the
head and neck is for the reconstruction of the nose and
the ears or for bone of the craniofacial region to correct
congenital deformities or to replace tissue after traumas
or tumor resections. Tissue engineering is a technology
based on developing biological substitutes for the repair,
reconstruction, regeneration or replacement of tissues. Its
long-term goal is to construct biomaterials that are biocompatible, biodegradable and capable of integrating
molecules (e.g. growth factors) or cells [Christenson et
al., 1997; Solchaga et al., 1999]. Currently, many different
ceramics, polymers of lactic and glycolic acid, collagen
gels, and other polymers have been tested in vitro and in
vivo [Christenson et al., 1997]. More recently, genetic
modifications have been included in tissue engineering
to optimize the healing process [Minsk, 1998]. Modified
cells are transplanted into injured tissue to effect the repair with the introduced gene.
Bone and cartilage are the tissues in which most tissue engineering techniques have been applied. Bone has
a high potential for repair. In large defects or when vascularization is impaired, however, augmentation with
scaffolds, genetically engineered cells, and/or growth
factors and cytokines can accelerate or enhance the
healing [Sakou, 1998]. Recently, autologous muscle tissue has been used as a delivery vehicle for growth factor
genes in the treatment of bone defects [Day et al.,
1999].
In contrast, cartilage has a poor intrinsic capacity for
healing and therefore a limited ability to regenerate [Solchaga et al., 1999]. Intense investigations have focused on
finding biomaterials that would be capable of repairing
cartilage defects, but no efficient therapeutic approaches
have yet been established [O’Driscoll, 1998, 1999], candidate materials include fibrin, collagen, ceramics, algi170
Cells Tissues Organs 2006;183:169–179
nate, polymers of lactic and glycolic acid, hyaluronic acid,
and synthetic materials.
Recently, the biology of embryonic and adult stem
cells has been intensively studied as these cells offer the
capacity for self-renewal and differentiation and carry
great potential for usage in tissue engineering. At this
stage, the use of adult stem cells and particularly of autologous cells would be the most readily acceptable form
of regenerating cell use in plastic surgery, as it avoids the
ethical problems associated with embryonic cell use
[Goessler et al., 2005].
Stem Cells in Tissue Engineering
A stem cell is defined by 3 main criteria: self-renewal,
ability to differentiate into multiple cell types and capability of in vivo reconstitution of a given tissue [Caplan,
1991]. The most primitive stem cell is the fertilized oocyte (the zygote). Once the fertilized egg starts dividing,
the descendants of the first 2 divisions are the totipotent
cells which are able to form the embryo and trophoblast.
After about 4 days, these totipotent cells form a hollow
ball of cells, the blastocyst, containing a cluster of cells
called the inner cell mass from which the embryo develops and embryonic stem cells (ESCs) are derived.
Lower down in the hierarchical tree are the multipotent or adult stem cells. Most adult tissues have multipotent stem cells that can produce a limited range of differentiated cell lineages appropriate to their location. At the
bottom of the hierarchical tree are the unipotent stem
cells, or committed progenitors, that generate one specific cell type.
Current sources of stem cells for tissue engineering
include ESCs and adult stem cells. ESCs are isolated from
the inner cell mass of a preimplantation blastocyst [Martin, 1981]. These cells have recently been given attention
because they are totipotent (i.e. they can normally form
all cells/tissues of an organism) and therefore may have
broad applications as they are able to differentiate into all
cells that arise from the 3 germ layers but not the embryo.
A unique network of transcription factors ensures selfrenewal and simultaneously suppresses the differentiation of ESCs. These include Nanog, octa-binding factor
3/4(OCT4) and Wnt [Polak and Hench, 2005]. Human
ESCs are characterized by their expression of SSEA3,
SSEA4, TRA-1–60 and TRA-1–81 antigens that are now
known to be downregulated during differentiation while
several other antigens are induced [Bhattacharya et al.,
Goessler /Riedel /Hörmann /Riedel
2004; Brandenberger et al., 2004]. Undifferentiated mesenchymal stem cells (MSCs) do not express immunologically relevant cell surface markers and, as such, appear
to be immunoprivileged [Hwang et al., 2004].
