Allogeneic transplantation as a replacement therapy for damaged or destroyed cells has potential in treatment of diseases such as type I diabetes or Parkinson's disease. Still, this procedure is limited by the need for chronic immunosuppressive therapy and the paucity of organ donors (16
). To overcome the shortage of available organ donors, one could potentially use pigs as an unlimited supply of transplantable tissue. Even so, the benefits of transplantation of porcine tissue, such as pancreatic islets, remain threatened by immunological destruction (10
). Engineering an immune-privileged cell, like the SC, that would survive transplantation without the need for immunosuppression to produce therapeutically relevant factor(s) is a novel approach to bypass these issues.
We previously explored the idea of genetically altering SC for transplantation by transplanting SC isolated from transgenic mice modified to stably express GFP (7
). These SC survived allogeneic transplantation and expressed GFP throughout the duration of the study (60 days) (7
). However, while this verified the immune-privileged status of genetically modified SC, it did not demonstrate their ability to either express a clinically relevant factor or to treat a disease. More recently, Triveldi et al. demonstrated short-term production of human neurotrophin-3 (hNT-3) by SC after allogeneic transplantation (30
). In that article, Lewis rat SC were transduced with an adenoviral vector that expressed hNT-3 under the control of the CMV promoter and transplanted as allografts to the injured spinal cord of Sprague-Dawley rats. Cell survival and hNT-3 production were examined. Allogeneic SC survived for at least 42 days, but hNT-3 production by these cells was only detectable for 3 days after transplantation. While these results support the concept of genetically modified SC surviving transplantation and expressing a transgene, they were unable to demonstrate in vivo function of the transgene.
In the current study, we explored the possibility of engineering SC to produce a therapeutically relevant protein; specifically, we modified them to express basal levels (nonglucose regulated) of insulin. SC isolated from neonatal pigs and pubertal mice were transduced with AdCMVhInsM and examined for the ability to express and secrete biologically active insulin. As shown in our results, these SC produced insulin mRNA and protein and secreted functional insulin. In addition, insulin was secreted at levels able to normalize blood glucose levels after transplantation into immune-compromised diabetic SCID mice. To the best of our knowledge, this is the first study to show genetically modified SC can express levels of a factor that can treat a disease in vivo.
Even so, in this study, the expression of insulin was transient. Because our current objective was to test the concept that SC can be genetically engineered as a vehicle for gene therapy, an adenoviral vector was used to drive insulin production. Use of an adenoviral vector as a delivery system was advantageous because of ease of use and the possibility of high levels of gene expression in the majority of transduced cells (26
). However, due to the epichromosomal location of the vector, combined with proliferation of the SC, we were only able to achieve transient gene expression (26
). There are currently other viral vectors and transgenic technologies (26
) that could be used to overcome the obstacle of transient gene expression and now that we have demonstrated that SC can express functional insulin, future studies will focus on creating stable gene expression in SC.
Another factor related to the transient decrease in blood glucose levels is that very large amounts of human insulin are needed to lower blood glucose levels in mice. If the amount of insulin secreted by the mSC or NPSC over 2 days of culture is converted to units of insulin and adjusted for the 20 million cells transplanted, this is equivalent to approximately 17 or 44 U/kg/day, respectively. This value seems excessive when compared to the amount of insulin typically used for treatment of humans with diabetes (0.5–1.0 U/kg). However, it is important to point out that human insulin has been shown to be less effective in rodents than in humans, where 20–40 U/kg was needed to normalize blood glucose levels in rats (21
). Taking this into consideration the amount of insulin secreted by the transduced mSC or NPSC and needed to lower blood glucose levels was similar to the previous reports.
The goal of gene therapy is to restore wild-type function in disease states by replacing defective or deficient proteins. While viral vectors are the most commonly used gene therapy method, because they generate sufficient expression of the gene of interest (26
), the use of these vectors raises additional concerns regarding stable gene expression, safety, toxicity, and immunogenicity (22
). As an alternative, generation of transgenic animals that express therapeutic factors could overcome the issues of transient gene expression and safety, but would still be subject to immune rejection. The use of SC, isolated from transgenic animals, engineered to express clinically relevant factors specifically in SC (using a SC specific promoter) presents a novel method by which gene therapy can overcome several of these issues, by providing long-term gene expression and an immune-privileged environment. Combining the results of the current study with our previous experiments using GFP transgenic mice (7
), our findings provide further support for this concept.
In theory, genetically modified SC could be designed to produce many different therapeutically relevant factors, such as insulin, glucagon-like peptide-1 (GLP-1), or factor VIII for the treatment of diabetes or hemophilia, respectively. The current study examined the production of insulin by SC after introduction of furin-modified human proinsulin cDNA. The addition of furin cleavage sites allowed SC to properly process and secrete functional insulin. While our goal was not to make this construct glucose responsive, but instead to provide proof of concept, this construct still has potential therapeutic value by providing continuous basal levels of insulin. According to a previous study, basal insulin production combined with conventional insulin treatment, results in near normal glucose metabolism without fasting hypoglycemia (4
). Glucose-regulated insulin secretion is also an area of great interest and active research, but is beyond the scope of this study. Currently, various methods such as glucose-regulated promoters and glucose-regulated production of furin have been investigated, but to date, no solution has been found (1
In this study, we demonstrate that both mouse and porcine SC can be altered to express a biologically active and therapeutically relevant factor, insulin, at levels applicable for the treatment of a disease. Combined with our previous results demonstrating long-term survival of allogeneic and xenogeneic SC (5
) and survival of allogeneic GFP-positive transgenic SC (7
), these data suggest that immune-privileged cells genetically modified to secrete clinically relevant proteins could be created as an unlimited source of tissue for transplantation. Future studies will examine the ability of these engineered cells to treat disease conditions after transplantation into immune-competent animals.