Periodic Reporting for period 2 - EYELETS (A regenerative medicine approach in diabetes.)
Berichtszeitraum: 2021-07-01 bis 2022-12-31
Diabetes mellitus has reached pandemic proportions and in Europe and besides the personal suffering the disease represents a substantial economic burden to society. Diabetes develops when the supply of insulin by pancreatic islets is insufficient to maintain blood glucose homeostasis. In insulin-dependent diabetes islet transplantation is considered a promising therapy. However, the outcome varies due to transplantation site, quality of islets and the fact that transplanted islets are affected by the same challenges as in situ islets. Tailor-making islets for transplantation by tissue engineering combined with a more favorable transplantation site that allows for both monitoring and local modulation of islet cells is thus instrumental. We have established the anterior chamber of the eye (ACE) as a favorable environment for long term survival of islet grafts and the cornea as a natural body window for non-invasive, longitudinal optical monitoring of islet function. ACE engrafted islets are able to maintain blood glucose homeostasis in diabetic animals. Tissue engineering of native islets is technically difficult. We will therefore apply genetically engineered islet organoids. This allows us to generate i) standardized material optimized for transplantation, function and survival, as well as ii) islet organoids suitable for monitoring (sensor islet organoids) and treating (metabolic islet organoids) insulin-dependent diabetes. Our overall aim is to create a platform allowing monitoring and treatment of insulin-dependent diabetes in mice that can be transferred to large animals for validation.
The overall objective of the present proposal is to design a novel strategy to settle the so far unmet scientific/clinical problems currently associated with islet transplantation. This will be achieved by taking a regenerative medicine approach involving tailor-making islet organoids by tissue engineering, transplanting these organoids to the ACE, a site that allows for both monitoring and local modulation of the graft and combine this approach with synthetic biology and the development of novel microelectronic/micro optical readout systems for islet cells.
The overall objective of the present proposal is to design a novel strategy to settle the so far unmet scientific/clinical problems currently associated with islet transplantation. This will be achieved by taking a regenerative medicine approach involving tailor-making islet organoids by tissue engineering, transplanting these organoids to the ACE, a site that allows for both monitoring and local modulation of the graft and combine this approach with synthetic biology and the development of novel microelectronic/micro optical readout systems for islet cells.
Aim 1. Sensor islet organoids for monitoring function and survival of metabolic islet organoids. The objective is to establish i) sensor islet organoids that monitor islet/β-cell function and survival, and ii) methodologies to efficiently engineer islet organoids to treat insulin-deficient diabetes. Sensor islet organoids are equipped with genetically engineered fluorescent biosensors that report on the status of metabolic islet organoids. So far, we equipped islet organoids with biosensors that report on changes in cytoplasmic free Ca2+ concentration, stimulus-induced gene transcription, and insulin resistance. Our studies validated the ‘sensor islet organoid’ approach for non-invasive, longitudinal monitoring of islet cell/β-cell function and survival in diabetic animal models. Established methodologies for genetic engineering of islet organoids include adenoviruses and adeno-associated viruses as gene delivery systems, inducible expression systems as well as protocols to down-regulate gene expression by siRNA or temporarily activate expression of endogenous genes by saRNA.
Aim 2. Islets organoids for treatment of insulin-dependent diabetes. The objective is to generate islet organoids that are suitable to treat insulin-dependent diabetes. As proof-of-concept we modify islet organoids to downregulate their expression of the beta-3 subunit of voltage-dependent L-type calcium channels. We have shown that its downregulation in islets improves function and survival when transplanted to diabetic mice. Other targets are downregulation of apolipoprotein CIII or activation of VIP and ectopic expression of GLP-1. To establish ‘metabolic transplantation’ to the ACE to treat insulin-dependent diabetes we are currently evaluating the number of islet organoids needed to treat insulin-dependent diabetes in a streptozotocine-diabetes mouse model and compare that to the number of genetically modified islet organoids.
Aim 3. Local intraocular treatment strategies for the modulation of islet organoid function. The objective is to combine metabolic transplantation with local ocular pharmacological treatment strategies. We showed that local intraocular administration of dexamethasone implants improves survival and function of islet allografts transplanted to the ACE of non-human primates. In addition to local immunosuppression, an eye-drop application of doxycycline allowed us to modulate gene expression in intraocular islet organoids.
