Final Report Summary - XENOISLET (Macroencapsulated Porcine Pancreatic Islets to cure Diabetes Mellitus type 1/2)
Executive Summary:
Allogeneic transplantation is today the only successful therapy for several life-threatening diseases. However, organ donation only partially meets the demand and many patients still die while waiting for transplantation. Cellular transplantation could represents a very successful tool to treat type 1 diabetes mellitus (T1DM) by transplantation of human islets. Therefore, the scientific community has today reconsidered the possibility of using porcine cells to cure specific diseases by xenogeneic cellular transplantation.
XENOISLET had therefore several objectives: (1) to select and screen for pathogens a pig line source to go to clinical trials and as such to develop an unlimited source of pig insulin producing cells; (2) to produce genetically modified pigs for improving the pig islet function; (3) to dramatically change the monitoring and the follow-up of patients since NO immunosuppression will be used owing to alginate macro-encapsulation devices, and (4) to develop all the necessary tools for regulatory and safety procedures to use pig cells instead of human cells.
During this four year project, more than 150 pigs have been screened for PERV and eventually, around twenty pigs have been selected since PERV C negative and particular low PERV A/B level ( in term of expression and copies number). These pigs insure thus a breeding colony which is acceptable by the regulator for clinical use. In addition to this result, partners of the consortium developed a kit which allows to test these levels of PERV A/B expression or copies number by PCR. This kit will be available for the network in the future but will also be put on the market for others customer.
Then, a deep comparison has been made between Adult Pig Islet (API) and Neonate Pancreatic Pig Cluster cells (NPPCs) from unmodified pigs. The conclusion of several types of assays allowed confirm today that NPPCs are the most appropriate source of insulin producing pig cells. In parallel, the network has developed the appropriate QC sampling and measurement standard operating procedures (SOPs) for a standardized pancreatic islet product usable for clinical patients.
Having the pig line and knowing that NPPCs will be used, we also developed a project with a PME (Pig for live, Belgium) to build a facility which will allow to keep these pigs into a DPF facility (CER, Marche en Famenne, Belgium). This facility will be finished in May 2018 and ready to accept the pig breeders.
One of the major discovery of XENOISLET network is certainly the fact that although correction of diabetes has been regularly obtained with pig adult cells in preclinical models, we clearly know that pig islets provide a lower response than human beta cells to glucose challenge. We thus studied deeply the possible ways to significantly improve and amplify the production of Insulin by pig cells. One natural pathway by which the pig islet function could be improved to respond to glucose challenge in vivo was the GLP- 1 pathway. Additionally, we also discovered in pig that a specific muscarinic receptor (M3R) for the beta cells could also be activated to amplify the insulin production (M3R) and eventually, we showed in vitro and ex vivo that the activation of both GLP1 and the M3R receptor improved significantly the production of insulin by pig cells allowing almost to reach the same level of production than a human islet cell.
This major discovery in fact changed an important part of our project, since by knowing that a double transgenesis of the pig islet could significantly improve the function, then ethically, we needed to adapt our plan and we choose to develop and clone a NEW pig colony which will express both transgenes under an insulin promotor. This choice, however, delayed the project since we needed to produce the good pig
As today, and owing to a major effort of several members of the team, a pig line with a SINGLE INTEGRATION into the safe harbour V2G of the double transgene GLP1/M3R under an insulin promotor exists and is viable. The analysis of the pig islets of these pigs are in fact “super pig islets” since their production of insulin following a glucose challenge are 7/8 fold higher than unmodified animals.
The transgenic pigs will then be introduced into the NEW DPF facility in Belgium in May 2018 and then the first pig islets could be isolated for human use after matching all the regulatory guidelines needed.
Project Context and Objectives:
Project context and main objectives
The main objectives of XENOISLETS was therefore to undertake specific final steps enabling successful transplantation of pig insulin-producing cells into human T1DM patients by using alginate macroencapsulation and pig transgenesis. So during this four year time line, several major objectives were defined as following:
Selection of a pig line fulfilling the safety requirement of xenotransplantation of pig insulin-producing cells into humans.
After contact in 2013 with regulator in Belgium (AFMPS) and EMA (since two members of AFMPS are also EMA representative) several major issues were raised to specify the work to be done.
First, Pancreatic Pig insulin-producing cells (PPIs) will be used from Neonate pancreatic pig cells (NPPCs). In fact the latter could be easier to use for establishing standard of procedure (SOP) and maturation of these cells during 7/8 days will not be considered as a major modification, so that NPPCs are considered as a DRUG and therefore follow the National and EMA guidelines for drug therapy. But since we will use xenogeneic cells, it will also refer to a specific guideline edited by EMA about xenogeneic tissue or cells. Between 18 and 36 months, we have achieved more than 150 harvesting and isolation of NPPCs and we have now all the guidelines to obtain a very reproducible method which allows to obtain each time 25/ 30.000IEQ from one neonate animal with a good viability and no contamination. During this second/third period we also have studied several growth factors which allows to evidence that adult pig islets and neonate pigs islets have the same level of pig insulin production after three weeks of maturation. As a consequence, appropriate SOPs have been established and proposed to the regulatory agencies for the use of NPPCs islets in humans (Annex: AFMPS Minutes: MAY 2017).
