1 s2.0 S0169409X98000209 Main PDF

Advanced Drug Delivery Reviews 33 (1998) 5369
Gene therapy for tissue repair and regeneration

a,
b
c
Jeffrey Bonadio *, Steven A. Goldstein , Robert J. Levy
a
University of Michigan Medical School, Department of Pathology, 1198 SE, 300 North Ingalls Building, Ann Arbor,
MI 48109 -0417, USA
b
University of Michigan Medical School, Department of Surgery, Orthopaedic Research Laboratories, Section of Orthopaedic Surgery,
400 North Ingalls Building, Room G-0161, Ann Arbor, MI 48109 -0486, USA
c
Children s Hospital of Philadelphia, Abramson Pediatric Research Center, 11 th Floor, 34 th and Civic Center Blvd., Philadelphia,
PA 19104, USA
Received 17 September 1997; received in revised form 10 December 1997; accepted 30 December 1997
Abstract
This review presents a current overview of the discipline of human gene therapy. In addition, a gene therapy method is
described in which plasmid genes are transferred from a structural matrix carrier into fresh wound sites so as to enhance
tissue repair and regeneration. The potential to develop a gene therapy for bone regeneration is discussed in detail. 1998
Elsevier Science B.V. All rights reserved.
Keywords: Fracture repair; Cytokine; Plasmid gene transfer; Granulation tissue; Fibroblast; Gene activated matrix
Contents
1. Introduction ............................................................................................................................................................................
2. Gene therapy ..........................................................................................................................................................................
2.1. Overview ........................................................................................................................................................................
2.2. Classes of disease under consideration ...............................................................................................................................
2.3. Inherited single gene disorders ..........................................................................................................................................
2.4. Cancer ............................................................................................................................................................................
2.5. Cardiovascular disease .....................................................................................................................................................
2.6. Infectious disease .............................................................................................................................................................
2.7. Vector systems .................................................................................................................................................................
2.8. Current status of gene therapy clinical trials .......................................................................................................................
2.9. Future directions ..............................................................................................................................................................
3. Bone growth gene therapy .......................................................................................................................................................
3.1. Overview ........................................................................................................................................................................
3.2. Clinical problems associated with fracture repair: current state-of-the-art .............................................................................
3.3. Synthetic materials as bone graft substitutes .......................................................................................................................
3.4. Recombinant cytokine bone graft substitutes ......................................................................................................................
3.5. Tissue engineering ...........................................................................................................................................................
3.6. Gene therapy for fracture repair ........................................................................................................................................
4. Concluding remarks: gene therapy for tissue repair and regeneration...........................................................................................
Acknowledgements ......................................................................................................................................................................
References ..................................................................................................................................................................................
*Corresponding author. Tel.: 1 1 734 6474774; Fax: 1 1 734 9360669; E-mail: jbonadio@umich.edu
0169-409X / 98 / $ see front matter 1998 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 98 )00020-9
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J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69
1. Introduction
The purpose of this review is twofold. First, the
review presents a summary of the discipline of gene
therapy. Second, a novel gene therapy method is
described in which plasmid genes are transferred
from a structural matrix carrier into fresh wound
`
sites. The implications of this method vis-a-vis
tissue
engineering and the development of a gene therapy
for bone regeneration are discussed.
2. Gene therapy
2.1. Overview
Gene therapy was originally conceived of as a way
to correct heritable gene defects in targeted populations of mutant cells [14]. Now, however, gene
therapy is more generally understood as a treatment
approach for several types of human disease based
on the transfer of genetic material (DNA) into an
individual. (For the purposes of this review, DNA
may refer to a recombinant plasmid gene, an oligonucleotide, or a ribozyme.) Somatic cells have been
the only targets of human gene therapy to date.
Therefore, gene therapy is designed to treat only the
patient in question and not, for example, their
offspring.
The initial effort toward developing somatic gene
therapy relied on an indirect means of introducing
genes into tissues, called ex vivo gene therapy or
autologous cell transfer. By this approach, target
cells were removed from the body, transduced with
viral vectors carrying recombinant genes, and then
reimplanted. [Small molecule drugs can be given to
modulate therapeutic gene expression once genetically-modified cells have been reintroduced into
the body (e.g., Refs. [5,6]).] Treatment protocols
based on ex vivo gene therapy methods have proposed for epithelial cells [7], endothelial cells [8],
hepatocytes [911], fibroblasts [1215], lymphocytes [16], hematopoietic stem cells [17], and skeletal muscle cells [18,19], among other cell types.
More recently, somatic gene therapy has relied on
the direct introduction of DNA into tissues. A large
number of methods have been developed for this
purpose. Thus, the gene gun technology has been
used to locally deliver plasmid gene constructs into

plants and animals, including humans [20,21]. DNA
has also been formulated in a liquid buffer (e.g.,
naked DNA) [2224] and with (proteo)liposome
carriers [25,26], and then delivered in vivo for DNAvaccination as well as other gene therapy applications. In terms of pharmaceutical delivery, DNA has
been encapsulated in controlled release synthetic
polymer particles for oral delivery [27] and for
intraarterial delivery [28,29]; DNA has been incorporated into hydrogels [30] and sustained release
polymer emulsions [31], both of which have been
employed as medical device coatings; and DNA has
been incorporated directly into structural matrix
carriers for implantation into tissues [32]. Finally, in
order to augment gene transfer efficiency and target
specific populations of cells, DNA has been complexed with agents such as cationic condensing
peptides [33,34]; endosomal-disrupting peptides
(e.g., Ref. [35]); cell surface receptor targeting
agents, e.g., fibroblast growth factor [36,37]; and
synthetic nuclear-targeting peptides [38]. Similar to
the situation with ex vivo gene therapy, small
molecule drugs may be administered to control the
therapeutic expression of DNA directly delivered to
tissues in vivo (e.g., Ref. [5]).
2.2. Classes of disease under consideration

