Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 131

viral replication. In order to grow the ∆ E1 and E4 adenoviral vectors, cell lines stably express-
ing both E1 and E4 have been generated. Given that E4 expression is toxic to cells, these stable
cell lines have to express the E4 region from an inducible promoter. Although second genera-
tion viruses can be propagated, their titers are usually lower than first generation viruses. Analysis
of gene transfer in vivo suggests that the toxicity and immunogenicity of the second generation
vectors has been reduced, allowing for longer term gene expression. However, the level of
transgene expression also appears to be reduced.
To generate adenoviral vectors that are even less immunogenic and toxic, third generation,
gutted vectors have been generated where all viral genes have been removed, leaving only the
viral terminal repeats and the packaging site needed in cis for viral replication and packaging.
In order to propagate the gutted vectors, a helper virus carrying the E2, E4 and late genes is
required. The difficulty with this type of vector is how to separate the recombinant gutted
vector from the helper viruses after propagation in the helper cell line. The use of packaging
compromised mutants in the helper virus have allowed for production of 3-4 logs more gutted
vector than helper virus, but more optimal production methods are still required.
Applications of Adenoviral Vectors to Bone Healing
Adenoviral vectors have been used to deliver genes encoding proteins able to enhance bone
healing in several different types of models. In addition, adenovirus has been used for both in
vivo gene transfer at the site of defect or ex vivo to modify cells in culture prior to implantation
into the defect. Currently, most studies have been performed with an adenovirus expressing
human bone morphogenetic protein (BMP) 2. Infection of a bone marrow stromal cell line
with Ad.BMP-2 followed by injection into large femoral defects resulted in enhanced and
improved healing. 39 Similarly, Ad.BMP-2 was used to infect muscle-derived stem cells that
were implanted into 5 mm diameter critical sized skull defect in the mouse, using a collagen
sponge. Expression of BMP-2 in the stem cells resulted in their differentiation into osteogenic
cells and improved healing of the critical sized defect. 40 In contrast to the modification of cells
in culture that are then implanted, several groups have directly injected the adenoviral vector.
Injection of an adenovirus carrying the luciferase marker gene into a segmental defect in a
rabbit femur resulted in expression only at the site of the defect and adjacent soft tissue. 41
Expression within the defect and surrounding muscle persisted for at least 6 weeks with no
expression observed in other tissues such as lung, liver and spleen. Injection of Ad.BMP-2
resulted in significant differences in healing as determined by radiological criteria, histology,
and biomechanical testing. 42 Taken together, these results suggest that adenoviral vectors are
well suited for transient delivery of therapeutic agents to either nondividing stem cells in cul-
ture or to cells within the defect in vivo. At least in animal models, there appears to be only
limited inflammation associated with direct injection of low doses of Ad.BMP-2. Thus aden-
oviral vectors may be well suited for orthopaedic applications where only transient, localized
gene expression is required to provide enhancement of the normal healing process.
Adeno-Associated Viral Vectors
Adeno-associated virus type 2 (AAV2) is a single-stranded DNA, nonenveloped, parvovirus
that has gathered much attention in the viral gene delivery arena. The only cis components
required to generate AAV2 vectors are the two 145-nucleotide inverted terminal repeats (for
review, see ref. 43).
AAV vectors have many advantages for gene therapy. First, they have no proven pathogenic-
ity in humans. 44 Secondly, they possess the capability of transducing a broad host of tissues, 45
although this could also be viewed as a liability in a bone fracture healing model. AAV vectors
transduce both proliferating and nonproliferating cells; 46,47 the DNA can be stably integrated
into cellular DNA and stable expression of the transgene was shown in animal models. 48 Low
risk of insertional mutagenesis, due to the weak activation of downstream genomic sequences
or intrinsic promoters by the inverted terminal repeats (ITR) was also noted. 44