Advanced Drug Delivery Reviews 44 (2000) 185194

www.elsevier.com / locate / drugdeliv

Tissue engineering via local gene delivery:

Update and future prospects for enhancing the technology
Jeffrey Bonadio*
Selective Genetics Inc., 6046 Cornerstone Court West, Suite 107, San Diego, CA 92121, USA
Received 19 April 2000; accepted 20 July 2000

Abstract
This review describes the status of a local plasmid-based gene transfer technology known as the gene activated matrix
(GAM). Studies over the past 6 years suggest that GAM may serve as a platform technology for local gene delivery in the
wound bed of various tissues and organs. These studies demonstrated that plasmid encoding genes can be delivered to
acutely injured tendon, ligament, bone, muscle, skin and nerve. Moreover, direct in vivo transfer of therapeutic plasmid
encoding genes in bone, skin and nerve was associated with a significant regenerative response relative to sham controls. The
review also describes new technology that should enhance the potential of local gene delivery in a manner consistent with
the riskbenefit profile associated with tissue engineering applications. 2000 Published by Elsevier Science B.V.
Keywords: Wound healing; Gene transfer; Gene activated matrix; Gene therapy vectors; Regulated gene transfer

Contents
1. Gene activated matrix (GAM) technology Update.................................................................................................................
2. Future prospects for enhancing the GAM technology .................................................................................................................
3. Summary ................................................................................................................................................................................
Acknowledgements ......................................................................................................................................................................
References ..................................................................................................................................................................................

1. Gene activated matrix (GAM) technology

Update
Wound healing in mammals is a highly evolved
and highly conserved process [1]. The cells that
participate in wound healing include platelets, acute
inflammatory cells, macrophages, fibroblasts, endo-

*Tel.: 1 1-858-625-8414; fax: 1 1-858-455-6822.

E-mail address: jbonadio@earthlink.net (J. Bonadio).

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thelial cells, pericytes and tissue-specific progenitor

cells. To co-ordinate the cellular response, cytokines
and growth factors act locally through wound-specific signal transduction cascades. The typical response to acute tissue injury initially focuses on
controlling hemorrhage and removing damaged cells.
Repair then begins with the formation of granulation
tissue and ends with scar formation or tissue regeneration. This injury-response sequence occurs following traumatic tissue injury (bone fracture),
pathology-induced injury (tissue necrosis following

0169-409X / 00 / $ see front matter 2000 Published by Elsevier Science B.V.

PII: S0169-409X( 00 )00094-6

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J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

infection), and iatrogenic injury (tissue injury associated with surgical procedures).
In the US alone, billions of dollars are spent
annually on the repair and replacement of human
tissues and organs [2]. Therapeutic strategies based
on bridging of lesions with biomaterial scaffolds
(that promote cell migration, proliferation and differentiation) have shown only limited success [3].
More recently, tissue engineering has presented an
alternative strategy based on transplantation of neoorgan constructs consisting of endogenous stem /
progenitor cells grown ex vivo within biodegradable
scaffolds [4]. The scaffold is resorbed over time, so
that the neo-organ eventually will consist of transplanted cells and the stroma they produce. Generally,
several weeks ex vivo are required to generate a
neo-organ; the neo-organ strategy therefore is most
appropriate for chronic tissue injury applications,
examples of which include replacement of defective
hyaline cartilage in osteoarthritis and repair of
diabetic skin ulcers.
Even though they are rational drug targets, the
effort to develop cytokine and growth factor
therapies for acute tissue injury has not yet shown
significant clinical benefit in excess of demonstrated
safety risks [5]. Toward this end, a second tissue
engineering strategy involves sustained local delivery
of recombinant cytokines directly into the wound
bed. Such a strategy would be expected to confer
both significant safety and efficacy advantages and
potentially lower costs and improved patient compliance [6]. However, the large size and fragile
three-dimensional structure of many recombinant
proteins has slowed clinical implementation of sustained local delivery technology [6,7]. Sustained
release systems are commonly based on microspheres made of poly(lactide-co-glycolide). The process to generate such a system typically involves
sonication as well as exposure to organic solvents,
high temperature and high concentrations of surfactants. These conditions may promote therapeutic
protein degradation, and in turn, decreased potency
and increased risk of immune toxicity. Moreover,
proteins successfully incorporated in microspheres
may undergo irreversible aggregation / deamidation /
oxidation (as a result of elevated levels of moisture
and the evolution of harsh microclimates), leading to
loss of full bioactivity. With few exceptions [810],