Through the manipulation of culture conditions, ESCs
have been shown to generate hematopoietic precursors
[Wiles and Keller, 1991], adipocytes [Dani et al., 1997],
muscle cells [Rohwedel et al., 1994], chondrocytes [Kramer et al., 2000] and other tissues. However, the interest
in these cells must be buffered by known drawbacks, including cell stability, oncogenicity and substantial ethical
and likely regulatory/legal limitations [Garry et al., 2003;
Vats et al., 2002].
In contrast to the ESCs, bone marrow-derived adult
stem cells have been studied for decades, are well characterized and have demonstrated clinical utility for bone
marrow reconstruction after radiation or chemotherapy
[Mandalam and Smith, 2002]. Bone marrow is the source
of 2 distinct adult stem cell populations. Hematopoietic
stem cells are responsible for the development of the entire blood cell line, including white cells, red cells and
platelets [Arai et al., 2005a, b]. The second population
encompasses stromal stem cells or MSCs, populations
that give rise to a variety of connective tissues (including
bone, cartilage and adipose) and muscle tissue in vitro
and in vivo [Hauner et al., 1987; Johnstone et al., 1998;
Wakitani et al., 1995]. The MSCs can be further categorized into 3 different types depending on the source: bone
marrow, niche-specific stem cells (from adult tissues) and
newer sources (umbilical chord blood, adipose tissue,
placenta and spleen).
MSCs of bone marrow have been discovered as several groups have shown the existence of a rare cell type
that can be cultured from bone marrow and other organs
called the multipotent adult progenitor cell or MAPC
[Lakshmipathy and Verfaillie, 2005; Raff, 2003; Verfaillie
et al., 2002]. These cells, that were technically demanding
to isolate, could differentiate to osteoblasts, chondrocytes
and adipocytes [Anjos-Afonso et al., 2004]. Under current in vitro culture conditions that include fetal bovine
serum, MSCs obtained from young donors can grow to
24–40 population doublings in vitro, while the proliferative potential of MSCs obtained from older donors is
more compromised [Stenderup et al., 2003]. As older individuals are most likely to require tissue engineering,
this senescence reduces the applicability of autologous
MSCs in tissue repair strategies.
The second adult stem cell type comprises the nichespecific stem cells (fig. 2). Adult tissues undergo continuous self-renewal and hence require resident stem cells
with the capacity for multipotent differentiation. These
stem cells are most often slow cycling and give rise to transient amplifying cells, which impart the majority of tissue
renewal in the setting of injury [Ivanova et al., 2002; Lemischka and Moore, 2003; Moore and Lemischka, 2006].
Unlike stem cells, which by definition have an unlimited
proliferative capacity, transient amplifying cells are restricted in their capacity to divide and cannot proliferate
indefinitely. Although stem cells and transient amplifying
cells are both self-renewing populations, the latter are
thought primarily to be the major progenitor population,
which give rise to the terminally differentiated cell types
of an adult organ [Lemischka and Moore, 2003].
In addition, many authors have recently reported the
differentiation of MSCs from other sites into multiple cell
lineages, and newly identified sources include fat [Wickham et al., 2003], the placenta [Yen et al., 2005] and the
spleen [Kodama et al., 2005]. There is some evidence in
animal models that bone marrow stem cells can engraft
in the lung and differentiate to pulmonary alveolar epithelium where they can repair injury [Sabatini et al.,
2005]. Such sites can be an abundant source of stem cells;
for example, a single gram of human adipose tissue has
been reported to yield more than 70,000 pluripotent adipose-derived adult stem cells after 24 h in culture [Wickham et al., 2003].
Four areas for the potential clinical use of MSCs have
been explored: local implantation for localized diseases,
systemic transplantation, combining stem cell therapy
with gene therapy, and use in tissue engineering protocols [Kraus and Kirker-Head, 2006; Le Blanc and Pittenger, 2005; Mauney et al., 2005].