Many types of human disease currently are under
consideration for somatic gene therapy, e.g., inherited disorders that arise from single gene mutations,
multifactorial disorders (e.g., cancer and cardiovascular disease, in which several genes contribute to etiology and pathogenesis), and infectious
diseases [14]. Those diseases that produce life
threatening situations have been investigated for the
most part. The number and variety of human diseases suitable for gene therapy should increase
rapidly as a consequence of the success of the human
genome project.
2.3. Inherited single gene disorders

Examples of single gene disorders amenable to
gene therapy include, but are not limited to, sickle
cell anemia, the hemophilias, inherited immune
deficiency disorders, familial hypercholesterolemia,
Gaucher disease, a1-antitrypsin deficiency, SCIDadenosine deaminase deficiency, and cystic fibrosis.
In those instances where a single gene disorder
results from the absence of protein, the replacement
of gene function should be curative: the delivery of a
normal clotting factor gene to a patient with
hemophilia is a relevant example [39]. In other
instances, the mutant protein participates less directly
in cellular pathology. An example of the latter is
sickle cell anemia, where a variant globin molecule
causes hemoglobin to undergo abnormal polymerization under low oxygen tension, leading to the
destruction of distorted red blood cells. In this
situation, expression of a normal globin chain following therapeutic gene transfer would be expected
to benefit the patient. A second example would be a
dominantly-inherited connective tissue disorder such
as osteogenesis imperfecta, in which the presence of
an abnormal collagen interferes with connective
tissue integrity. Here, selective silencing of the
mutant gene (e.g., via ribozyme gene transfer) may
be of benefit to the patient [40], although the basis
for this assertion is still quite theoretical and, for all
intents and purposes, unproven, and formidable
challenges exist.
Gene correction strategies for inherited disorders
have yet to achieve a meaningful clinical outcome,
and several reasons have been postulated to explain
why this is so [4]. First, it is accepted that the
regulation of most genes is complex (i.e., under
homeostatic control) and that at present, long-term
physiological control over the expression of exogenous transgenes is not possible. (A concern, therefore,
is that unphysiological transgene expression either
may not be efficacious or may lead to tissue toxicity.) Second, the expression of therapeutic transgenes
tends to be extinguished in vivo a few days to weeks
after delivery, which certainly represents a formidable barrier to curative therapy in individuals with
inherited disease mutations. In fact, much is known
regarding the DNA sequences that direct the highlevel, tissue-specific expression of genes in cultured
cells and in transgenic mice. In practice however,
long-term physiological gene expression in somatic
cells is difficult to achieve. These difficulties may be
due to undefined cellular mechanisms that repress
virally introduced genes, a subtle selective disadvantage of stem cells that express transferred genes, or
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the failure to include appropriate regulatory sequences in gene therapy vectors.
2.4. Cancer
A variety of cancers are being treated by gene
therapy. Cancer often arises through a multistage
process that includes the somatic mutation of cellular
genes and the selection of mutant cells with increasingly aberrant growth and differentiation properties
[41,42]. The (aberrant) expression of mutant protooncogenes, tumor suppressor genes, and DNA repair
genes has been shown to contribute to this multistage
process.
The introduction into cancer cells of a therapeutic
gene that will alter or inhibit the malignant phenotype is an appealing concept, based in part on
experimental data showing that the introduction of
normal copies of tumor suppressor genes into cancer
cell lines in vitro restores normal growth (e.g., p53;
see Ref. [43]). For several reasons, however, gene
correction strategies have yet to achieve a meaningful clinical outcome. First, some cancers arise following mutations in which the gene product has a
dominant effect. Consequently, the transfer of a
normal copy of the exogenous transgene into an
affected cell may have little, if any, impact. Second,
the number of cells within a clinically detectable
cancer is large ( . 10 9 ) and the mutation rate within
them is high. Therefore, mutations may arise in the
transgene of a subset of transfected cells, inactivating
therapeutic action and, perhaps inevitably, causing
the regrowth of the tumor. Third, present technologies do not permit the transfection of every
malignant cell within a tumor, a technological deficit
that may result in tumor regrowth. Fourth, current
gene transfer technology is incapable of reaching
cells that have metastasized far and wide, which is
the major clinical complication of cancer.
Because of these formidable problems, other gene
therapy approaches to cancer treatment have been
investigated. Thus, tumor infiltrating lymphocytes or
other immune effector cells have been transduced ex
vivo to increase their ability to attack tumor cells
[44]. Cytokine genes and other immune-modulatory
products have also been delivered to cancer cells,
either outside the body (ex vivo) or directly into the
patient, in order to stimulate the immune rejection of
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tumor cells [45]. Mutant adenoviruses have been