Second, AFMPS clearly stated that the pig line should be characterized deeply for pathogens and that an accredited pig facility should be proposed, in order to raise and keep a pig colony breeding in DPF (designated pathogen free) conditions.
As such, a complete list of pathogens including viruses, bacteria, parasites, and fungi was proposed from the regulator authority which gathers all the pathogens analysed today for pig food additional to all pathogens which could be specific for XENOTRANSPLANTATION. This list is shown in WP7 and laboratories which may analyse these agents have been identified either in Belgium, UK or Italy. Although several pathogens may be detected in several basic laboratories, viruses should be deeply analysed and GCU/AVD have done for that a major work to specify PERV and other viruses in hundreds of pig samples send by AVT/UCL. The priority has been to screen pigs for low PERV-A/B and lacking PERV-C retrovirus dangerous loci. During this screening, both AVD and CGU have developed qualified and validated assays for pathogens. In fact, a commercial kit for PERV A,B,C has been developed by AVD and is now in a period of internal validation before to get access to the market. As such we defined criteria which should be respected to be proposed to regulator:
Our criteria for a suitable/acceptable donor animal, which exceeds that of Diatranz Otsuka Ltd (DOL) are as follows:
• PERV C env negative
• Less than 30 copies of PERV per genome
• Low PERV A and PERV B copy number
• Low level of expression in fibroblasts
• Null by in vitro cell culture
• Lack of pathogens specified in lists in report
During the project, we have studied whether the PERV expression is variable between PBMCs and several tissues as well as between the whole pancreas and the islet preparation (N.Mourad et al. Xenotransplantation, 2017). We confirmed in a very large number of samples (from at least 40 pigs) that the level of PERV expression is lower in the whole pancreas than in other tissues such as lung, kidney, spleen, serum. The level of expression and copy number in ISLETs is however, higher than in the pancreas but remains low in comparison with PBMCs or several other organs.
Today, we have selected several breeders both female and male which are PERV C negative and have a low level of PERVA/B. We have therefore two small colonies in both Cremona (AVT) and Belgium (UCL) and these animals are under reproduction to have a breeding colony in both places.
Third, the AFMPS also clearly stated that identification of a pig facility which is able to house a pig bred under DPF guidelines was needed. A solution has been also established in Belgium where a pig facility of 500m² will be built to reach the safety level of DPF at Marloie Center of Research (CER), Marche-en-Famenne, Belgium. As seen in WP6, this facility should be built for May 2018 and thus the first piglets will be introduced under sterile conditions after hysterotomy in June 2018 in a brand new facility. This facility will be used as a new company which has been created for this project and is named PIG for LIFE (P4L).With this small facility, we could raise 200 piglets per year in DPF conditions.
Now that we have decided that NPPCs will be used, all the SOP and QC have been adapted and written during the last year of the project. The in vitro culture period will allow us to implement all safety and quality control (QC) procedures necessary for the release of pig islet batches for the putative future transplantation in humans. The XENOISLET project will develop the safety and quality control methods via partners GCU and AVD who have developed the necessary tools (DNA arrays, traditional and digital PCR reagent kits) for identifying pig pathogens but mainly viruses which need to be controlled prior to clinical trials. BIOT has developed the appropriate QC sampling and measurement standard operating procedures (SOPs) for a standardized pancreatic islet product. This new approach might lower the requirement (and the costs) of having a DPF pig facility since the safety can be assured batch by batch examinations during the in vitro culture period prior to transplantation.
Production of a genetically engineered pig expressing GLP1 proteins and Muscarinic 3 receptor at the level of pig islets (under a pig insulin promoter)
Although correction of diabetes has been regularly obtained with pig adult cells, we clearly know that pig islets provide a lower response than human beta cells to glucose challenge. Indeed, a careful in vitro comparison between pig and human islets function clearly evidenced that while basal insulin secretion was not different, it was only in the presence of 1 μM forskolin or glucagon that glucose or amino acids could increase the secretory rate in pig islets to levels similar to those observed in human islets tested without forskolin. These observations and the improvement of insulin secretion by cAMP-raising agents suggest that low cAMP levels in pig β-cells may explain the poor insulin secretion. In addition, a structural hypothesis could partially explain this dysfunction since porcine glucagon cells are especially located in the islet periphery and may be partially destroyed at the time of enzymatic isolation. Pre-clinical data strongly suggested that to obtain pancreatic pig islets which respond to glucose challenge in the same physiological manner as humans, the introduction of transgenes such as glucagon, or glucoincretin hormone glucagon-like peptide (GLP-1) protein would thus be beneficial. A first construct for GLP-1 protein under a porcine insulin promoter has been designed in 2012. The first line of GLP-1 transgenic pigs on a Gal-/- background was available in 2013 in Cremona piglets have been produced by AVT by SCNT thereby confirming that the transgenic animals are viable. This first line however was not efficient for increasing the insulin production.
