Feasibility of percutaneous gene transfer to an atrophic nonunion in a rabbit.

Treatment of atrophic nonunions is a challenge to orthopaedic surgeons. Growth factors potentially are valuable factors for improvement of tissue healing. The use of growth factors, however, is limited by their short half-lives. Gene therapy has the potential to improve the treatment. This study aimed to establish and validate an atrophic nonunion model in a rabbit for the use of a percutaneous in vivo gene therapy protocol. An atrophic tibial nonunion was established in 24 New Zealand White rabbits. Radiologic and histologic followup was for 64 weeks. The rabbit tibias showed no radiologic or histologic signs of healing. In addition, an adenoviral vector carrying a marker gene was injected percutaneously into the nonunion site in 12 rabbits. Expression of the marker gene was assessed for as many as 4 weeks. The percutaneous gene delivery resulted in transgene expression in the nonunion site for as many as 4 weeks. The described model reliably leads to an atrophic tibial nonunion in rabbits. Adenoviral percutaneous gene delivery into the nonunion site is feasible and leads to transgene expression locally for at least 1 month. This study provides investigators with a reliable and reproducible model of an atrophic nonunion.

Gene transfer to the tendon-bone insertion site.

This study investigated whether gene transfer to the tendon-bone insertion site is possible during early tendon-transplant healing using viral vectors. In addition, we evaluated the optimal gene delivery technique for an in vivo adenoviral gene transfer to a tendon-bone insertion site in a bone tunnel. Twenty-six rabbits underwent a bilateral transfer of the flexor digitorum longus tendon into a bone canal in the calcaneus. The animals were divided into two groups. The first group (n=18) received a direct injection of an adenoviral vector carrying the luciferase marker gene into the tendon on the left side, while on the right side the adenoviral vector was first injected into the bone trough and the tendon was later inserted into the trough. The analysis of this experiment showed that over a 4-week period a higher luciferase activity was achieved using the bone trough immersion technique. In the second group (n=8) we therefore used the qualitative marker virus (Ad/-LacZ) with the bone trough immersion technique in order to show the site of gene expression. The histological analysis of this experiment demonstrated the presence of beta-galactosidase positive cells within the tendon-bone interface over a 4-week period. Therefore we showed in the first part of this study that the bone canal provides a more efficient target for direct adenoviral gene delivery than the tendon. In the second part of the study we demonstrated the feasibility of the bone trough immersion technique since sustained gene expression within the tendon-bone interface was obtained for up to 4 weeks. This study has shown the feasibility of gene delivery to the tendon-bone interface and provides the basis for the application adenoviral delivery of growth factor genes to the tendon-bone insertion site.

Tissue distribution of liposome-mediated epidermal growth factor receptor antisense gene therapy.

Despite the widespread use of liposome-mediated gene transfer in cancer therapy protocols, little is known about the tissue distribution of intralesionally administered DNA. We have previously shown that antisense gene therapy targeting the epidermal growth factor receptor (EGFR) inhibited tumor growth in a human head and neck squamous cell carcinoma (HNSCC) xenograft model. Further investigation demonstrated lack of systemic toxicity with intramuscular or intratumoral administration of this liposomal-DNA complex. In the present study, we compared two approaches to determine the presence of exogenous DNA in the plasma and tissues of mice treated with intramuscular injection of EGFR antisense gene therapy. PCR analysis using genomic DNA plus plasmid DNA as template was 83-fold more sensitive than PCR using a mixture of total RNA and plasmid DNA as template. With the more sensitive method (able to detect fewer than 500 molecules of EGFR antisense DNA in 1 microg of genomic DNA), foreign DNA was detected in all organs up to 1 month following a single injection. In contrast, using RNA plus plasmid DNA as template, exogenous DNA was only detected at the injection site at 1 week, and was undetectable at 1 month. Optical imaging studies demonstrated plasmid DNA only at the injection site. Although less sensitive than PCCR, Southern blot hybridization showed no evidence of integration of foreign DNA into the host genome in vitro or in vivo. These results emphasize the importance of defining the assays used to detect foreign DNA and suggest that the ability to detect intralesionally administered liposomal gene therapy, in organs distant from the injection site, is directly correlated with the sensitivity of the method employed.