introduced into p53-deficient human cancer cells in
order to lyse them in vivo [46]. A recent proposal is
to use chemoresistance genes for somatic gene
transfer [47]; possible applications include protection
of bone marrow and other organs from the adverse
effects of cancer chemotherapy. Finally, genes encoding viral or bacterial enzymes involved in the
conversion of nontoxic prodrugs to their active
molecules have been delivered to cancer cells to
facilitate tumor rejection. In one instance involving
the latter approach, the thymidine kinase gene from
herpes simplex virus was transferred into brain and
liver cancer cells, rendering them more susceptible to
the drug ganciclovir [48]. However, although they
have shown promise in preclinical models none of
these strategies has yet been shown to be safe and
effective in humans.
2.5. Cardiovascular disease

Bypass surgery and percutaneous revascularization
(balloon angioplasty) are currently used to improve
blood flow to ischemic (atherosclerotic) tissues.
However, many patients with symptomatic cardiovascular and central nervous system disease cannot take advantage of these treatment modalities
because of the extensive multifocal nature of the
occlusive disease [49] and because of the morbidity
associated with the manipulation of diseased blood
vessels. Coronary stents may be used to improve the
immediate and midterm outcome following coronary
angioplasty because of the ability of stents to control
blood vessel patency [50,51]. Clinical reports have
also suggested that a similar benefit will apply
following stent deployment in the aorta and iliac
arteries.
The major clinical complications of bypass
surgery and percutaneous revascularization (discussed in Ref. [52]) include unpredictable vessel /
vascular graft closure, mural dissection, and abrupt
occlusion of the vessel / vascular graft lumen from
the pathological growth of neointimal tissue (restenosis). An area of intense investigation, the mechanism of pathological neointima formation is complex and remains unclear at the present time [53].
Several arterial gene therapy strategies have been
employed to help ensure the safety and efficacy of
bypass surgery and percutaneous revascularization,

although none as yet have resulted in a meaningful
improvement in patient care. These strategies include
an attempt to limit neointima formation by inhibiting
the proliferation of vascular wall cells [5464], an
attempt to stimulate angiogenesis and endothelial cell
proliferation at sites of partial occlusion [30,6572],
and an attempt to modify the stentvascular wall
interface so as to promote biocompatibility [73,74].
Genes have been delivered directly and indirectly
using a wide variety of vector strategies and delivery
approaches. A more recent development is based on
the perceived tactical advantage of direct intramuscular plasmid gene transfer at sites of partial occlusion
and tissue ischemia [52,75]. This approach, which
targets the adventitial as opposed to the endothelial
side of the atherosclerotic vessel, has been proposed
as an alternative treatment strategy for patients with
extensive disease that are not candidates for intravascular catheter-based gene transfer.
2.6. Infectious disease

Among the infectious diseases being treated by
gene therapy, HIV infection and AIDS have received
the most attention [14,76], although it can be
anticipated that many other pathogens will be investigated in the future. Recent technological and
conceptual developments include the ability to target
genes to the surface of Kaposis sarcoma cells [77]
and combination gene therapy [78]. In the clinic,
current efforts are focused on two general areas:
postexposure vaccination to boost the host immune
response to infection, and expression of genes in
target cells that prevent infection or virus replication.
Although several trials are ongoing at present, they
are in the very early stages, and only the first results
have been presented [79].
In vaccination trials, modified HIV genes have
been introduced directly into the infected individual
following ex vivo treatment of target CD4 1 or
precursor cells, typically with retroviral vectors that
express genes encoding antiviral products. Products
currently being tested include single chain antibodies
that prevent key HIV enzymes from functioning [80]
as well as mutant proteins that inhibit virus replication, antisense RNA that blocks translation of HIV
gene products or causes destruction of the HIV
genome, ribozymes that attack HIV RNA at specific

unique sites, and decoy RNAs that efficiently
compete for binding of viral proteins.
Although each of these approaches block HIV
replication in cell culture systems, serious obstacles
to their practical application remain [81]. For example, it is not yet known what cell types are best to
target, or how the target cells will be isolated,
treated, and returned to the patient. An important
uncertainty is whether resistant mutants, which now
appear to be a major obstacle to successful multidrug
chemotherapy, will also present a serious problem
for gene therapy of HIV infection and AIDS.
2.7. Vector systems