NPPCs from the first line of GLP1 transgenic pigs were equally tested for their secretory response but no evidence of GLP1 production or increased insulin secretion was found, thereby driving us to reconsider a new vector.
As broadly described in WP3, a new construct containing a Furin, as a secretion sequence and a Serine in position 8 to extend the half live of GLP1 have been designed BUT more importantly and additionally to GLP1, it has been discovered that acting on the muscarinic receptor PKA/Epac2 pathway amplifies significantly porcine beta-cell secretory response to glucose. Therefore a bicistronic vector containing both new GLP1 sequence and muscarinic receptor activator has been designed and evaluated in Mouse Min Cell Line (beta cell line) and in pig islets by viral transfection. The results shown in WP3 clearly demonstrate that this transgenesis amplify significantly (five to seven times) the response to hyperglycaemia. Therefore a new line of double transgenic pig has been produced by AVT.
As seen in WP3, the islets harvested from the pancreas of the first line of these double transgenic pigs (May 2016) confirmed that NPPCs from these pigs significantly produced more insulin that unmodified pigs. In some cases, the production of insulin in “ex vivo” conditions were similar or even superior to human islets. It seemed therefore that this project needed to be adapted in order to produce these double transgenic pigs for human uses. Therefore, an amendment has been asked to EU for prolonging this EU consortium form 36 to 48 months.
The adjusted objectives were to produce for the end of this period (August 2017) a brand new LINE of clean pigs (PERV C negative and low PERV A/B) which have been transfected with the double transgene GLP1 and Muscarinic 3 under an insulin promotor.
Whether DPF pig colony is found/selected and approved by regulator, then a meeting with the regulator AFMPS can be organized as well as the submission of IMPD .
With the help of RLM consulting, we have prepared the file to be propose to AFMPS and EMA in order to fulfil all the criteria. The product under development is a combined ATMP composed of maturated piglet pancreatic islets encapsulated in alginate and assembled in a subcutaneous patch with a pig collagen matrix, a filter and surgical clips. These three elements have the certification CE which is required for medical devices. As the islets are isolated from a genetically modified pig strain, they are also considered as GTMP.
Additionally to these requirements, work has been done at the level of the patch itself and mainly at the level of alginate to demonstrate the stability for safety reasons. As proposed in XENOISLET, specific in vitro assays have been achieved (WP4) and analyses first whether proteins of viruses or pig cells may go out of the patch and as such diffuses into the human body. In other words, the study of the permeability of the alginate for other proteins and peptides than insulin (viral sequences) has been done and patches containing pig islets seemed to be safe and did not release viral particles. Additionally, ex vivo work showed us that pig islets survived better in alginate when they are seeded on a porcine extracellular matrix called PERMACOL and which is already used for clinical study. Eventually, a robot has been delivered by QUIMESIS for obtaining a reproducible monolayer depot of encapsulated cells on the ECM patch.
Project Results:
As shown already in the publishable summary and main objectives, XENOISLETS mainly delivered the following MAJOR results:
1) We selected, screened more than 150 pigs to OBTAIN today a pig colony (n=20) which is PERV C negative and low in PERV A/B (under 30 copies/genome).These pigs therefore were used to CLONE our NEW double transgenic pig line which today respond to the criteria asked by the regulator AFMPS (and EMA).
2) From this colony, we produced double transgenic pigs GLP1/M3R under the promotor of insulin which have ONE SINGLE INTEGRATION in a SAFE HARBOUR V2G as requested by the regulator.
3) The pig islets from these pigs are “SUPERISLETS” since they produce 7 to 8 fold more insulin than islets from unmodified pig islets and as such approach the efficiency under Glucose 15% challenge, THAN Human Islets OR Embryonic Human Stem cells.
4) We demonstrated that Neonates pig islets are the most appropriate islets for going into human use, since the isolation is safe, sterile, and reproducible. In addition, the maturation in vitro of these islets may increase significantly the content of insulin within 3 weeks of in vitro maturation into alginate patch. Therefore, QC and SOPs for neonate pig islets have been elaborated in this project and submitted to the regulator.
5) We have shown in vitro that SUPERISLETS were safe in vitro for 6 weeks culture since these islets encapsulated into alginate did not release any pig viral particles.
6) We have designed a robot which is able to reproduce the monolayer delivery of pig cells onto a pig extra cellular matrix (PERMACOL) in order to go into human application.
7) We have shown that islets are safe in term of viral transmission especially neonates pig islets which allows the regulator to propose us to characterize the pig line but mainly also the final product which is a number of pig islets well characterized for pathogens and with a definite level of function (insulin production for a glucose challenge)
8) We created a company in Belgium (Pig for live) which is building right now a pig facility which will have all the requested characteristics for keeping pigs into DPF conditions and as such serve as a facility which can deliver pig cells for human use.