therefore, it has been difficult to maintain full

bioactivity of systems designed to sustain the release
of recombinant cytokines and growth factors. As a
consequence, dosage forms consisting of milligram
amounts of recombinant cytokine have been required
to show significant efficacy advantages in pre-clinical model systems and human clinical trials [11].
Product prototypes with this amount of a biologic
substance are expensive to manufacture and present
an additional safety risk: high-dose local delivery has
been associated with cytokine diffusion from the
wound bed into the bloodstream (dose dumping).
The major concern with dose dumping is that too
little of the cytokine drug will be delivered to the
diseased tissue to be effective, while too much of the
cytokine will be delivered to bystander tissues
(expressing cognate cell surface receptor) to be safe.
Taken together, the challenges identified above
provide a mechanism for the relatively narrow
therapeutic window shown by many wound healing
cytokines and growth factors in vivo [5]. Since 1993,
my research group has explored the possibility that
these delivery barriers could be overcome if cytokines and growth factors were delivered not as
recombinant proteins but as plasmid-genes [12]. An
important assumption was that, following gene transfer, the recombinant cytokine would be expressed at
more nearly physiological levels but for a prolonged
period of time by local wound healing cells. This
pattern of expression was expected to avoid local
toxicity. Moreover, plasmid diffusion from the delivery site was not expected to cause systemic toxicity
because of the high efficiency of DNA catabolism in
the bloodstream [13] and the low transfection efficiency of plasmids in normal (non-wounded) tissue
[14]. Additional potential advantages included the
fact that plasmid DNA was inherently stable and
flexible, and therefore likely to be compatible with
established sustained delivery systems. Finally, we
assumed that plasmid DNA could be manufactured
using simple protocols [15] that would be expected
to yield kilogram amounts of sterile clinical-grade
material on a routine basis at significantly less cost
than the typical bulk protein reagent.
We eventually developed a local gene delivery
system referred to as the gene activated matrix or
GAM [16]. At its most basic, a GAM consists of two
ingredients: plasmid DNA and a biodegradable struc-

J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

tural matrix carrier. The GAM matrix carrier serves

as a scaffold that holds DNA in situ until endogenous wound healing fibroblasts arrive. Once transfected, fibroblasts in the matrix carrier act as local in
vivo bioreactors, secreting plasmid-encoded proteins
that augment tissue repair and regeneration. This is
an unusual method of DNA delivery to cells. In
traditional gene delivery, the drug substance finds
the target cells. In a GAM, on the other hand, the
target cells find the DNA drug. Therefore, GAMs
do not follow a drug delivery paradigm in the
traditional sense of this term. By taking advantage of
the natural propensity of granulation tissue to grow
into the wound, GAMs allow for the physical
targeting of repair fibroblasts and other cells for
direct in vivo plasmid gene transfer.
GAMs have provided unexpected levels of transfection in vivo. Using a collagen sponge GAM
formulation, 2050% of available canine wound
healing cells were reproducibly transfected 3 weeks
after implantation, as determined by semi-quantitative substrate utilization assays and immunohistochemistry [17]. Plasmid-gene retention and expression (RNA) by fibroblasts was documented for at
least 6 weeks, and 1.0 mg of plasmid DNA yielded
picogram amounts of recombinant peptide expressed
over at least a 2-week period. Similar results with
collagen-plasmid DNA formulations were reported
recently by independent groups [18,19]. The molecular basis of local gene transfer via GAM is largely
unexplored at this point. However, the efficiency of
GAM gene transfer may well be influenced by
biological factors (e.g. high-level mitotic activity of
wound healing cells) as well as physical factors (e.g.
GAM matrices provide a large surface area for gene
transfer) (see [16,20,21] for related references).
The mechanism of action of GAM plasmid-gene
transfer is tied to the normal sequence of events
associated with wound healing. Given the near-universal nature of the wound healing sequence, it was
hypothesized that physical targeting of repair cells by
GAM would occur in fresh wounds of various tissues
and organs and across species. In support of this
idea, direct GAM plasmid-gene transfer to repair
cells has been reported in both rat and canine model
systems and in bone [17,22], skin [23], tendon /
ligament [24], heart / skeletal muscle [25], and cranial
nerve [26]. In these and other experiments, GAM