Somatic gene therapy entails two critical steps,
namely, the delivery of DNA to the appropriate cells
(either ex vivo or in vivo) followed by the therapeutic expression of transgene-encoded proteins [14].
Ordinarily, the intent is to transfer a gene into host
cells for as long as would be required for efficacy.
Several gene therapy vector systems are in use or
under consideration, including viruses (e.g., retroviruses and adenoviruses), pure DNA molecules (either
double- or single-stranded), and whole chromosomes. Each vector system has perceived advantages
and disadvantages.
The biology of retroviruses is understood best [1].
Accordingly, retroviral vectors have been employed
in the majority of human clinical gene therapy
protocols. Retroviruses appear to be most suitable for
permanent correction of genetic diseases: among
their advantages are the ability to efficiently enter
dividing cells and then integrate the recombinant
retroviral genome into the host genome. Retroviruses
may be unsuited for certain other applications,
however, because they infect and integrate only
dividing cells. Moreover, experience has also shown
that it is cumbersome, expensive and technically
difficult to prepare pure recombinant virus at relatively high titer; that there are size constraints on
inserted genes; that there are difficulties in controlling or ensuring transgene expression; and that the
potential for genetic damage exists following the
random integration of retroviral DNA within the host
genome.
The adenovirus vector system has also been
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intensely studied. Among its advantages are the

capacity to infect nondividing cells, the efficient
infection of many types of human cells, the ability to
achieve high-level expression following transfection,
and the ability to produce high titer stocks [82,83].
Experience has shown, however, that it is expensive
and technically difficult to produce pure recombinant
virus stocks. Additionally, the relatively high immunogenicity of adenoviral proteins [84,85], the
potential for autoimmune reactions to transgene-encoded proteins [86], and the potential for oncogenicity (e.g., Ref. [87]) pose additional challenges to the
development of adenoviral-based products for human
use. The mechanism of the autoimmune response is
unknown. Taken together, this evidence of tissue
toxicity is perhaps not too surprising given that
adenoviruses are human pathogens against which
elaborate defense mechanisms naturally exist.
The direct administration in the clinic of pure
DNA (or pure DNA complexes) has several advantages. These include the virtually unlimited size of
recombinant plasmid constructs, the ready availability of low-cost straightforward methods for DNA
production, the established stability of DNA under a
wide variety of conditions, the ability to readily
combine DNA with a variety of established pharmaceutical delivery systems and carriers, and the
proven safety of pure DNA in vivo (especially so
long as plasmid DNA is manufactured properly
[88,89]). However, pure DNA is transferred into
tissues at low efficiency, which suggests that only
those diseases that require low-level therapeutic gene
expression will be suitable targets for a pure DNA
gene therapy approach. In this regard, the use of
naked DNA for peripheral neovascularization [30,66]
and for in vivo vaccination [90,91] now appears
feasible and highly promising. Indeed, the development of human DNA vaccines, should they be
realized, will represent a milestone for modern
medicine, i.e., one that may be compared to the
development of antimicrobial drugs or the ability to
overcome barriers to tissue transplantation.
Studies of yeast cells have defined many components necessary for maintaining chromosomes
within cells. The development of artificial human
chromosomes as vectors [92] may allow genes to be
transferred without random insertion of foreign
sequences into the host genome. The efficient intro-
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duction of these vectors into cells, however, may

represent a formidable obstacle to their use for gene
therapy, at least in the near term.
2.8. Current status of gene therapy clinical trials

Studies of retrovirus-based gene transfer into
hematopoietic stem cells have demonstrated how
some of the perceived problems with gene therapy
are manifest in actual experiments. In mice, for
example, current protocols permit the transfer of
genes into a substantial fraction of stem cells following retroviral infection of marrow cells ex vivo.
However, gene transfer into marrow stem cells of
other species (e.g., canines, nonhuman primates, and
humans) has been less efficient, with 10% or fewer
cells transduced. It is generally accepted that the low
efficiency of gene transfer has compromised the
demonstration in the clinic of therapeutic efficacy as
well as gene transfer efficiency. In this regard, some
have concluded that both the clinical benefit and the
scientific value of clinical trials have been compromised [4].
More than 1000 individuals in experiments involving more than two dozen diseases, have now participated in clinical protocols for gene therapy ( . 200
human gene therapy protocols approved by the U.S.
Food and Drug Administration; e.g., Ref. [93]).
Retroviruses have been employed as gene transfer
vehicles in the majority of protocols, which have
focused on cancer.
Although widely referred to as clinical trials,
gene transfer protocols to date have been, as would
be expected with any revolutionary therapeutic advance, small scale exploratory studies meant to test
the feasibility and safety of particular vectors and to
evaluate the effect of expressing specific gene products [4]. These studies have been criticized, however,
because of a lack of sufficient controls to evaluate
efficacy or compare gene therapy with more conventional approaches to the same disease. For example,
administration of PEGADA (a preparation of adenosine deaminase) to patients with adenosine deaminase deficiency, though clinically appropriate in light
of its demonstrated efficacy, complicated the evaluation of patients that were treated with retrovirally
transduced lymphocytes [16]. As a second example,

treatment results in five patients with homozygous
familial hypercholesterolemia were judged to be
inconsistent and disappointing, with only slight or no
changes in cholesterol metabolism and levels
[94,95]. Although not established for any gene
therapy protocol to date, it is likely that clinical
efficacy will be demonstrated sometime in the next
25 years.
Adverse short-term effects related to gene transfer
protocols appear to vary depending on the nature of
the vector system and the patient. For example,
retroviral delivery in patients with adenosine deaminase deficiency (and in marker studies) has not been
associated with any obvious adverse effects. On the
other hand, the administration of high titer adenovirus vectors to patients with cystic fibrosis [96,97]
and atherosclerotic cardiovascular disease [98] has
been associated with severe host inflammatory responses. Administration of plasmid DNA associated
with liposomes has also resulted in significant toxic
reactions in pulmonary airways.
2.9. Future directions