9) We developed a KIT for screening PERV A/B/C by PCR techniques and as such this kit will be released on the market after validation by several laboratories.
10) We have protected our double transgenic pig lines by patent or process of demand as well as the site of integration of the transgene (V2G harbour).
Potential Impact:
Potential impact and exploitation of the results
Although the goal of the project was to achieve all the objectives within 3 to 4 years, we have been surprised that we could significantly improve the function of the pig islets by genetic manipulations.
In fact, the discovery that a double transgene under an insulin promotor showed a 7/8 fold increase into the insulin production by the pig islets provoked a MAJOR CHANGE in our project and this was NOT really foresee. In fact, the XENOISLET project was mainly based of the work to be done on UNMODIFIED and wild type pig cells, but the discovery that a transgenic pig would do significantly better complicated the work in term of delay. But at the end, we have now generated all the tools to continue and go ahead with a maximal deadline of two more years for arriving into the pilot clinical study.
We indeed, have today the appropriate PIG line (both male and female pigs) and we need to wait that these pigs are able to reproduce themselves by crossing them with wild type pigs having a low PERV A/B as we have in our farm. In May 2018, these transgenic pigs will be bred with low PERV pigs which are in Belgium and we will wait the 4 months to obtain the bird of piglets. These piglets will be introduce after hysterotomy into our NEW FACILITY in Marche en Famenne, Belgium. As such, we can predict to have a higher number of piglets in the DFP facility around October 2018. We will then allow these piglets to grow and will as such have 9 months after low PERV double transgenic pigs into a DPF facility, ready to breed and produce piglets with “SUPERISLETS” for clinical use.
THE MAJOR IMPACT of this project will thus be to have produced a SAFE and EFFICIENT pig colony which will continuously produce “superislets” able to be grafted into humans under the form of a subcutaneous patch WITHOUT IMMUNOSUPPRESSION. Since the whole file has been already discussed with the regulator, we know very well how we need to act in the following months to be ready for going into humans without any problems. Whether the first pilot study succeeds, then we could also envision to collaborate with bigger enterprise in order to scale up our facility or to sale our superislets as we could sale human or embryonic islets. We have already contact with OTSUKA pharmaceutical for that part, but we need to expand our colony first and demonstrate the stability of the transgene. No worry is really about that since the animals have been cloned by AVT.
In addition to the fact that we could reproduce such breeding colony here in Belgium, AVT has also all the cells in BANKING so that we could also collaborate in different countries by providing them under contract, the original cells in order to create everywhere such pig colonies.
One other Major FACT is that in vitro, our superislets produced after a period of three weeks of maturation, as much insulin as Embryonic human stem cells, almost as much than Human Islets and maybe IPs, but for the latter we have no published data allowing us to compare.
Eventually, this project is a great success since many patients could afford to have access to a new type of treatment.
Potential impact for type 1 diabetic patients
The ultimate aim would be to improve the lives of patients with type 1 diabetes by providing a complete physiological treatment. In comparision with current treatments for type 1 diabetes, the bioartificial pancreas intends to bring several benefits and impacts significantly improving
the quality of life for many patients with diabetes, allowing thus:
- The control of diabetes in a physiological way, without requiring an external supply of insulin and without the continuous monitoring of blood sugar,
- The transplantation of pig pancreatic islets without immunosuppressive treatment. Thus, this therapy could be extended to a larger number of patients,
- A simple routine visit to a diabetology unit every 3 to 6 months is required to replace the transplanted cells, in the case those are exhausted. Thus, the old cells are replaced by new ones by a simple drainage/filling action through entry/exit ports,
- The reduction of long-term complications of diabetes through a normalization of blood sugar,
- Access to pig cells in infinite amounts. Thus, this solution will allow treating a larger number of diabetic patients and alleviating the organ shortage.
IP and business Plan
As proposed in XENOISLET program, IP and business plan have also been developed. UCL has patented the discovery that a transgene expressing both GLP1 and muscarinic receptor significantly increases the insulin secretion in response to glycaemic challenge (EP14164372A and EP1419991A/ WO 2015/107176). AVT on his side has patented a V2G locus which allows target a high level expression of a transgene in pigs and this location should be used to insert the UCL bicistronic gene into our pig line. Analysis of patentable pig line was studied by BIOT as seen in WP6, but there is no possibility to protect such pig line in EU. Thus the double transgenic animal will be patented as a source of insulin producing pig cells for human use. AVD still needs to define if patent would be of interest for their PERV Kit, but the decision is not yet taken. Moreover, AVD developed in the meantime an assay for testing mycoplasma which is an added value to the consortium since today the Kit is commercialised and used for our own analysis. In addition, both UCL and AVANTEA are founder member of two new SME (PIG for Live and XENOTHERA, respectively) which are devoted to SPF facilities in both Belgium and France, respectively.
Simultaneously to this work, and as reported in WP4, AVD is developing a second kit for identifying several others important viruses such as exposed in WP4. Progresses have been made, and the development of this assay as well as the validation was being achieved.
