Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 135

endocytosis, phagocytosis, pinocytosis and direct fusion may all contribute to the liposome-DNA
crossing the cellular barrier. 110 To facilitate uptake into the cell as endosomes, targeting pro-
teins have been included in liposomes, for example, anti-MHC antibody 111 and transferrin. 112
This method of introducing DNA has been used in clinical trials for the treatment of cancer
and cystic fibrosis, 113,114 lung, 115 brain, 116 tumor, 117,118 and skin 119 by local administration or
to vascular endothelial cells 120,121 after systemic, intravenous injection.
Cationic polymers include poly-L-lysine (PLL), poly-L-ornithine, polyethylenimine (PEI),
chitosan, and starburst dendrimer. In addition to the cationic nature of these polymers they
complex the DNA and condense it, both of these characteristics facilitate the endocytic uptake
of the DNA and increase the transfection efficiencies.
The low molecular weight PEI showed favorable results in in vitro and in vivo applications
and with the least toxicity among the cationic polymers group. It has been used successfully in
vivo to transfer genes in mice brain 122 and rat kidney. 123 Another desirable characteristic of
using PEI as a gene delivery vehicle is the capability of buffering the endosome/lysosome com-
partment, thus avoiding DNA degradation. Many parameters have been modified to optimize
the PEI transfection efficiency. Some of these parameters include: different PEI concentra-
tion, 124 PEI nitrogen:DNA phosphate ratios, 123 PEI molecular weight 125 and improved pack-
ing of PEI. 126
Immunogenicity of Plasmid DNA
Plasmid DNA complex can elicit an immune response and cells of the immune system
recognize the DNA as an extrinsic antigen. Phagocytosis by the mononuclear phagocytes ap-
pears to be the primary defense mechanism. Krieg et al 127 has reported that an unmethylated
CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines induces immune reac-
tions. This property has been used to improve genetic vaccination. However, when the purpose
of plasmid DNA delivery is to produce a therapeutic effect, the immune reaction to the
unmethylated CpG is a critical obstacle to the success of nonviral gene therapy. Efforts are
underway to engineer vectors that minimize the presence of the CpG motif.
Applications of Nonviral Vectors to Bone Healing
In vivo direct plasmid DNA delivery was successfully reported by Fang et al and Bonadio et
al. 128,129 The investigators coined the term gene activated matrices (GAM) for the methodol-
ogy they used. GAM consists of a biodegradable scaffold where the plasmid DNA is entrapped
and locally delivered to the wound site in a sustained manner. The released DNA will transfect
the recruited cells during the granulation phase of bone repair. The newly transfected cells will
regulate the expression of the transfect gene. To stimulate or enhance bone formation, the
choice of an osteoinductive gene is critical. Fang et al 128 delivered either a bone morphogenetic
protein-4 (BMP-4) plasmid or a plasmid coding for a fragment of parathyroid hormone (amino
acids 1-34) (PTH 1-34 ) in both cases, a biological response was shown. In addition, it appears
that when both BMP-4 and PTH 1-34 are delivered, bone formation occurs at more rapid rate
than either factor alone. Bonadio et al 129 used a tibial defect model where collagen and PTH 1-34
were delivered. The local delivery of PTH 1-34 was shown to substantially increase bone forma-
tion whereas the control containing only the matrix produced no bone.
Local gene delivery of plasmid DNA was also used in a spinal fusion model with PTH 1-34 as
the gene of choice. The authors reported significant bone infiltration in the experimental group
whereas the control showed moderate bone fill. Five of the seven specimens analyzed showed
anterior fusion mass.
In conclusion, non-viral gene delivery is showing great promise for therapeutic purposes.
Much research is underway to optimize the transfection efficiency. As mentioned above, there
are applications for which nonviral vector would be better suited. In particular, applications
that require a transient expression and repetitive administration of vectors. Repetitive adminis-
tration of viral vectors poses the risk of developing an immune response against the viral pro-
teins and, hence, the vector. Nonviral DNA delivery vehicles offer—at least in principle—a
nonimmunogenic alternative.