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XENOISLET Report Summary

Project ID: 601827
Funded under: FP7-HEALTH
Country: Belgium

Periodic Report Summary 2 - XENOISLET (Macroencapsulated Porcine Pancreatic Islets to cure Diabetes Mellitus type 1/2)

Project Context and Objectives:
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 represent a very successful tool to treat type 1 diabetes mellitus (T1DM) by transplantation of human islets. Unfortunately, islet allotransplantation suffers from comparable limitations which are further aggravated by the fact that more than one donor is regularly needed to treat one T1DM recipient. Although human embryonic stem cells may solve these problems in the near future, there are still several major hurdles that preclude their use for clinical applications. Therefore, the scientific community has today reconsidered the possibility of using porcine cells to cure specific diseases by xenogeneic cellular transplantation. In fact, (i) pig islet cells have a stable function/differentiation pattern and are not tumorigenic; (ii) pig cells have been shown to meet the physiological needs in large animal models; (iii) the source of pig cells can be scaled-up to meet all demands on a highly standardized manner, in the respect of animal welfare rules; (iv) Designated Pathogen-Free (DPF) pig lines can be produced and could result in a higher safety profile than allotransplantation itself; (v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk and (vi) the pig insulin has been used during decades for treating T1DM patients since its differs from human insulin only at one amino-acid out of 52.
The use of xenogeneic cells, however, raises a major difficulty which is the need for a heavy systemic immunosuppression (IS). In order to avoid this heavy IS, mechanical immunoprotection has been developed and has been proven to be successful solution in pre-clinical models. In fact, we have convincingly demonstrated in Non-Human Primates (NHP) that a complete control of induced type 1 diabetes is routinely obtained for 6 months after transplantation of a subcutaneous alginate macroencapsulated porcine islets xenograft without the use of any immunosuppression (Transplantation 2010, Cell Transplantation 2013 and PCT/EP2007055857).

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/or pig transgenesis. So during this four year time line, several major objectives were defined as following:

1) Selection of a pig line fulfilling the safety requirement of xenotransplantation of pig insulin-producing cells into humans.

2) Development of QC and SOP for production of pig pancreatic products

3) Production of a genetically engineered pig expressing GLP1 proteins at the level of pig islets (under a pig insulin promoter)

4) Whether DPF pig colony is found/selected and approved by regulator, then a pilot study in humans could be initiated at the end of XENOISLET project after having obtained Ethical Committee, National and EMA approvals, submission of IMPD and after procedures are standardized.

Project Results:
1) 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 several major issues were raised to specify the work to be done.
Pancreatic Pig insulin-producing cells will be used from Neonate Pancreatic Pig Cells (NPPCs). Between 18 and 36 months, we have achieved more than 150 isolations of NPPCs and, have now a very reproducible method which allows obtain each time 25.000IEQ from one animal. By using several growth factors we also showed that after 3 weeks the same production of insulin with neonate pigs islets than with pig adult islets was obtained (WP3: Fig 3). As such we are now able to propose SOP to the regulatory agencies for the use of NPPCs islets in humans (36 to 48 months).
AFMPS requested also that the pig line should be characterized deeply for pathogens and a complete list of pathogens was proposed from the regulator authority. This list is shown in WP7 and laboratories which may analyse these agents have been identified. The priority has been to screen pigs for low PERV A/B and lacking PERV C retrovirus dangerous loci. A commercial kit for PERV A,B,C developed by AVD is now in a period of internal validation before to get access to the market. During the last 18 months, we have studied PERV characteristics and showed in more than 40 samples, that the level of PERV expression is lower in the whole pancreas than in other tissues. The level of expression and copy number in isolated 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.
Finally, AFMPS requested also the use of a pig facility under DPF guidelines. A facility was identified in Brescia, Italy. An additional solution has been also established in Belgium where a pig facility of 500m² will be built at Marloie Center of Research (CER). As seen in WP6, this facility should be built for May 2017 and thus the first piglets will be introduced under sterile conditions after hysterotomy in June 2017. With these two small facilities, we could raise 500 piglets per year in DPF conditions.

2) Development of QC and SOP for production of pig pancreatic products
Now that we have decided that NPPCs will be used, all the SOP and QC will be adapted and written during the last year of the project.