List of Websites:
http://xenoislet.eu/
Allogeneic transplantation is today the only successful therapy for several life-threatening diseases. However, organ donation only partially meets the demand and many patients still die while waiting for transplantation. Cellular transplantation could represents a very successful tool to treat type 1 diabetes mellitus (T1DM) by transplantation of human islets. Therefore, the scientific community has today reconsidered the possibility of using porcine cells to cure specific diseases by xenogeneic cellular transplantation.
XENOISLET had therefore several objectives: (1) to select and screen for pathogens a pig line source to go to clinical trials and as such to develop an unlimited source of pig insulin producing cells; (2) to produce genetically modified pigs for improving the pig islet function; (3) to dramatically change the monitoring and the follow-up of patients since NO immunosuppression will be used owing to alginate macro-encapsulation devices, and (4) to develop all the necessary tools for regulatory and safety procedures to use pig cells instead of human cells.
During this four year project, more than 150 pigs have been screened for PERV and eventually, around twenty pigs have been selected since PERV C negative and particular low PERV A/B level ( in term of expression and copies number). These pigs insure thus a breeding colony which is acceptable by the regulator for clinical use. In addition to this result, partners of the consortium developed a kit which allows to test these levels of PERV A/B expression or copies number by PCR. This kit will be available for the network in the future but will also be put on the market for others customer.
Then, a deep comparison has been made between Adult Pig Islet (API) and Neonate Pancreatic Pig Cluster cells (NPPCs) from unmodified pigs. The conclusion of several types of assays allowed confirm today that NPPCs are the most appropriate source of insulin producing pig cells. In parallel, the network has developed the appropriate QC sampling and measurement standard operating procedures (SOPs) for a standardized pancreatic islet product usable for clinical patients.
Having the pig line and knowing that NPPCs will be used, we also developed a project with a PME (Pig for live, Belgium) to build a facility which will allow to keep these pigs into a DPF facility (CER, Marche en Famenne, Belgium). This facility will be finished in May 2018 and ready to accept the pig breeders.
One of the major discovery of XENOISLET network is certainly the fact that although correction of diabetes has been regularly obtained with pig adult cells in preclinical models, we clearly know that pig islets provide a lower response than human beta cells to glucose challenge. We thus studied deeply the possible ways to significantly improve and amplify the production of Insulin by pig cells. One natural pathway by which the pig islet function could be improved to respond to glucose challenge in vivo was the GLP- 1 pathway. Additionally, we also discovered in pig that a specific muscarinic receptor (M3R) for the beta cells could also be activated to amplify the insulin production (M3R) and eventually, we showed in vitro and ex vivo that the activation of both GLP1 and the M3R receptor improved significantly the production of insulin by pig cells allowing almost to reach the same level of production than a human islet cell.
This major discovery in fact changed an important part of our project, since by knowing that a double transgenesis of the pig islet could significantly improve the function, then ethically, we needed to adapt our plan and we choose to develop and clone a NEW pig colony which will express both transgenes under an insulin promotor. This choice, however, delayed the project since we needed to produce the good pig
As today, and owing to a major effort of several members of the team, a pig line with a SINGLE INTEGRATION into the safe harbour V2G of the double transgene GLP1/M3R under an insulin promotor exists and is viable. The analysis of the pig islets of these pigs are in fact “super pig islets” since their production of insulin following a glucose challenge are 7/8 fold higher than unmodified animals.
The transgenic pigs will then be introduced into the NEW DPF facility in Belgium in May 2018 and then the first pig islets could be isolated for human use after matching all the regulatory guidelines needed.
Project Context and Objectives:
Project context and main objectives
The main objectives of XENOISLETS was therefore to undertake specific final steps enabling successful transplantation of pig insulin-producing cells into human T1DM patients by using alginate macroencapsulation and pig transgenesis. So during this four year time line, several major objectives were defined as following:
Selection of a pig line fulfilling the safety requirement of xenotransplantation of pig insulin-producing cells into humans.
After contact in 2013 with regulator in Belgium (AFMPS) and EMA (since two members of AFMPS are also EMA representative) several major issues were raised to specify the work to be done.
First, Pancreatic Pig insulin-producing cells (PPIs) will be used from Neonate pancreatic pig cells (NPPCs). In fact the latter could be easier to use for establishing standard of procedure (SOP) and maturation of these cells during 7/8 days will not be considered as a major modification, so that NPPCs are considered as a DRUG and therefore follow the National and EMA guidelines for drug therapy. But since we will use xenogeneic cells, it will also refer to a specific guideline edited by EMA about xenogeneic tissue or cells. Between 18 and 36 months, we have achieved more than 150 harvesting and isolation of NPPCs and we have now all the guidelines to obtain a very reproducible method which allows to obtain each time 25/ 30.000IEQ from one neonate animal with a good viability and no contamination. During this second/third period we also have studied several growth factors which allows to evidence that adult pig islets and neonate pigs islets have the same level of pig insulin production after three weeks of maturation. As a consequence, appropriate SOPs have been established and proposed to the regulatory agencies for the use of NPPCs islets in humans (Annex: AFMPS Minutes: MAY 2017).
