Many of the presently approved somatic gene therapy protocols involve reimplantation of genetically engineered autologous cells into a patient. A potentially more cost-effective approach to the delivery of therapeutic gene products is the use of a universal recombinant cell line that can be implanted into a number of patients with the same product requirements. Enclosure of these non-autologous cells inside a permselective microcapsule membrane would permit the diffusion of the recombinant product but prevent entry of the host’s immune mediators. The clinical efficacy of this approach has been demonstrated by the implantation of recombinant fibroblasts and myoblasts to correct mutant phenotypes in murine models of diseases such as dwarfism (Al-Hendy et al., 1995) and lysosomal storage disease (Bastedo, 1994). In the first part of this thesis, a new microcapsule type was created that incorporated a combination of traits from both alginate-poly-L-lysine-alginate and barium-alginate microcapsules. The new, barium-poly-L-lysine-alginate microcapsule was cross-linked with BaCl2 and received a poly-L-lysine, and a second alginate coat. The three different types of microcapsules were compared with respect to encapsulated cell viability, proliferation and secretion in vitro. Results of these analyses demonstrated that cells inside alginate-poly-L-lysinealginate microcapsules had higher viability and a greater proliferation rate than did cells inside either barium-alginate or barium-poly-L-lysine alginate microcapsules. However, secretion from the alginate-poly-L-lysine alginate microcapsules was lower than from either of the barium-alginate types, and the two types of barium-alginate microcapsules, formulated with a higher alginate concentration, were more resistant to well-defined fluid shearing forces, than was the calcium alginate microcapsule. No significant difference in any of the parameters measured was observed between the barium-alginate and bariumpoly-L-lysine alginate microcapsule types.
In the second part of this thesis the three different types of microcapsules, each containing canine MDCK cells secreting ~20 ng/106 cells/hr of human growth hormone (hGH) were implanted into the peritoneal cavities of a large animal model. The microencapsulated cells were able to deliver recombinant human growth hormone to the circulation of dogs at levels nearly 100 % higher than human physiological levels. In contrast, implantation of unencapsulated recombinant cells resulted only in short-term delivery of hGH to the dogs. The level of titre of anti-hGH antibodies was monitored in the experimental and control animals, and its increase was determined to be associated with the disappearance of the human growth hormone from the circulation of the dogs. The BaCl2 cross-linked capsules with the higher alginate concentration lasted longer in vivo, confirming their superior mechanical integrity relative to the alginate-poly-L-lysine alginate type. The presence of the microcapsules in the peritoneum of the dogs was associated with localized inflammation of the omentum, and mild lymphadenitis. This pathology, combined with varying degrees of fibrotic overgrowth of the microcapsules with increasing time in vivo, suggests that modifications must be made in order to improve the biocompatibility of alginate microcapsules.
In conclusion, modifications of alginate microcapsules, such as the cross-linking with barium cations, the use of higher alginate concentrations and lamination with polyL-lysine alginate have contributed to the mechanical stability of the capsules and permitted the long-term delivery of recombinant gene products using non-autologous cells. This study has highlighted some of the issues to be addressed during pre-clinical studies in large animal models, in order to determine the efficacy of this new technology for humans. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/30038 |
| Date | 09 1900 |
| Creators | Peirone, Michael |
| Contributors | Chang, P. L., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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