The recommendations of the OrkinMotulsky
panel [4] that were made in 1995 are still largely
applicable today. The panel concluded that (a)
Somatic gene therapy was a logical and natural
progression in the application of fundamental biomedical science to medicine and offers extraordinary
potential, in the long-term, for the management and
correction of human disease . . . ; (b) While the
expectations and the promise of gene therapy are
great, clinical efficacy has not been definitively
demonstrated at this time in any gene therapy
protocol . . . ; and (c) Significant problems remain
in all basic aspects of gene therapy. Major difficulties at the basic level of understanding include
shortcomings in all current gene transfer vectors and
an inadequate understanding of the biological interaction of these vectors with the host.
It seems clear from these conclusions that more
workboth basic and applied, and all of it fascinatingneeds to be done before the promise of gene
therapy can be realized.
3. Bone growth gene therapy
3.1. Overview
This section of the review describes a novel gene
transfer method for bone growth. The impetus to
develop the method was derived initially from a
long-standing effort to improve the quality of life of
individuals with osteogenesis imperfecta, an inherited disease characterized by frequent bone fractures
[99,100]. It time, however, it became clear that gene
therapy could be applied to a broad range of clinical
orthopedic indications. Therefore, the novel gene
transfer method will be presented and discussed from
a broad clinical orthopedic perspective related to the
need for therapies that stimulate rapid bone regeneration.
3.2. Clinical problems associated with fracture

repair: current state-of-the-art
In dentistry and orthopedics, the current treatment
of a fracture or osteotomy is based on the control of
pain and stabilization of the defect [101]. A challenge arises with complex fractures that heal with
difficulty, e.g., comminuted fractures, segmental
defects, and crush fractures, that may occur at sites
of marginal vascularity or may be associated with
large areas of tissue loss [102]. Here, the chance of
developing nonuniona significant clinical concern
associated with severe and disabling pain, reduced
productivity, degradation of the quality of life, and
expensive chronic careincreases with the severity
of the fracture. Large open fractures with gross
contamination and extensive soft tissue damage are
at highest risk for nonunion: in a series of type II and
type III open tibial fractures, for example, Velazco
and Fleming [103] reported a 14% incidence of
nonunion. While the clinical definition varies somewhat in the literature, nonunion generally is declared
if, in the opinion of the attending oral / orthopedic
surgeon, further conservative management will fail
to unite a fracture site after 8 months of nonsurgical
care [104]. More than 6.0 million fractures occur in
the U.S. per year [105,106], and | 1 000 000 of
these are categorized as complex and therefore at
risk to develop nonunion.
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The treatment of a complex fracture is designed to

mitigate against nonunion [107]. For closed uninfected complex fractures with anatomical alignment,
new bone formation is often achieved by placement
of autologous bone graft. Cancellous bone is the
graft material of choice, and union rates of 87100%
have been reported [108]. However, experienced
surgeons say that there is never enough autologous
material to harvest, that bone grafts are hard to
contour, and that the harvest procedure is morbid,
i.e., is associated with prolonged, debilitating pain.
Moreover, new bone growth may be slow (i.e., up to
18 months or more may be required to achieve
complete functionality), and, therefore, a successful treatment may still be associated with expensive
long-term care, during which time patients must deal
with chronic pain and immobilization. Finally, bone
grafts may become devitalized because they fail to
develop an adequate blood supply, and thus are
prone to delayed healing and mechanical degradation. Therefore, the need to develop a bone graft
substitute capable of stimulating new bone growth in
vivo clearly still exists.
Large open complex fractures present an even
greater challenge, e.g., they often require surgical
debridement and fixation, as well as soft tissue
coverage [104]. External fixation is a preferred
method of stabilizing such fractures: Blick et al.
[109] and Behrens et al. [110] reported union rates of
about 90% when bone grafting was combined with
external fixation. Many traumatologists believe that
once fixation is employed, it should be continued
until union occurs. Over time, however, instability of
the fixator may lead to abnormal, excessive loading
of the fracture site and poor fracture healing. Pin
track infections and tissue necrosis (pin toggling)
also are common time-dependent complications of
fracture fixation. Therefore, a faster method of bone
healing would allow for the early removal of fixation
devices and a decrease in morbidity. Rapid bone
growth within the fracture site also would help avoid
the muscular atrophy and soft tissue contracture that
result from prolonged immobilization.
To paraphrase from Goulet and Templemann
[102], The ultimate goal of a functional lower
extremity should not be overlooked in the attempt to
achieve bone union. If heroic efforts must be taken,
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and yet the outlook for a functional result seems

grim, amputation should be viewed as a reasonable
alternative to the pursuit of union. Moreover, as
Einhorn [106] has concluded, . . . there are several
conditions under which enhancement of the (fracture) repair process would be of great benefit to
ensure rapid restoration of skeletal function. The
ability of injured patients to return to the work force
or to recreational activities early would not only have
a substantial economic impact on society but would
also improve the overall physical and mental wellbeing of the patients.
Therefore, a compelling need still exists for a safe
and effective rapid bone growth therapy.
3.3. Synthetic materials as bone graft substitutes