Second, AFMPS clearly stated that the pig line should be characterized deeply for pathogens and that an accredited pig facility should be proposed, in order to raise and keep a pig colony breeding in DPF (designated pathogen free) conditions.
As such, a complete list of pathogens including viruses, bacteria, parasites, and fungi was proposed from the regulator authority which gathers all the pathogens analysed today for pig food additional to all pathogens which could be specific for XENOTRANSPLANTATION. This list is shown in WP7 and laboratories which may analyse these agents have been identified either in Belgium, UK or Italy. Although several pathogens may be detected in several basic laboratories, viruses should be deeply analysed and GCU/AVD have done for that a major work to specify PERV and other viruses in hundreds of pig samples send by AVT/UCL. The priority has been to screen pigs for low PERV-A/B and lacking PERV-C retrovirus dangerous loci. During this screening, both AVD and CGU have developed qualified and validated assays for pathogens. In fact, a commercial kit for PERV A,B,C has been developed by AVD and is now in a period of internal validation before to get access to the market. As such we defined criteria which should be respected to be proposed to regulator:
Our criteria for a suitable/acceptable donor animal, which exceeds that of Diatranz Otsuka Ltd (DOL) are as follows:
• PERV C env negative
• Less than 30 copies of PERV per genome
• Low PERV A and PERV B copy number
• Low level of expression in fibroblasts
• Null by in vitro cell culture
• Lack of pathogens specified in lists in report
During the project, we have studied whether the PERV expression is variable between PBMCs and several tissues as well as between the whole pancreas and the islet preparation (N.Mourad et al. Xenotransplantation, 2017). We confirmed in a very large number of samples (from at least 40 pigs) that the level of PERV expression is lower in the whole pancreas than in other tissues such as lung, kidney, spleen, serum. The level of expression and copy number in ISLETs is however, higher than in the pancreas but remains low in comparison with PBMCs or several other organs.
Today, we have selected several breeders both female and male which are PERV C negative and have a low level of PERVA/B. We have therefore two small colonies in both Cremona (AVT) and Belgium (UCL) and these animals are under reproduction to have a breeding colony in both places.
Third, the AFMPS also clearly stated that identification of a pig facility which is able to house a pig bred under DPF guidelines was needed. A solution has been also established in Belgium where a pig facility of 500m² will be built to reach the safety level of DPF at Marloie Center of Research (CER), Marche-en-Famenne, Belgium. As seen in WP6, this facility should be built for May 2018 and thus the first piglets will be introduced under sterile conditions after hysterotomy in June 2018 in a brand new facility. This facility will be used as a new company which has been created for this project and is named PIG for LIFE (P4L).With this small facility, we could raise 200 piglets per year in DPF conditions.
Now that we have decided that NPPCs will be used, all the SOP and QC have been adapted and written during the last year of the project. The in vitro culture period will allow us to implement all safety and quality control (QC) procedures necessary for the release of pig islet batches for the putative future transplantation in humans. The XENOISLET project will develop the safety and quality control methods via partners GCU and AVD who have developed the necessary tools (DNA arrays, traditional and digital PCR reagent kits) for identifying pig pathogens but mainly viruses which need to be controlled prior to clinical trials. BIOT has developed the appropriate QC sampling and measurement standard operating procedures (SOPs) for a standardized pancreatic islet product. This new approach might lower the requirement (and the costs) of having a DPF pig facility since the safety can be assured batch by batch examinations during the in vitro culture period prior to transplantation.
Production of a genetically engineered pig expressing GLP1 proteins and Muscarinic 3 receptor at the level of pig islets (under a pig insulin promoter)
Although correction of diabetes has been regularly obtained with pig adult cells, we clearly know that pig islets provide a lower response than human beta cells to glucose challenge. Indeed, a careful in vitro comparison between pig and human islets function clearly evidenced that while basal insulin secretion was not different, it was only in the presence of 1 μM forskolin or glucagon that glucose or amino acids could increase the secretory rate in pig islets to levels similar to those observed in human islets tested without forskolin. These observations and the improvement of insulin secretion by cAMP-raising agents suggest that low cAMP levels in pig β-cells may explain the poor insulin secretion. In addition, a structural hypothesis could partially explain this dysfunction since porcine glucagon cells are especially located in the islet periphery and may be partially destroyed at the time of enzymatic isolation. Pre-clinical data strongly suggested that to obtain pancreatic pig islets which respond to glucose challenge in the same physiological manner as humans, the introduction of transgenes such as glucagon, or glucoincretin hormone glucagon-like peptide (GLP-1) protein would thus be beneficial. A first construct for GLP-1 protein under a porcine insulin promoter has been designed in 2012. The first line of GLP-1 transgenic pigs on a Gal-/- background was available in 2013 in Cremona piglets have been produced by AVT by SCNT thereby confirming that the transgenic animals are viable. This first line however was not efficient for increasing the insulin production.