187

formulations have utilized collagen (i.e. as a lyophile

sponge, paste and gel), hyaluronan, alginate, carboxymethylcellulose, and poly(lactide-co-glycolide)
(i.e. as a sponge and a medical device coating) as
matrix carriers of plasmid DNA.
To assess regeneration potential, the first GAM
feasibility study involved direct plasmid-gene transfer to repair cells participating in bone repair in the
adult rat [22]. GAMs with either a bone morphogenetic protein-4 plasmid or a plasmid coding for
a secreted fragment of parathyroid hormone (PTH)
induced in a biological response of new bone filling
the defect. In a second experiment, implantation of
two-plasmid GAMs encoding both bone morphogenetic protein-4 and the PTH peptide, which act
synergistically in vitro, caused new bone to form
faster than with either factor alone. This study
demonstrated for the first time that repair cells in
bone could be genetically manipulated in vivo.
Bonadio et al. [17] further investigated potency,
using canine bone regeneration as the endpoint in
vivo. To regenerate bone, GAM implants were again
formulated with a plasmid-gene encoding PTH peptide in a collagen sponge. A DNA dose-escalation
design was employed using a critical-sized defect
(2.0 3 0.8 3 0.8 cm) in the beagle tibia. Bone regeneration was not induced at lower DNA doses of
1.020.0 mg per defect. In contrast, a significant
bone regenerative response relative to controls was
observed in all dogs receiving a higher dose of 40.0
and 100.0 mg DNA. In progressively smaller defects,
GAMs containing 100.0 mg DNA induced new bone
formation and complete healing over a period of 6
months. Altogether, it was concluded that local PTH
expression induced the growth of centimeters of
normal new bone in a safe, stable, and reproducible
manner that was both dose- and time-dependent.
Beyond the effort to regenerate bone, Shea et al.
[23] investigated the potency of GAM plasmid-gene
delivery from a sustained-release polymer matrix in
rat skin. A high-pressure gas foaming process,
developed in the laboratory of D. Mooney, efficiently
incorporated supercoiled DNA into a three-dimensional porous matrix. Cumulative release of DNA
incorporated in the matrix occurred over a monthlong period in vitro. Following implantation in rat
dermis, in vivo delivery of a plasmid encoding
platelet derived growth factor (PDGF-B) led to a 3-

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J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

to 4-fold increase in granulation tissue relative to

sham controls. This result was contrasted with direct
injection of the PDGF-B plasmid, which did not
significantly enhance local tissue formation, a result
that emphasized the utility of the matrix carrier.
Finally, Berry et al. [26] investigated the potency of
GAM plasmid-gene delivery to sites of cranial nerve
injury. When GAMs consisting of plasmid DNA in a
collagen paste were placed between the proximal and
distal ends of severed rat optic nerve, DNA was
taken up and transported in retrograde fashion to the
nerve cell body in the retina. The investigators also
showed that plasmid-gene uptake was enhanced by
linking recombinant FGF2 to plasmid DNA. Following uptake, production of DNA, RNA, and recombinant protein in the retina was demonstrated. Moreover, GAMs with growth factor plasmid-genes promoted retinal ganglion cell survival for more than 3
months after injury relative to controls.
Taken together, these studies demonstrate that
wound healing fibroblasts can be genetically manipulated via GAM in vivo in a safe, reproducible and
effective manner; that plasmid DNA can be delivered
to fibroblasts in skin from a biodegradable tissue
engineering matrix; and that plasmid delivery from a
matrix can be targeted to specific cell populations in
nerve via growth factors such as FGF2. Similar
results with collagen-based formulations were reported recently by independent groups [18,27].