Bioinert, resorbable, porous, and bioactive metals
and ceramics have been tried, but no completely
effective bone graft substitute for rapid, stable bone
growth currently is available for human use (see Ref.
[111] for review). Alumina prostheses have been
used in a variety of dental and orthopedic procedures
because of their minimal interaction with surrounding tissues, low coefficients of friction, and low wear
rates. Porous materials (pore size . 100 mm) allow
bone ingrowth, which strengthens the union between
implant and bone, but not as rapidly as needed. In
general, it has been difficult to define and manufacture a polymer with optimal strength and degradation
properties. Finally, bone growth has been induced
(creeping substitution) when cells are grown on
synthetic polymers and ceramics [112,113], but
again, at a suboptimal rate.
3.4. Recombinant cytokine bone graft substitutes

Knowledge of the cellular and molecular events
associated with fracture repair have stimulated an
intense effort worldwide to develop medical (drug)
bone growth therapies that effectively substitute for
autologous bone grafts. Normal bone tissue is
thought to possess a relatively unique capacity to
regenerate following fracture. The complex but
ordered fracture repair sequence includes hemostasis,
clot dissolution, granulation tissue ingrowth, formation of a callus, and remodeling of the callus to an
optimized structure [114]. The cells that participate
in this process include platelets, inflammatory cells,

fibroblasts, endothelial cells, pericytes, osteoclasts,
and osteogenic progenitors. It is generally accepted
that the cells involved in fracture repair respond to
extracellular signals (i.e., small regulatory molecules
such as cytokines and hormones) which govern cell
behavior and gene expression (e.g., cell proliferation,
migration and differentiation, and the biosynthetic
activity of repair cells).
While the list is by no means complete, a large
number of cytokines have been identified that control
the cellular events associated with bone formation
and repair [115]. Two important examples of these
factors include bone morphogenetic protein (BMP)
and transforming growth factor-beta (TGF-b), which
are both members of the TGF-b superfamily of
genes and proteins. BMPs are soluble extracellular
factors that control osteogenic cell fate: BMP genes
are normally expressed by fetal osteoblasts in vitro
[116] and in vivo [117119], recombinant BMP
proteins initiate cartilage and bone progenitor cell
differentiation [120124], BMP delivery induces a
bone formation sequence similar to endochondral
bone formation [125], and BMP gene expression is
upregulated early in the process of fracture repair
[126]. Osteogenic protein-1, a member of a family of
molecules related to the BMPs [127], is capable of
similar effects in vitro and in vivo [128,129]. TGF-b
has also been shown to stimulate cartilage and bone
formation in vivo [130,131].
The work cited above certainly suggests a
rationale for the use of osteogenic cytokines as bone
growth therapies. However, more than three decades
have passed since the pioneering work of Urist and
coworkers first identified a bone morphogenetic
activity in tissue extracts, and still no effective
cytokine therapy is available. Clues as to why
progress has been slow may be derived from an
understanding of cytokine structure and biosynthesis,
which are complex. To illustrate this point, a brief
description of the structure and biosynthesis of TGFb is presented below.
TGF-b is initially synthesized as a | 400 amino
acid, glycosylated precursor that consists of a signal
peptide, an amino-terminal propeptide, and a carboxy-terminal mature growth factor [132]. Two precursor chains associate to form a disulfide-bonded
dimer with latent activity. The full length dimer
eventually is cleaved at an endoproteolytic cleavage

motif, and the propeptide dimer and mature growth
factor dimer remain noncovalently associated to form
a latent TGF-b complex. The disulfide-bonded structure of TGF-b certainly must be maintained for full
functional activity. Latent complexes are associated
with one of several known binding proteins; of these,
the latent TGF-b binding protein (LTBP) is the best
understood (e.g., Refs. [133,134]). Inside of cells,
LTBP binds and regulates the assembly of the latent
TGF-b complex, and then targets the complex to the
pericellular matrix, where it is stored. LTBP also
modulates the activation and release of intact mature
TGF-b from its storage site.
The complexity of the TGF-b biosynthetic pathway probably is related to the need to regulate
TGF-b activity in vivo. At sites of fracture, TGF-b is
a multifunctional growth factor with the ability to
serve as a chemoattractant for macrophages and
other inflammatory cells; to modulate immune and
inflammatory responses; and to promote angiogenesis and extracellular matrix production. Given these
important and wide-ranging effects, TGF-b activity
must be regulated with precision: previous studies
have shown that the aberrant production of TGF-b
can be associated with a potentially lethal immune
dysfunction as well as fibrosis [135,136]. Intuitively,
a complex biosynthetic pathway provides numerous
checkpoints at which bioactivity can be controlled. A
similar need exists to regulate the activity of BMPs.
While the complicated structure and biosynthetic
pathway of cytokines may facilitate Natures need
for precise physiological control, these factors certainly may prove to be the nemesis of the bone
growth product developer, especially if the goal is to
produce the cytokine as a recombinant protein (ex
vivo) for therapeutic purposes. First, cytokines produced ex vivo generally are not associated with
cognate binding proteins, the absence of which may
lead to errors in protein assembly (or other aspects of
biosynthesis) as well as a loss of functional activity.
Second, despite more than a decade of research and
development it still is not possible to combine
recombinant cytokines with flexible, user-friendly
pharmaceutical delivery systems and still maintain
structural integrity and functional activity in vivo.
This technical deficit has diminished patient care.
Third, the delivery of exogenous cytokines is in-
61
efficient: pharmacologic doses of recombinant proteins must be used to induce bone formation in
preclinical animal models (e.g., Refs. [129,130]) and
in humans. One explanation for this may be that
recombinant cytokines are quickly turned over ( | 30
min [137] in the harsh environment of a fracture site
undergoing repair) and therefore are degraded before
cells bearing cytokine signaling receptors on their
surface appear in significant numbers [114,138].
Attempts have been made to limit the administration
of high dosage forms through immobilization of
therapeutic protein at the target site, but this approach complicates the readministration of the protein (for repeated dosing). Together, these observations suggest that the current cytokine therapies for
fracture repair are flawed because of the complexities
associated with developing drugs from natural cytokine molecules.
3.5. Tissue engineering