For clinical applications of stem cell transplantation
therapy, the direct manipulation of cells and their interactions would be desirable. Hitherto, establishing distinct protocols for precisely inducing and maintaining
cellular differentiation with a defined phenotype and
function has been extremely challenging [Corti et al.,
2005]. It is imperative that in vitro culture protocols
should be devoid of animal or human products to avoid
potential contamination with pathogens [Corti et al.,
2005]. The avoidance of products of animal or human
origin would also reduce variability within the culture
milieu and provide a more stringent level of quality control. Moreover, supplemented animal or human proteins
may adhere onto the surface of cultured stem cells, which
could possibly enhance their antigenicity on transplantation [Ryan et al., 2005]. Hence, the ideal culture milieu
for promoting the differentiation of stem cells in vitro
should be chemically defined and either be serum free or
Gene Therapy in Stem Cell Tissue
Engineering
Cells Tissues Organs 2006;183:169–179
171
Fig. 1. Stem cells are clonogenic, have a
self-renewal capacity throughout their
lifetime and give rise to terminally differentiated cells of various cell lineages. Their
differentiation pathway is unidirectional,
passing through the stage of lineage commitment and finally generating terminally
differentiated cells. The most primitive is
the fertilized oocyte (the zygote). Lower
down in the hierarchical tree are the multipotent cells. Most adult tissues have multipotent stem cells that can produce a limited range of differentiated cell lineages
appropriate to their location. At the bottom of the hierarchical tree are the unipotent stem cells, or committed progenitors,
that generate one specific cell type.
Fig. 2. There are 3 different sources for
adult stem cells. First, stem cells can be derived from bone marrow. The second adult
stem cell type comprises the niche-specific stem cells. Adult tissues undergo continuous self-renewal and hence require
resident stem cells with the capacity for
multipotent differentiation. Many authors
have recently reported a third type of stem
cells from other sources including fat, the
placenta and the spleen.
use synthetic serum replacements with the possible supplementation of specific recombinant cytokines and
growth factors if required [Wong and Tuan, 1993, 1995].
The major problem with culturing stem cells under
serum-free conditions is that cells generally tend to have
172
Cells Tissues Organs 2006;183:169–179
a lower mitotic index, become apoptotic and display poor
adhesion in the absence of serum [Pochampally et al.,
2004]. The removal of serum has been reported to slow
down the proliferation rate of MSCs [Ramoshebi et al.,
2002].
Goessler /Riedel /Hörmann /Riedel
The use of exogenous cytokines and growth factors is
another step forward in the development of a defined culture milieu for directing the osteogenic differentiation of
stem cells. The typical cytokines for the induction of the
chondrogenic differentiation of MSCs are TGF-3, BMP6, and IGF-1; the growth factor combination for the osteoinduction from human MSCs includes various isoforms
of BMPs, IL-6, growth hormone, leptin, sortilin and
transglutaminase [Canalis et al., 2003; Kroger et al., 1997;
Maeda et al., 2002; Nurminskaya et al., 2003; Ramoshebi
et al., 2002; Taguchi et al., 1998; Thomas et al., 1999].
In addition to protein-based cytokines and growth
factors, a number of nonproteinaceous chemical compounds have also been shown to promote stem cell differentiation in vitro. Such chemicals tend to be less labile,
with a longer active half-life in solution compared with
protein-based cytokines and growth factors [Kristensen
et al., 2005]. This would be advantageous for a prolonged
in vitro cell culture over several days or even weeks.
Moreover, unlike proteins that have to be synthesized in
living cells and subjected to complex posttranslational
modifications (i.e. glycosylation, peptide splicing, conformational folding), non-protein-based chemical compounds can be manufactured by chemical reactions in
the laboratory and therefore are more structurally and
chemically defined compared with proteins.
Adding to the complexity, the process of cellular differentiation has to be embedded into the context of cellcell and cell-matrix interactions found, for example, in
wound healing or tissue regeneration.