NPPCs from the first line of GLP1 transgenic pigs were equally tested for their secretory response but no evidence of GLP1 production or increased insulin secretion was found, thereby driving us to reconsider a new vector.
As broadly described in WP3, a new construct containing a Furin, as a secretion sequence and a Serine in position 8 to extend the half live of GLP1 have been designed BUT more importantly and additionally to GLP1, it has been discovered that acting on the muscarinic receptor PKA/Epac2 pathway amplifies significantly porcine beta-cell secretory response to glucose. Therefore a bicistronic vector containing both new GLP1 sequence and muscarinic receptor activator has been designed and evaluated in Mouse Min Cell Line (beta cell line) and in pig islets by viral transfection. The results shown in WP3 clearly demonstrate that this transgenesis amplify significantly (five to seven times) the response to hyperglycaemia. Therefore a new line of double transgenic pig has been produced by AVT.
As seen in WP3, the islets harvested from the pancreas of the first line of these double transgenic pigs (May 2016) confirmed that NPPCs from these pigs significantly produced more insulin that unmodified pigs. In some cases, the production of insulin in “ex vivo” conditions were similar or even superior to human islets. It seemed therefore that this project needed to be adapted in order to produce these double transgenic pigs for human uses. Therefore, an amendment has been asked to EU for prolonging this EU consortium form 36 to 48 months.
The adjusted objectives were to produce for the end of this period (August 2017) a brand new LINE of clean pigs (PERV C negative and low PERV A/B) which have been transfected with the double transgene GLP1 and Muscarinic 3 under an insulin promotor.
Whether DPF pig colony is found/selected and approved by regulator, then a meeting with the regulator AFMPS can be organized as well as the submission of IMPD .
With the help of RLM consulting, we have prepared the file to be propose to AFMPS and EMA in order to fulfil all the criteria. The product under development is a combined ATMP composed of maturated piglet pancreatic islets encapsulated in alginate and assembled in a subcutaneous patch with a pig collagen matrix, a filter and surgical clips. These three elements have the certification CE which is required for medical devices. As the islets are isolated from a genetically modified pig strain, they are also considered as GTMP.
Additionally to these requirements, work has been done at the level of the patch itself and mainly at the level of alginate to demonstrate the stability for safety reasons. As proposed in XENOISLET, specific in vitro assays have been achieved (WP4) and analyses first whether proteins of viruses or pig cells may go out of the patch and as such diffuses into the human body. In other words, the study of the permeability of the alginate for other proteins and peptides than insulin (viral sequences) has been done and patches containing pig islets seemed to be safe and did not release viral particles. Additionally, ex vivo work showed us that pig islets survived better in alginate when they are seeded on a porcine extracellular matrix called PERMACOL and which is already used for clinical study. Eventually, a robot has been delivered by QUIMESIS for obtaining a reproducible monolayer depot of encapsulated cells on the ECM patch.
Project Results:
As shown already in the publishable summary and main objectives, XENOISLETS mainly delivered the following MAJOR results:
1) We selected, screened more than 150 pigs to OBTAIN today a pig colony (n=20) which is PERV C negative and low in PERV A/B (under 30 copies/genome).These pigs therefore were used to CLONE our NEW double transgenic pig line which today respond to the criteria asked by the regulator AFMPS (and EMA).
2) From this colony, we produced double transgenic pigs GLP1/M3R under the promotor of insulin which have ONE SINGLE INTEGRATION in a SAFE HARBOUR V2G as requested by the regulator.
3) The pig islets from these pigs are “SUPERISLETS” since they produce 7 to 8 fold more insulin than islets from unmodified pig islets and as such approach the efficiency under Glucose 15% challenge, THAN Human Islets OR Embryonic Human Stem cells.
4) We demonstrated that Neonates pig islets are the most appropriate islets for going into human use, since the isolation is safe, sterile, and reproducible. In addition, the maturation in vitro of these islets may increase significantly the content of insulin within 3 weeks of in vitro maturation into alginate patch. Therefore, QC and SOPs for neonate pig islets have been elaborated in this project and submitted to the regulator.
5) We have shown in vitro that SUPERISLETS were safe in vitro for 6 weeks culture since these islets encapsulated into alginate did not release any pig viral particles.
6) We have designed a robot which is able to reproduce the monolayer delivery of pig cells onto a pig extra cellular matrix (PERMACOL) in order to go into human application.
7) We have shown that islets are safe in term of viral transmission especially neonates pig islets which allows the regulator to propose us to characterize the pig line but mainly also the final product which is a number of pig islets well characterized for pathogens and with a definite level of function (insulin production for a glucose challenge)
8) We created a company in Belgium (Pig for live) which is building right now a pig facility which will have all the requested characteristics for keeping pigs into DPF conditions and as such serve as a facility which can deliver pig cells for human use.
9) We developed a KIT for screening PERV A/B/C by PCR techniques and as such this kit will be released on the market after validation by several laboratories.
