1085. In Vivo Imaging of Microencapsulated Gene Therapy
Authors: Anna Li, Donna Hou, Feng Shen, Eric P. Seidlitz, Patricia L. Chang, Murray A. Potter
Journal: Molecular Therapy (2006)

Microencapsulated cells engineered to secrete therapeutic proteins have been effectively applied in treating several genetic disorders such as dwarfism, lysosomal storage diseases, hemophilia, and cancer in mouse models. While various classes of microcapsules have been developed for use in this type of gene therapy, the most commonly used and studied have been the alginate-poly-L-lysine-alginate (APA) microcapsules. Once these microcapsules are administered to an animal, however, the fate of the implanted microcapsules and their engineered-cell payload can only be directly ascertained by surgical retrieval. Our group has previously established a novel method to quantitatively monitor the microcapsules in vivo with MRI by incorporating ferrofluid into the microcapsule. However there is limited availability of MRI for small animal studies and this technique does not provide information about the cells contained in the microcapsule. To overcome these limitations, we have developed a system to track the implanted microcapsules and their cells with in vivo bioluminescent imaging (BLI).We have developed a cell line expressing luciferase (pMONO- Luci & pC3B.sp.Luci with signal peptide) that allows us to monitor microcapsule integrity and encapsulated cell viability in vivo. BLI has become widely available as a research tool, making this technique more accessible than other imaging modalities. In our experiment, luciferase-expressing microencapsulated cells were imaged with a cooled charge-coupled device (CCCD) camera in vitro after exposure to luciferin substrate for 30 seconds, and in vivo after implantation into the peritoneal cavity of BalB/C mice aged 6–8 weeks (3 ml capsules per mouse in 2 ml saline). In vitro experiments showed that capsules loaded in 96-well-plate can be monitored individually and quantitatively, both for single or multi-layered loading. In vivo experiments confirmed that at various time points (4, 24, 48, 72, 96 hrs), with the injection of the substrate luciferin (injected i.p. at 150 mg/kg), the luciferase signal could be detected for the duration of the study (5 days). The strongest signal was seen at 4 hours post- implantation, with a subsequent fivefold decrease by 24 hours. The luciferase signal then increased consistently till the last time point of our study (96 hours) when the signal was 73% of the maximum. We postulate that the decrease of the luciferase signal from 4 hours to 24 hours post-implantation might be due to the initial diffusion of the expressed protein from the transfected cells into the peritoneal fluid and the blood. There was no indication of distribution of luciferase into the organs (liver, spleen, kidney, lung, heart, muscle and brain). The consistent increase of the signal from 24 hours to 96 hours likely reflects the proliferation of the encapsulated cells and increased expressed luciferase.Our data indicates that luciferase could be developed as a marker for microencapsulated cells to monitor the status of microcapsules and encapsulated cells after 24 hours post-implantation. Microencapsulated cells engineered to secrete therapeutic proteins have been effectively applied in treating several genetic disorders such as dwarfism, lysosomal storage diseases, hemophilia, and cancer in mouse models. While various classes of microcapsules have been developed for use in this type of gene therapy, the most commonly used and studied have been the alginate-poly-L-lysine-alginate (APA) microcapsules. Once these microcapsules are administered to an animal, however, the fate of the implanted microcapsules and their engineered-cell payload can only be directly ascertained by surgical retrieval. Our group has previously established a novel method to quantitatively monitor the microcapsules in vivo with MRI by incorporating ferrofluid into the microcapsule. However there is limited availability of MRI for small animal studies and this technique does not provide information about the cells contained in the microcapsule. To overcome these limitations, we have developed a system to track the implanted microcapsules and their cells with in vivo bioluminescent imaging (BLI). We have developed a cell line expressing luciferase (pMONO- Luci & pC3B.sp.Luci with signal peptide) that allows us to monitor microcapsule integrity and encapsulated cell viability in vivo. BLI has become widely available as a research tool, making this technique more accessible than other imaging modalities. In our experiment, luciferase-expressing microencapsulated cells were imaged with a cooled charge-coupled device (CCCD) camera in vitro after exposure to luciferin substrate for 30 seconds, and in vivo after implantation into the peritoneal cavity of BalB/C mice aged 6–8 weeks (3 ml capsules per mouse in 2 ml saline). In vitro experiments showed that capsules loaded in 96-well-plate can be monitored individually and quantitatively, both for single or multi-layered loading. In vivo experiments confirmed that at various time points (4, 24, 48, 72, 96 hrs), with the injection of the substrate luciferin (injected i.p. at 150 mg/kg), the luciferase signal could be detected for the duration of the study (5 days). The strongest signal was seen at 4 hours post- implantation, with a subsequent fivefold decrease by 24 hours. The luciferase signal then increased consistently till the last time point of our study (96 hours) when the signal was 73% of the maximum. We postulate that the decrease of the luciferase signal from 4 hours to 24 hours post-implantation might be due to the initial diffusion of the expressed protein from the transfected cells into the peritoneal fluid and the blood. There was no indication of distribution of luciferase into the organs (liver, spleen, kidney, lung, heart, muscle and brain). The consistent increase of the signal from 24 hours to 96 hours likely reflects the proliferation of the encapsulated cells and increased expressed luciferase. Our data indicates that luciferase could be developed as a marker for microencapsulated cells to monitor the status of microcapsules and encapsulated cells after 24 hours post-implantation.