Growth Factors and Tissue Healing
Various growth factors effect musculoskeletal tissue
healing (table 1) [Trippel, 1997]. These growth factors are
small proteins that can be synthesized both by resident
cells at the injury site (e.g. fibroblasts, endothelial cells
and MSCs) and by infiltrating repair or inflammatory
cells (e.g. platelets, macrophages and monocytes). The
proteins are capable of stimulating cell proliferation, migration, and differentiation as well as matrix synthesis
[Collier and Ghosh, 1995; Scherping et al., 1997], and
their effects have been shown on different tissues [Hunziker and Rosenberg, 1996; Kasemkijwattana et al., 1998;
Linkhart et al., 1996; Luyten, 1995; Robbins and Ghivizzani, 1998; Sakou, 1998; Sellers et al., 1997; Spindler et al.,
1995]. The genes encoding most of the known growth
factors have been determined. Using the recombinant deoxyribonucleic acid (DNA) technology, researchers can
Gene Therapy in Stem Cell Tissue
Engineering
Table 1. Effect of growth factors in muscu-
loskeletal tissues
Growth factor
Hyaline cartilage Bone
IGF-1
bFGF
NGF
PDGF
EGF
TGF-
TGF-
BMP-2
+
+
+
+
+
+
+
+
+
+ = Positive effect; blank = not tested;
IGF-1 = insulin-like growth factor 1;
bFGF = basic fibroblast growth factor;
NGF = nerve growth factor; PDGF = platelet-derived growth factor; EGF = epidermal growth factor; TGF = transforming
growth factor; BMP-2 = bone morphogenetic protein 2.
produce large quantities of proteins for their use in treatment [Trippel, 1997].
Although the direct application of recombinant human proteins has some beneficial effects on healing [Kasemkijwattana et al., 1998], their relatively short halflives in vivo often require high doses and repeated injections. Another major limitation of using growth factors
to promote healing is the mode of delivery to the injury
[Evans and Robbins, 1995]. Many strategies – including
polymers, pumps and heparin – have been tested in attempts to achieve consistent growth factor levels at the
injured site [Gospodarowicz and Cheng, 1986; Mitchell
et al., 1996]. Despite the improved local persistence of
growth factor proteins with various approaches, the results of these delivery techniques remain limited. Among
the methods developed for the local administration of
growth factors, gene transfer techniques have proven the
most promising [Mulligan, 1993].
Definition of Gene Therapy
Gene therapy is the science of the transfer of genetic
material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Gene therapy applied to medicine includes the transfer
of defined genes (such as those encoding growth factors
or antibiotics) into the target tissue. Thus, a successful
Cells Tissues Organs 2006;183:169–179
173
ic modification of the cells ex vivo and the return of the
cells to the patient. Of the 2 approaches, the in vivo method is technically simpler to perform in a clinical setting,
giving it greater potential utility. The ex vivo techniques
may be more complex, but they are relatively safer. Additionally, this method allows for a selection of the cells
that express the therapeutic gene at higher levels [Cheng
et al., 1993]. The choice of method requires one to take
into account the disease to be treated, the gene to be delivered to treat the disease and the vector used to deliver
the gene [Oligino et al., 2000].
Relevant genes
introduced into vectors
Vectors:
Adenovirus
Retrovirus
Herpes simplex virus
Adeno-associated virus
Plasmid DNA
Vectors
Systemic delivery
Vector with gene
injected into
bloodstream
Local delivery
ex vivo
Cell culture of
modified cells to be
injected
in vivo
Direct injection of
vector
Gene inserted into target tissue (e.g. cartilage)
Fig. 3. Delivery strategies for gene transfer.
application of gene therapy would promote the production of therapeutic levels of desired proteins by the transformed cells at the site of injury or inflammation.