2. Future prospects for enhancing the GAM

technology
Compared with diseases such as cancer and AIDS,
tissue engineering applications generally have a low
clinical riskbenefit ratio [16], and the safety of new
tissue engineering technology must be well established before use in man. The initial development of
the GAM technology was guided in part by a risk
benefit consideration such as this; the first GAM
formulations to be tested in humans will likely
consist of ingredients with a well-established record
of safety (i.e. plasmid DNA in a neutralized, bovine
collagen matrix). However, plasmid DNA is a relatively inefficient gene therapy vector whose application may be limited by pharmacoeconomic (i.e.
medical reimbursement) considerations. Therefore,

what opportunities exist for significantly enhancing

the efficiency of local gene delivery beyond what has
been achieved thus far? What are the relative risks of
deploying these new technologies?
Potential barriers to efficient gene delivery and
expression involve degradation and inactivation of
the vector upon exposure to blood [2831], poor
vector attachment to plasma membranes [32], nonspecific vector internalization, vector degradation in
endosomes [33,34], poor vector nuclear localization
[3537], and vector degradation in the nucleus [38].
Synthetic gene delivery vectors [39] are molecular
conjugates that consist either of DNA and cationic
polymers or peptides (polyplexes) or DNA and
cationic lipid (lipoplexes). Considerable effort has
been expended in the design of synthetic gene
delivery vectors that overcome one or more of the
barriers to efficient gene delivery [4043]. For
example, cationic dendrimers, peptides, proteins and
lipids bind non-covalently to plasmid DNA, providing a mechanism to increase plasmid stability and
gene transfer efficiency [3337,4453]. Properly
formulated DNA / cationic carrier complexes consist
of particles that resist degradation during formulation
in vitro (e.g. induced by sonication) as well as
degradation in vivo by serum nuclease [54]. Control
of the surface properties of complexes with PEG
may also generate particles with a longer half-life in
vivo [30]. It is reasonable to expect that plasmid
DNA should be repelled at the plasma membrane
because of its size / structure / surface charge, but
DNA-cation complexes are positively charged and
therefore capable of binding plasma membranes
through non-specific electrostatic interactions [55].
The presence of a cell surface receptor ligand may
allow binding of complexes to specific populations
of cells. In this regard, bacteriophage-display technology promises to yield additional peptide-ligands
without the necessity of identifying a tissue-specific
receptor beforehand [56,57]. Cell specific promoter
elements are feasible. Finally, incorporation of
fusogenic peptides and pH-responsive polymers,
which mimic the endosome-disruptive properties of
viruses and toxins, represents an important strategy
to significantly increase gene transfer efficiency at
this step [5860].
The gain in gene transfer efficiency associated
with polyplex- and lipoplex-based strategies certainly