3) Production of a genetically engineered pig expressing GLP1 proteins at the level of pig islets (under a pig insulin promoter)
We clearly know that pig islets provide a lower response than human beta cells to glucose challenge. Therefore a bicistronic vector containing both new GLP1 sequence and muscarinic receptor activator has been designed and evaluated. The results shown in WP3 clearly demonstrate that this transgenesis amplify significantly 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 confirmed that NPPCs from these pigs significantly produced more insulin that unmodified pigs. It seemed therefore that this project needed to be adapted in order to produce these double transgenic pigs for human uses. The adjusted objectives now is 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.

4) Whether DPF pig colony is found/selected and approved by regulator, then a pilot study in humans could be initiated at the end of XENOISLET project.
We are now preparing the file to be proposed to AFMPS and EMA. The product under development is a combined ATMP and 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 mainly to demonstrate the alginate stability for safety reasons. Additionally, ex vivo work showed us that pig islets survived better in alginate when they are seeded on a porcine extracellular matrix called PERMACOL (already used in surgery). Eventually, a robot is under construction by QUIMESIS for obtaining a reproducible monolayer depot of encapsulated cells on the ECM patch.
IP and business plan have also been developed. UCL has patented a transgene expressing both GLP1 and muscarinic receptor (EP14164372A and EP1419991A/ WO 2015/107176). AVT has patented a V2G locus which allows target a high level expression of a transgene in pigs. 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.

Potential Impact:
The expected final result is the successful transplantation of pig insulin-producing cells into human T1DM patients by using alginate macroencapsulation and/or pig transgenesis.
Diabetes is one of the costliest health problems in the world. More than 220 million people worldwide are affected. The International Diabetes Federation (IDF) estimates that the direct annual healthcare cost of diabetes in 2007 globally for people aged 20–79 is 160–295 billion €. There are also indirect costs: lost productivity due to the inability to work, sickness, absence, disability, premature retirement or premature death.
The combination of the key technical components, such as porcine islet cells (TG or not), macroencapsulation, and safety arrays will provide a new and innovative solution to major medical and societal problems such as (1) the lack of cell supply for human cellular transplantation, (2) the need for chronic immunosuppression following transplantation, and (3) complicated surgical procedures. Indeed, this innovative therapy could open the way to a better treatment of millions of patients who are not even considered for transplantation. The source of porcine cells would be, effectively, infinite and limited only by the animal welfare guidelines, safety and QC procedures. Since no systemic immunosuppression is needed, the recipient’s immune system, being intact, will significantly decrease the potential risk of zoonosis when using xenogeneic cells, and moreover give access to treatments for more fragile people (younger and older patients). Although xenotransplantation of pig islets will, at first, only benefit a percentage of these patients, the possible market to provide a product such as “engineered pig cells” is enormous.

A central issue is the development of consistent and validated assays for global use for PERV analysis. The WHO recommends that Laboratories leading in the development and practice of testing methods for xenozoonosis, PERV in particular, are exchanging samples and rely on each other’s published methods to progress. As such, we will endeavour to ensure that developed methods meet the requirements of the WHO and involve other relevant individuals to ensure optimum development– XENOISLET will be part of this crucial and internationally organised work.

XENOISLET would provide a better management in comparison to the current therapies to treat T1DM (and the end-treatment of 20% of the patients suffering from T2 Diabetes), as the macro-encapsulated patch containing pig insulin–producing cells would have numerous potential clinical and quality-of-life advantages and impacts. First, the patch developed, will improve the security of adequate insulin delivery avoiding both unwanted hypo- and hyperglycaemic episodes and thereby, improve the quality of life of the diabetic patients, through a more physiological regulation of glycaemia by insulin secretory islets/cells and a high autonomy for several months. Second, the patients would thus need less glucose monitoring, less or no multiple daily insulin injection and time-fixed meals. Third, it is likely that this better control will directly influence the occurrence of side-effects due to mismanaged diabetes and this is even more likely for less developed countries. Four, we expect that one subcutaneous patch will allow a patient to be stabilised for one year, which would clearly also decrease the costs. Five, these diabetic patients would not need chronic immunosuppression which is normally necessary to maintain a transplant but also has a major daily cost in terms of drugs and induces several medical complications. Overall, these advantages will be sufficiently valuable to make the proposed xenotransplantation approach financially viable over the current treatments and the other competing technologies under development.

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