Basic Principles of Genetic Modulation
By employing techniques of gene therapy, genes can be
used therapeutically to produce proteins to treat and potentially cure acute and chronic conditions [Salyapongse
et al., 1999]. There are 2 general ways to perform gene
therapy: (1) a direct in vivo method and (2) an indirect ex
vivo method (fig. 3). The direct method involves transferring the genetic material into the target somatic cells
in vivo [Crystal, 1995]. The indirect technique involves
the removal of cells from the patient followed by a genet174
Cells Tissues Organs 2006;183:169–179
In order for target cells to manufacture the protein
products of the introduced gene, the exogenous genetic
material must be delivered to the cell’s nucleus. This process of transfection exists in 2 classes of vectors: viral and
nonviral (table 2). The viral technique is associated with
increased technical demands and an increased risk of virus-associated toxicity [Salyapongse et al., 1999]. However, viral vectors have been engineered for safety by
making them replication incompetent [Robbins and
Ghivizzani, 1998]. It is the viral ability to efficiently infect
cells and in this process to transfer DNA to the host without invoking an immune response that makes viruses attractive as vectors (fig. 4). These altered viruses can be
propagated in cell lines specialized to provide the necessary absent viral functions [Krougliak and Graham,
1995].
In general, retroviruses have been used for ex vivo
gene therapy applications as they are unable to efficiently
infect nondividing cells [Danos and Heard, 1992; Robbins and Ghivizzani, 1998]. Adenovirus, herpes simplex
virus and adeno-associated virus, as well as the nonviral
vectors may be used for either direct in vivo or ex vivo
delivery [Oligino et al., 2000]. Retroviruses are RNA viruses that carry a gene for a reverse transcriptase that
transcribes the viral genetic material into a doublestranded DNA intermediate. This DNA intermediate is
then incorporated into the host DNA allowing the host
cell machinery to produce all the necessary viral components. Additionally, because the viral genome is stably
integrated into the host DNA, any modification that has
been made will be passed to all daughter cells that are derived from the transfected cell [Goff and Lobel, 1987; Oligino et al., 2000].
Currently, the most common retrovirus used is derived from the murine leukemia virus. The majority of
Goessler /Riedel /Hörmann /Riedel
Table 2. Vectors used for gene delivery into cells and their characteristics
Modality
Advantages
Disadvantages
Direct injection
(naked DNA/plasmids)
Technically simple
Local delivery
Can act on nondividing cells
Nontoxic
Only applicable to tissues accessible by direct injection
Unable to target specific cells
Low transfection efficiency
Low long-term transfection rates
Liposomes
Technically simple
Local delivery
Can deliver large amounts of DNA
No immunogenicity
Only applicable to tissues accessible by direct injection
No targeting
Relatively low transfection efficiency
Low long-term transfection rates
Electroporation
Nontoxic
Can deliver large amounts of DNA
Can transfect nonreplicating cells
Nonspecific
Need for electrical pulses
Complex equipment
Difficult in vivo
Low transfection efficiency
Particle bombardment
(gene gun)
Can deliver large amounts of DNA
Technically simple
Nonspecific
Potential damage by bombardment
Potential foreign body reaction to particles
Relatively low transfection efficiency
Low long-term transfection rates
Antisense
oligonucleotides
Technically simple
Sequences can be ordered commercially
Nontoxic
Not always successful in decreasing gene expression
Finding ideal sequence for decreased gene expression is
hit or miss
Very short term
Nonspecific
Adenoviruses
Can infect virtually all cell types, as well as
dividing/nondividing cells
High transfection efficiency
Relatively long-term expression
Not integrated into host genome
Can deliver large amounts of DNA
Complicated techniques needed to produce and grow
Immune response
Inflammatory reaction
Lack of permanent expression
Potential wild-type breakthrough
Adeno-associated viruses
Can infect dividing and nondividing cells
Tropism for chromosome 19
Long-term expression
Relatively high transfection efficiency
Small DNA insert size
Potential for insertional mutagenesis
Potential wild-type breakthrough
Possible immune response
Possible inflammatory reaction
Social stigma in clinical uses
Retroviruses
Selectively infect proliferating cells
Long-term expression
Simple to make
Large DNA inserts possible
Potential insertional mutagenesis
Potential inflammatory/immune response
Difficult to grow and concentrate
Relatively low transfection efficiency
Herpes simplex virus
Neurotropic
Large DNA inserts possible
Long-term expression
Neurotropism limits use for other tissues
Potential wild-type breakthrough
Complex life cycle and large genome size complicate
technical production
Relatively low transfection efficiency
Gene Therapy in Stem Cell Tissue
Engineering
Cells Tissues Organs 2006;183:169–179
175
Fig. 4. Gene therapy can be used to stimulate the healing of injured tissue. Gene transfer can be accomplished by using a viral
vector. Genes to be inserted (here a growth factor gene) are incorporated into a truncated viral genome and packaged into a virus
particle. Virus particles infect the target cells, where the growth
factor gene is transported to the nucleus and is either integrated
into the host chromosomes or maintained as an episome. With
the help of viral and cellular proteins, the gene is transcribed into
messenger RNA. The messenger RNA is then translated into
growth factor proteins by the cellular machinery and the ribosomes. Growth factor proteins are then secreted and exert their
effect on the surrounding tissues.