J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

can be impressive, although evidence suggests it is

less than an order of magnitude relative to what is
achievable with naked plasmid-gene vectors [61].
This advantage must be balanced against the general
tendency of cationic agents to cause tissue necrosis,
which can be dramatic when DNA / cationic carrier
complexes are delivered locally in high concentration. DNA-cation complexes may also disassemble shortly after in vivo gene delivery. Disassembly
in the extracellular environment of course will
significantly limit efficacy and may contribute to
local toxicity. While several successful strategies to
stabilize DNA / cationic carrier complexes have been
proposed, care must be taken so that the complex
will readily disassemble once in the nucleus or
else the transgene may effectively be sequestered
from the transcription apparatus [62]. Strategies
involving the use of chemical crosslinking agents
must also be deployed with care or the DNA
complex may bind irreversibly to the GAM structural
matrix.
Beyond the barriers described above, the efficiency of plasmid vectors may be limited because
of their short-lived persistence in the nuclei of
replicating repair fibroblasts. In this regard, plasmid
vectors have been developed that show impressive
mitotic stability and long-term gene expression. For
example, Lipps and colleagues [63,64] have shown
that a simian virus 40 origin of replication linked to a
scaffold / matrix-attached region (that specifically
binds the nuclear matrix and the chromosome scaffold of mammalian cells) allows sustained episomal
replication that is independent of the virally encoded
large T-antigen. This vector has been maintained in
culture as an autonomous, self-replicating, functional
episome for more than a hundred cell-generations.
More recently, transposon technology was used for
the non-homologous insertion of a plasmid-gene into
the genome of adult mammalian cells [65]. The
efficiency of this approach depends in part on the
efficiency of chromosomal transposition. In vivo,
transfection of 5 to 6% of mouse liver cells and
chromosome integration resulted in transgene expression at levels that were considered therapeutic in a
mouse model of hemophilia B for more than 5
months.
Looking forward, the potential advantages of
persistent expression of plasmid encoding therapeutic

189

genes that code for wound healing cytokines must be

weighed against the potential for oncogenesis, e.g.
either from sustained local growth factor expression
or because of insertional mutagenesis (a long-recognized safety issue that nevertheless is poorly quantified). A second concern involves the backbone of
standard plasmid vectors, which contain CpG motifs
[6672] capable of immune suppression as well as
lymphocyte activation (in a manner reminiscent of
endotoxin). Plasmid DNA administration to animals
was recently associated with development of pathogenic antibodies to DNA and renal disease, leading
to premature death [73].
Viral vectors, which maximize gene transfer efficiency, may be formulated with structural matrices
as alternative GAM formulations [16,27]. Viral vectors derived from retroviruses, adenovirus, and
herpes simplex virus have shown considerable potential for in vivo therapeutic gene delivery [74].
However, production and safety of these vectors has
proved to be problematical. Specifically, processes
for vector preparation and purification remain to be
established, may be costly, and may be scaled only
with considerable difficulty. Although designed to be
replication-defective, recombination between the
viral vector and helper virus on occasion have led to
unwanted by-products [75]. Furthermore, certain
viral vectors induce an immune response that diminishes efficacy and increases toxicity [7678].
Among a multiplicity of alternatives, promising viral
vector technology includes the recombinant adenoassociated viral (rAAV) vectors [79]. Small-scale
procedures are available, allowing the manufacture
of pure, sterile rAAV preparations at titers of . 10 12
particles / ml [80]. Additionally, rAAV particles are
physically stable and small (2025 nm), implying
that vector particles will traverse tissue barriers with
relative ease; the particles efficiently transduce both
dividing and non-dividing cells; and rAAV genomes
persist as integrated tandem repeats in chromosomal
DNA. Elimination of AAV coding sequences from
rAAV vectors prevents the generation of wild-type
helper virus and the possibility of an immune
response to viral gene products [81,82]. Together,
host chromosome integration and the absence of a
cytotoxic T lymphocyte response provide viable
mechanisms for establishing long-term transgene
expression: both marker and therapeutic transgenes

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J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