Tissue engineering is a multidisciplinary branch of
science that draws from biochemistry, cell and
molecular biology, materials science, engineering,
medical implant science, and transplantation science
[139,140]. The goal of tissue engineering is to
develop innovative, three dimensional biological
substitutes with structurefunction properties that
restore, maintain and improve tissue function. The
principles of tissue engineering have been used to
develop orofacial implants, orthopedic implants,
and bone graft substitutes. Advances in engineering
materials have allowed biodegradable polymers to be
used for guided tissue regeneration in the treatment
of oral, dental, and orthopedic diseases and traumatic
bone defects (e.g., Refs. [141149]). For example,
Vacanti, Vacanti and coworkers have seeded periosteal cells (among other cell types) into polymer
matrices in vitro (reviewed in Ref. [141]; also see
Refs. [150155]). The authors showed that the
culture supernatant taken at the time of implantation
contained osteocalcin and that cells within the polymer matrix were appropriately responsive to 1,25
dihydroxy Vitamin D3. Ectopic implantation of these
constructs resulted in the generation of cartilage that
then matured into new bone and bone marrow.
Similar results were obtained following implantation
62
into orthopedic sites (cranial and femoral osteotomy

sites).
Experience has shown that it is relatively easy to
generate thin tissue-engineered implants, but thick
implants tend to undergo central ischemic necrosis.
In fact, there are many clinical circumstances in
which large masses of cells for tissue engineering
need to be kept alive, both in vitro and in vivo. While
the mechanism is generally understood, the development of systems and strategies to maintain cell mass
continues to be an enormous and fundamental challenge related to mass transfer in vitro and in vivo
[156]. This challenge will be especially critical for
complex fractures that, as stated above, tend to be
large segmental skeletal defects.
3.6. Gene therapy for fracture repair

Only very recently has gene therapy for nonlifethreatening diseases been considered. In this regard,
recent efforts by the authors and colleagues have led
to the development of a method to transfer plasmid
DNA directly into mammalian repair cells involved
in fracture repair. The method involves the placement of an implant (a gene activated matrix or
GAM) into a fresh wound site. The method targets
granulation tissue fibroblasts for plasmid gene transfer. These fibroblasts normally originate in viable
tissue surrounding the fracture, where they proliferate and migrate into the GAM along with an
appropriate capillary vasculature. Once in the GAM,
the fibroblasts encounter, take up, and transiently
express plasmid DNA. The GAM matrix has two
functions: it holds plasmid DNA in the wound site
(until cells arrive), and it acts as a scaffolding that
promotes cell ingrowth and the accumulation of
fibroblasts near the DNA. While in the matrix,
transfected fibroblasts act as bioreactors within the
wound site, producing plasmid-encoded proteins that
stimulate bone regeneration. In an initial published
study [32], GAMs were implanted into segmental
gaps created in the adult rat femur. Implantation of
GAMs containing beta-galactosidase or luciferase
plasmids led to plasmid DNA uptake and functional
recombinant enzyme expression by granulation tissue
fibroblasts. Implantation of a GAM containing either
a BMP-4 plasmid or a parathyroid hormone (PTH)
134 plasmid resulted in a biological response of
new bone filling the gap. Finally, implantation of a