clinical trials have utilized vectors based on this virus
[Marshall, 2000, 2002]. The murine leukemia virus has a
number of characteristics that make it attractive as a gene
therapy vector. It can be considered fairly safe, since it is
nonpathogenic in humans. Additionally, because it has
little homology with human retroviruses, the risk of recombination between the vector and any resident human
viruses is low [Danos and Heard, 1992].
A strength of retroviral vectors is that they can stably
transduce target cells with long-term transgene expression, whereas no viral genes are expressed in the transduced cells. Consequently, an immune response to the
vector is not problematic with the use of retroviral vectors
compared to other vectors.
In contrast to retroviruses, the adenovirus does not
integrate its genome into the host genome. Instead, the
adenoviral genome remains in the nucleus as an episomal element after the infection of the host cell. The advan176
Cells Tissues Organs 2006;183:169–179
tages common to all adenoviral vectors include the ease
of purification and concentration and the high efficiency
rate of host cell infection of various cell types, dividing
or nondividing [Oligino et al., 2000]. These advantages
make adenoviral vectors a good candidate for direct in
vivo gene transfer. The usefulness of these vectors is limited by 2 factors. In most tissues, the duration of transgene expression is limited to a few days to a week [Robbins and Ghivizzani, 1998] and viral genes are also transduced and expressed, eliciting an immune response to
the transduced cells that ultimately results in their clearance [Ding et al., 2005].
The advantages of the herpes simplex virus are its large
size, the wide spectrum of action and the continuous expression of genes from long-lived infection [Oligino et al.,
2000]. Similar to the adenovirus, there is little risk of insertional mutagenesis with the herpes simplex virus because it remains outside the nucleus (episomal). Unfortunately, the herpes simplex virus also has its limitations,
which include low infection efficiency, wild-type breakthrough, and a large genome size that makes it more difficult to manipulate than other viral vectors. Finally, herpes simplex virus’s tropism for neural cells limits its action, but some researchers are trying to exploit this feature
to target neurons [Krisky et al., 1998a, b].
Nonviral vectors are much cheaper and easier to produce in large amounts. These vectors have a limited immunogenicity, which allows for potential redosing, and
they are considered safe, since there is no possibility of recombination that would result in a competent virus that
could potentially cause disease [Oligino et al., 2000]. Nonviral vectors are, however, put at a severe disadvantage
when compared to viral vectors when taking into account
their markedly less efficient gene transfer rate [Salyapongse et al., 1999]. The use of nonviral vectors can be in
the form of injections of naked DNA (usually plasmids),
liposomes or particle-mediated gene transfer (‘the gene
gun’). The genetic material can be placed in liposomes in
order to increase the DNA uptake in tissue culture. The
last of these vectors uses a process by which the microprojectiles (e.g. gold or tungsten) are coated with DNA and
then accelerated by either helium pressure or a high-voltage electrical discharge, thus carrying enough energy to
penetrate the cell membrane [Oligino et al., 2000].
A novel strategy of nonviral gene transfer is to load
cDNA onto a porous biomaterial scaffold and pack it directly into a wound with the subsequent transfer of the
gene into endogenous cells migrating into the site [Bonadio, 2000, 2002; Warren et al., 2002]. This technique is
called gene-activated matrix and is an extension of reGoessler /Riedel /Hörmann /Riedel
search producing biodegradable polymers appropriate
for tissue engineering. For example, Bonadio et al. [1999]
used a collagen sponge/parathyroid hormone gene-activated matrix to improve bone formation in a dose-dependent fashion in canine tibia and femur defect models.