Aim 2. Islets organoids for treatment of insulin-dependent diabetes. The objective is to generate islet organoids that are suitable to treat insulin-dependent diabetes. As proof-of-concept we modify islet organoids to downregulate their expression of the beta-3 subunit of voltage-dependent L-type calcium channels. We have shown that its downregulation in islets improves function and survival when transplanted to diabetic mice. Other targets are downregulation of apolipoprotein CIII or activation of VIP and ectopic expression of GLP-1. To establish ‘metabolic transplantation’ to the ACE to treat insulin-dependent diabetes we are currently evaluating the number of islet organoids needed to treat insulin-dependent diabetes in a streptozotocine-diabetes mouse model and compare that to the number of genetically modified islet organoids.
Aim 3. Local intraocular treatment strategies for the modulation of islet organoid function. The objective is to combine metabolic transplantation with local ocular pharmacological treatment strategies. We showed that local intraocular administration of dexamethasone implants improves survival and function of islet allografts transplanted to the ACE of non-human primates. In addition to local immunosuppression, an eye-drop application of doxycycline allowed us to modulate gene expression in intraocular islet organoids.
In the course of addressing aim 1 (Sensor islet organoids for monitoring function and survival of metabolic islet organoids), we established sensor islet organoids equipped with biosensors reporting on changes in cytoplasmic free Ca2+ concentration, stimulus-induced gene transcription, and insulin resistance. Additional biosensors we are currently testing report on insulin secretion, glucose metabolism and β-cell replication. Until the end of the project we will within this aim further optimize organoid engraftment and function in vivo and establish novel methodologies for efficient genetic engineering of imageable sensor islet organoids and islet organoids for treatment.
Addressing aim 2 (Islets organoids for treatment of insulin-dependent diabetes) we will engineer islet organoids with improved functional quality and that are able to withstand the hostile milieu of the diabetic recipient. Until the end of the project we expect to be able to prepare ‘super’ islet organoids, as compared to normal primary islets, and to genetically equip them for improved function and survival in vivo. A major hurdle for a successful world-wide islet transplantation program is the limited amounts of transplantable high performing islets. To overcome this roadblock in the amount of available human islets as well as the variability in islet quality, our future attention will be focused on creating organoids originating from human stem/islet progenitor cells.
With regard to aim 3 (Local intraocular treatment strategies for the modulation of islet organoid function), we will use the advantage that the ACE represents as a transplantation site constituting a well-regulated confined space allowing for local ocular/intraocular treatment strategies. As proof-of-concept, we showed that intraocular administration of immune-suppressive drugs delays rejection of islet allografts. Until the end of the project we expect within this aim to be able to improve metabolic allograft survival, delay graft immune-destruction and modulate graft function.
The final ambitious overall future goal of this project is to establish a highly sophisticated and efficient clinical platform comprising genetically engineered human pancreatic islet organoids superior to normal islets for transplantation into the ACE of diabetic patients, reporter human islet organoids that specifically report on the status of the endocrine pancreas, and state of the art delivery and measuring systems for human islet organoids in the ACE.
Addressing aim 2 (Islets organoids for treatment of insulin-dependent diabetes) we will engineer islet organoids with improved functional quality and that are able to withstand the hostile milieu of the diabetic recipient. Until the end of the project we expect to be able to prepare ‘super’ islet organoids, as compared to normal primary islets, and to genetically equip them for improved function and survival in vivo. A major hurdle for a successful world-wide islet transplantation program is the limited amounts of transplantable high performing islets. To overcome this roadblock in the amount of available human islets as well as the variability in islet quality, our future attention will be focused on creating organoids originating from human stem/islet progenitor cells.
With regard to aim 3 (Local intraocular treatment strategies for the modulation of islet organoid function), we will use the advantage that the ACE represents as a transplantation site constituting a well-regulated confined space allowing for local ocular/intraocular treatment strategies. As proof-of-concept, we showed that intraocular administration of immune-suppressive drugs delays rejection of islet allografts. Until the end of the project we expect within this aim to be able to improve metabolic allograft survival, delay graft immune-destruction and modulate graft function.
The final ambitious overall future goal of this project is to establish a highly sophisticated and efficient clinical platform comprising genetically engineered human pancreatic islet organoids superior to normal islets for transplantation into the ACE of diabetic patients, reporter human islet organoids that specifically report on the status of the endocrine pancreas, and state of the art delivery and measuring systems for human islet organoids in the ACE.