have been expressed constitutively for years in

skeletal muscle [8386] and brain [87] of immunocompetent animals. Moreover, the doseresponse of
rAAV vectors in pre-clinical animal models is linear
over a broad range of input vector. Consistent with
these observations, initial attempts to administer
rAAV2 to human subjects appears safe, with no
evidence of germline transmission, local tissue toxicity, or immune system perturbation [88]. Finally, it is
worth noting that rAAV2 vectors provide a mechanism to actively target wound healing fibroblasts,
given that the vectors bind to high-affinity FGF2 cell
surface receptors [8991] which are over-expressed
by wound healing fibroblasts.
In head-to-head comparisons in animal models,
rAAV appears to be 1 to 2 orders of magnitude more
effective than naked plasmid-gene vectors [83], an
exciting result because of the rigor and reproducibility of the experiments. Nevertheless, the great potential advantage of rAAV vectors must be balanced
against potential unwanted consequences associated
with vector integration (insertional mutagenesis) and
the fact that | 80% of the US population is immunized against parental AAV strains. Early results have
consistently shown that a humoral response to rAAV
does not interfere with transgene expression, even
upon vector re-administration [83,92]. On the other
hand, the consequences of chronic stimulation of
humoral immunity remain to be explored.
Finally, it is worth re-emphasizing that wound
healing occurs in both time and space, involving the
participation of heterogeneous populations of inflammatory, repair, and progenitor cells. Thus, GAMS
formulated with more than one gene [20] provide a
mechanism by which specific genes guide the behavior of specific cell populations. In theory, the
location of genes within scaffolds can be patterned
via printing technology [93]. Moreover, the bioavailability of different genes can be varied from scaffold
composites made from materials with different intrinsic dissolution rate constants. Another mechanism
to achieve the same end involves strategic positioning of microfabricated devices within scaffolds for
controlled DNA vector delivery (e.g. [94,95]). Finally, biopolymer matrices with a pre-design that
mimics structures in nature offer the potential to
convert the GAM matrix into an active participant in
wound healing, for example by influencing the
migration of wound repair cells.

3. Summary
DNA-based therapies for tissue regeneration blend
the technologies of gene therapy and tissue engineering. Local gene transfer technologies such as the
gene activated matrix (GAM) provide a mechanism
to passively target wound healing cells for sustained
production of therapeutic proteins. GAMs combine
DNA vectors with porous biomaterial scaffolds. The
scaffold fills the wound bed, holding DNA in situ
until endogenous wound healing cells arrive. Wound
healing cells migrate along the scaffold; once transfected, these cells essentially become local in vivo
bioreactors that produce the therapeutic cytokine /
growth factor encoded by the DNA vector. As
described here, first generation GAM technology
should be considerably enhanced by several exciting
developments in plasmid-gene and viral vector technology.
A key challenge is safety [96]. Important safety
parameters include the interaction between the genebased therapy and the tissue at the delivery site;
vector persistence and bioavailability; the interaction
between the gene-based therapy and the immune
system; and the pharmacokinetics and pharmacodynamics of the vector-encoded protein, both at the
site of delivery and system-wide.
It is hard to imagine a tissue engineering product
that does not involve regulated therapeutic gene
expression as a way to avoid toxicity and still
respond to the evolving nature of disease. To qualify,
a system conferring regulated gene expression should
feature low baseline transgene expression, a high
induction ratio, and positive control by a small
molecule drug. In this regard, promising technology
includes a system based on transgene expression of
chimeric human-derived proteins reconstituted into a
transcription factor complex following oral administration of rapamycin. The complex specifically drives
expression of a transgene coding for a therapeutic
protein. Proof of principle has been established
following somatic cell gene transfer with plasmid
vectors [97] as well as direct in vivo gene transfer
with rAAV [98]. Together, the studies demonstrate
that precise control of therapeutic protein expression
is achievable from cells engineered to respond to
rapamycin.
Local gene transfer technologies such as GAM are
early-stage and considerable work on their mecha-

J. Bonadio / Advanced Drug Delivery Reviews 44 (2000) 185 194

nism of action must be done before tissue engineering products will emerge into the marketplace.
Examples of relevant control parameters required for
both technology categories include the variation
associated with the site and extent of tissue injury;
the rate constants for cell proliferation-migration
within the porous biomaterial scaffold; gene transfer
efficiency; the rate constants for therapeutic protein
production by transfected cells; the pharmacokinetics
and pharmacodynamics of the vector-encoded protein; the safety factors (see above) that influence
gene transfer efficiency; and transgene silencing
[99]. The effort to understand these parameters
should provide a basis for reliable products that
significantly impact patient care.

Acknowledgements
To JCCJA.

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