10) We have protected our double transgenic pig lines by patent or process of demand as well as the site of integration of the transgene (V2G harbour).
Potential Impact:
Potential impact and exploitation of the results
Although the goal of the project was to achieve all the objectives within 3 to 4 years, we have been surprised that we could significantly improve the function of the pig islets by genetic manipulations.
In fact, the discovery that a double transgene under an insulin promotor showed a 7/8 fold increase into the insulin production by the pig islets provoked a MAJOR CHANGE in our project and this was NOT really foresee. In fact, the XENOISLET project was mainly based of the work to be done on UNMODIFIED and wild type pig cells, but the discovery that a transgenic pig would do significantly better complicated the work in term of delay. But at the end, we have now generated all the tools to continue and go ahead with a maximal deadline of two more years for arriving into the pilot clinical study.
We indeed, have today the appropriate PIG line (both male and female pigs) and we need to wait that these pigs are able to reproduce themselves by crossing them with wild type pigs having a low PERV A/B as we have in our farm. In May 2018, these transgenic pigs will be bred with low PERV pigs which are in Belgium and we will wait the 4 months to obtain the bird of piglets. These piglets will be introduce after hysterotomy into our NEW FACILITY in Marche en Famenne, Belgium. As such, we can predict to have a higher number of piglets in the DFP facility around October 2018. We will then allow these piglets to grow and will as such have 9 months after low PERV double transgenic pigs into a DPF facility, ready to breed and produce piglets with “SUPERISLETS” for clinical use.
THE MAJOR IMPACT of this project will thus be to have produced a SAFE and EFFICIENT pig colony which will continuously produce “superislets” able to be grafted into humans under the form of a subcutaneous patch WITHOUT IMMUNOSUPPRESSION. Since the whole file has been already discussed with the regulator, we know very well how we need to act in the following months to be ready for going into humans without any problems. Whether the first pilot study succeeds, then we could also envision to collaborate with bigger enterprise in order to scale up our facility or to sale our superislets as we could sale human or embryonic islets. We have already contact with OTSUKA pharmaceutical for that part, but we need to expand our colony first and demonstrate the stability of the transgene. No worry is really about that since the animals have been cloned by AVT.
In addition to the fact that we could reproduce such breeding colony here in Belgium, AVT has also all the cells in BANKING so that we could also collaborate in different countries by providing them under contract, the original cells in order to create everywhere such pig colonies.
One other Major FACT is that in vitro, our superislets produced after a period of three weeks of maturation, as much insulin as Embryonic human stem cells, almost as much than Human Islets and maybe IPs, but for the latter we have no published data allowing us to compare.
Eventually, this project is a great success since many patients could afford to have access to a new type of treatment.
Potential impact for type 1 diabetic patients
The ultimate aim would be to improve the lives of patients with type 1 diabetes by providing a complete physiological treatment. In comparision with current treatments for type 1 diabetes, the bioartificial pancreas intends to bring several benefits and impacts significantly improving
the quality of life for many patients with diabetes, allowing thus:
- The control of diabetes in a physiological way, without requiring an external supply of insulin and without the continuous monitoring of blood sugar,
- The transplantation of pig pancreatic islets without immunosuppressive treatment. Thus, this therapy could be extended to a larger number of patients,
- A simple routine visit to a diabetology unit every 3 to 6 months is required to replace the transplanted cells, in the case those are exhausted. Thus, the old cells are replaced by new ones by a simple drainage/filling action through entry/exit ports,
- The reduction of long-term complications of diabetes through a normalization of blood sugar,
- Access to pig cells in infinite amounts. Thus, this solution will allow treating a larger number of diabetic patients and alleviating the organ shortage.
IP and business Plan
As proposed in XENOISLET program, IP and business plan have also been developed. UCL has patented the discovery that a transgene expressing both GLP1 and muscarinic receptor significantly increases the insulin secretion in response to glycaemic challenge (EP14164372A and EP1419991A/ WO 2015/107176). AVT on his side has patented a V2G locus which allows target a high level expression of a transgene in pigs and this location should be used to insert the UCL bicistronic gene into our pig line. Analysis of patentable pig line was studied by BIOT as seen in WP6, but there is no possibility to protect such pig line in EU. Thus the double transgenic animal will be patented as a source of insulin producing pig cells for human use. AVD still needs to define if patent would be of interest for their PERV Kit, but the decision is not yet taken. Moreover, AVD developed in the meantime an assay for testing mycoplasma which is an added value to the consortium since today the Kit is commercialised and used for our own analysis. In addition, both UCL and AVANTEA are founder member of two new SME (PIG for Live and XENOTHERA, respectively) which are devoted to SPF facilities in both Belgium and France, respectively.
Simultaneously to this work, and as reported in WP4, AVD is developing a second kit for identifying several others important viruses such as exposed in WP4. Progresses have been made, and the development of this assay as well as the validation was being achieved.
List of Websites:
http://xenoislet.eu/