two-plasmid GAM (BMP-4 plus PTH 134, which
act synergistically) caused new bone to form faster
than with either factor alone. This work showed for
the first time that new bone will form rapidly in vivo
following direct osteoinductive plasmid gene transfer
to fibroblasts involved in skeletal repair. Similar
results were achieved recently in a canine preclinical
model (unpublished).
The GAM approach is consistent with the field of
tissue engineering. As Vacanti and Vacanti have
stated, In essence, new functional living tissue is
fabricated using living cells which are usually associated in one way or another to a matrix or scaffolding
which can be natural, man made, or a composite of
both. The living cells can migrate into the implant
after implantation or can be associated with the
matrix in cell culture after implantation. [140].
Certainly, this definition of tissue engineering is
consistent with the proposed mechanism of action of
GAM gene transfer. GAM constructs are also relevant to the field of tissue engineering in that a
specific biological activity (osteogenic plasmid
DNA) is designed directly into a biomaterial. Recent
data from a canine preclinical model (unpublished)
showed that GAM plasmid gene transfer lasts for at
least 6 weeks in vivo. The sustained local delivery of
osteogenic molecules by repair cells represents a
distinct advantage of gene therapy over recombinant
protein therapies. Finally, given that the method
involves gene transfer into well-vascularized granulation tissue, it will be of interest to see if GAM
implants will mitigate against the challenge of
central ischemic necrosis.
To our knowledge, the study by Fang et al. [32]
was the first to show that PTH 134 may have a
local osteogenic effect when delivered via a GAM
implant. PTH 134 actually has many interesting
attributes as a potential bone growth promoting
therapy. Endogenous PTH is an 84 amino acid
systemic calcium-regulating hormone that binds to
the PTH-1 [PTH / PTH-related protein (PTHrP)] receptor in target tissues of bone and kidney [157].
Knowledge that PTH can stimulate bone formation
has been recognized for more than 50 years ( [158];
see also Refs. [32,159179]). The mechanism of
action of this effect remains uncertain [180], however, even though several theories have been pro-
posed (e.g., Refs. [158,165,181]). The PTH-1 receptor is a member of a large family of G-proteinlinked, seven transmembrane domain receptors
[182,183]. (The PTH-2 receptor has been isolated
from brain [184], and there is direct evidence for
additional receptors that bind the PTH C-terminus
and the midregion of PTHrP. Nevertheless, the PTH1 receptor appears to be the main receptor for PTH
bioactivity in osteoblasts.) Circulating PTH levels
(1055 ng / l or 15 pM / l, [185]) are directly related
to serum calcium levels. PTH has a short halflife
(24 min.), which is similar to the halflife of
osteoinductive cytokines such as TGF-b and BMP,
which is adequate to bind PTH-1 receptors and
evoke biological responses. The major pathway of
PTH signal transduction appears to be mediated via
cAMP production [182,183]. It also is clear from the
literature cited above that the anabolic effects of
PTH are dependent on the dosage regime (intermittent is optimal) and that anabolic effects are dependent on the amino terminus of PTH (AA 134).
Future studies will be required to understand the
mechanism of the local osteogenic effects of the
PTH 134 peptide. These studies may support the
development of a safe and effective therapy capable
of stimulating rapid bone growth in humans. Of
course, considerable interest also exists in the relative osteogenic effects of other cytokine molecules
delivered by GAM implantation.
4. Concluding remarks: gene therapy for tissue

repair and regeneration
Certainly this is an exciting time in modern
medicine, in no small part because of the possibilities afforded by somatic gene therapy, which is a
logical extension of recent advances in molecular
biology and molecular medicine. The power and
reach of somatic gene therapy will be extended
considerably by advances in the human genome
project as well as technology advances that overcome some of the hurdles now faced. The GAM
approach is exciting to us because it may represent a
form of gene therapy that can take advantage of
recent advances in materials science and tissue
engineering, which should create broad opportunities
in the areas of wound repair and tissue regeneration.
63
Acknowledgements
We are grateful to our colleagues Jianming Fang,
Blake Roessler, Bev Davidson, Vinod Labhasetwar,
Laurie McCauley, Kevin Rice and David Mooney.
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Advanced Drug Delivery Reviews 33 (1998) 5369

Gene therapy for tissue repair and regeneration

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

used to locally deliver plasmid gene constructs into

2.2. Classes of disease under consideration

2.3. Inherited single gene disorders

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

the failure to include appropriate regulatory sequences in gene therapy vectors.

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

tumor cells [45]. Mutant adenoviruses have been

2.5. Cardiovascular disease

bypass surgery and percutaneous revascularization,

2.6. Infectious disease

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

genome, ribozymes that attack HIV RNA at specific

2.7. Vector systems

intensely studied. Among its advantages are the

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

duction of these vectors into cells, however, may

2.8. Current status of gene therapy clinical trials

transduced lymphocytes [16]. As a second example,

2.9. Future directions

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

3. Bone growth gene therapy

3.2. Clinical problems associated with fracture

The treatment of a complex fracture is designed to

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

and yet the outlook for a functional result seems

3.3. Synthetic materials as bone graft substitutes

3.4. Recombinant cytokine bone graft substitutes

in this process include platelets, inflammatory cells,

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

eventually is cleaved at an endoproteolytic cleavage

3.5. Tissue engineering

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

into orthopedic sites (cranial and femoral osteotomy

3.6. Gene therapy for fracture repair

new bone filling the gap. Finally, implantation of a

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

4. Concluding remarks: gene therapy for tissue

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

[14] D.S. Anson, R.A. Hock, D. Austen, et al., Towards gene

formation by direct transfer of osteoinductive plasmid genes,

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

of vascular smooth muscle cell proliferation and neointimal

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

[84] Y. Yang, F.A. Nines, K. Berencsi, E.E. Furth, E. Gonczol,

chains of type I procollagen produce osteogenesis imperfecta

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

cloning and developmental expression, Biochim. Biophys.

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

[153] W.C. Puelacher, D. Mooney, R. Langer, J. Upton, J.P.

J. Bonadio et al. / Advanced Drug Delivery Reviews 33 (1998) 53 69

Raicz, G.A. Rodan (Eds.), Principles of Bone Biology,

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