Limitations of Gene Therapy
For the treatment of injuries, the major concern for using gene therapy is safety. While gene therapy may represent a ‘last chance’ treatment option for severe disorders
such as cancer, Duchenne muscular dystrophy, Gaucher’s
disease or cystic fibrosis, the risk of side effects may be
unacceptable in elective reconstructive surgery. In addition, the integration of viral vectors into the host genome
carries the risk of insertional mutagenesis [Crystal, 1995].
An abnormal regulation of cell growth, toxicity from
chronic overexpression of the growth factor and cytokines, and malignancy are all theoretically possible, but
no cases have been reported. However, there is no guarantee that integrated DNA sequences will not cause mutations or malignancies years later. For this reason, longterm records of all human trials in gene therapy need to
be kept and exchanged among the research groups. Most
clinical trials of gene therapy are using the ex vivo approach, so the virus is not directly introduced into patients
and cells can be extensively tested before implantation.
Loss of expression of the transferred gene after a few
weeks is a common and not fully understood phenomenon. However, temporary and self-limiting gene expression could be useful in the treatment of musculoskeletal
injuries, in which only transient high levels of growth factors are needed to promote a healing response. Present
research is also focusing on the development of specific
inducible promoters that regulate the messenger RNA
transcription. Promoters are DNA sequences that are adjacent to the functional genes and are required for the
expression and regulation of gene transcription. Such inducible promoters could help to control the expression of
the transferred gene; they could modulate implanted
genes as well as turn them on and off. Although these
systems are very attractive, they remain under extensive
investigation in many laboratories and are not yet ready
for clinical trials.
Although great strides in gene therapy techniques
have taken place, they still have not become established
treatments, partly because of the lack of appropriate gene
vectors. Many laboratories successfully focus on developing therapeutic viral and nonviral vectors. Consequently,
Gene Therapy in Stem Cell Tissue
Engineering
major advances in vector development can be expected in
the near future [Robbins and Ghivizzani, 1998].
Future Directions for Gene Therapy in Tissue
Engineering
Cellular therapy has become an important strategy of
regenerative medicine. The cellular component of the tissue engineering paradigm is arguably the most important piece of the complex task of regenerating or repairing
damaged or diseased tissue. Critical to the development
of clinical strategies is the need for reliable sources of
multipotent cells that can be obtained with limited morbidity and can be precisely influenced and integrated into
tissue. The adult stem cell population may be well suited
for this task. Ahead lies the challenge to master the creation of a defined local milieu with regard to the unique
culture environments for different cell types.
Genetic engineering may hold the key to identifying
and modifying genes critical to cellular development and
differentiation. Although it is not yet established as an
approved therapeutic technique, a great potential exists
for the treatment of musculoskeletal injuries in the future. Currently, only a few effective gene therapy techniques for cartilage reconstruction have been tested on
human joints [Evans and Robbins, 1995]. At the experimental level, many studies have successfully been performed to prove the feasibility of gene delivery into different tissues of the musculoskeletal system. Beyond this
stage, initial experimental studies have demonstrated the
positive effects of transduced genes (especially BMP-2,
IGF-1, TGF-) in vitro and in vivo. The main obstacle
today seems to be the availability of vectors carrying effective genes, and some concerns about the safety of viral
vectors after the death of a patient in a recent gene therapy trial. However, great progress has been noticed in
many laboratories working on the engineering of these
vectors.
In general, we believe that the combination of gene
therapy and tissue engineering will help us develop effective therapies for tissues that have a low healing capacity
(i.e. cartilage) and for other disorders such as osseous defects. With these new technologies, however, a large number of basic science and preclinical studies still needs to
be performed before the efficiency necessary for plastic
and reconstructive surgery applications and guaranteed
safety are reached [Mulligan, 1993]. But with more and
more questions answered, adult stem cell biology will
likely transition from bench top to clinical reality.
Cells Tissues Organs 2006;183:169–179
177
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