Periodic Reporting for period 5 - INTENS (INtestinal Tissue ENgineering Solution)
Berichtszeitraum: 2022-01-01 bis 2022-09-30
Short Bowel Syndrome (SBS) is a condition that occurs when part or the entire small intestine is missing or has been removed during surgery. This condition renders the bowel incapable of fulfilling its nutritional function (intestinal failure).
SBS is a chronically debilitating disorder without a cure. Intravenous feeding offers a survival rate of 70% at 5 years in newborn infants. However, in the most severe cases, when only 10% of expected intestinal length is present, 5-year survival is reduced to around 20%. In particular we will focus on developing an INtestinal Tissue ENgineering Solution (INTENS) for children with SBS.
The objective of this programme is to deliver a functional bowel reconstruction to patients with SBS through an autologous tissue engineering strategy, overcoming the shortage of organs, and avoiding the need for immunosuppression. The work is designed to lead directly to a clinical trial for the application of the optimal protocol for tissue-engineered intestine.
SBS is a chronically debilitating disorder without a cure. Intravenous feeding offers a survival rate of 70% at 5 years in newborn infants. However, in the most severe cases, when only 10% of expected intestinal length is present, 5-year survival is reduced to around 20%. In particular we will focus on developing an INtestinal Tissue ENgineering Solution (INTENS) for children with SBS.
The objective of this programme is to deliver a functional bowel reconstruction to patients with SBS through an autologous tissue engineering strategy, overcoming the shortage of organs, and avoiding the need for immunosuppression. The work is designed to lead directly to a clinical trial for the application of the optimal protocol for tissue-engineered intestine.
INTENS consortium have developed and tested a number of protocols and recommendations to establish translational pathway, including:
• Protocol to engineer human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds
• Protocol utilising organoids (3D mini-organs) as a good manufacturing practice-compliant system
• Demonstrated tissue engineered intestine (TEI) implantation in a mouse model.
We have begun pre-clinical work in a large animal model, developing an immunosupression protocol which was demonstrated to improve growth of the TEI. Further work is now needed to demonstrate safety in a pig model of the engineered graft – this has been delayed primarily due to the impact of COVID-19 pandemic. Early engagement with the EMA Taskforce has been established to determined feasible next steps to progress towards the clinical trial. Patient have also been engaged through various opportunities at festivals, workshops for schools, open research days. We also published outputs of a health economic study which demonstrated that patient-level cost of care analysis is feasible applied to SBS.
WP 1
Across this work package a number of consortium members have made seminal contributions. This has enabled the development of scaffolds in the subsequent work packages. In WP1, this includes the development and optimisation of protocols for the derivation of different cell types found in the small intestine from both patient biopsies and pluripotent stem cells. To ascertain the correct cellular identities and the appropriateness of these cell types in downstream implementations, we have developed tests to prove the functional properties of the different cell derivatives and also aspects relevant for their safety.
WP 2
We designed a chemically-defined low-defect thiol-Michael addition (LDTM) hydrogels that can be formed at low polymer content without loss of mechanical integrity. These hydrogels promote the development of patterned mouse and human intestinal organoids, the latter in the absence of any animal-derived components, and thus provide a substitute for ill-defined Matrigel for successful translation of organoid technology from the lab to the clinic.
WP3
We have successfully generated mucosal grafts, with organoids alone and fibroblasts and organoids, using RAFT® scaffolds. This is advantageous as, unlike decellularized tissue scaffold, there is an unlimited supply and both seeding density (of organoids and fibroblasts generated in WP1) and thickness of RAFT can be adjusted.
Organoids on grafts organised into a columnar epithelium and, most importantly, we have optimised a protocol to turn epithelial layer to form crypt-villus structures and differentiate mucosal graft to give rise to all intestinal epithelial cell lineages. We plan to go on to assess the digestive and absorptive function of these grafts in vitro and utilise them for orthotopic transplantation in vivo.
A protocol, detailing work utilising decellularized native intestine to generate jejunal mucosal grafts, in vitro, has been published in Nature Protocols “Bio-engineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds”. The teams will carry on working on further developments to eventually translate the use of engineered mucosa for first-in-human trial.
WP4
The INTENS program has resulted in the development of culture conditions allowing the production of intestinal cells in vitro in conditions compatible with clinical application including cell-based therapy. These conditions can be used with a diversity of human induced Pluripotent Stem Cell lines including cells lines generated in GMP conditions. Finally, we have generated hiPSC line lacking key immune molecule using genome engineering. These immune silent cells will avoid the use of heavy immune suppressive treatments since they will not be recognised by the immune system.
The possibility to generate human intestinal organoids, mini-organs in a dish, has opened tremendous opportunities for regenerative medicine. However, the growth of these organoids relies on the use of animal-derived (growth) factors and poorly defined 3D Matrices, excluding clinical application. In WP4, we developed and tested the ideal culturing conditions allowing optimal, safe and good manufacturing practice (cGMP)-level expansion of human adult stem cell derived intestinal organoids.
WP5
This work package has generated several major advances towards potential tissue-engineered solutions to Short Bowel Syndrome:
• Development of scaffolding materials that will serve as the essential substrate and support for engineered intestine.
• Demonstration that human tissue-engineered intestine can readily be implanted in immunocompromised or immunosuppressed mouse and pig models in order to study potential translational use.
• Identification of a key challenge (transport of intestinal human organoids to distant sites) that will need to be addressed for successful translation of the technology.
WP6
Dynamic public-facing website developed, updated and integrated with eurostemcell.org.
- Development and dissemination of digital engagement tools. Ongoing dissemination secured through integration with eurogct.org.
- Built capacity within the INTENS consortia for public engagement through establishing a network of communicators, the production of tools to communicate digitally and through face-to-face engagement.
- Two online Q&As with patient families
- INTENS researchers received training at Hydra Summer School on Stem Cell Biology and Regenerative Medicine.
WP7 and WP8
WP7 activities covered all aspects of project monitoring, reporting, financial and contractual administration in accordance with the Commission’s rules, ensuring proper communication within the consortium and implementing the project governance’s decisions.
Copies of ethics approvals and renewals were collected and checked by Ethic External Experts. First submission to EMA Innovation Task Force has been achieved and the consortium is waiting for feedback.
• Protocol to engineer human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds
• Protocol utilising organoids (3D mini-organs) as a good manufacturing practice-compliant system
• Demonstrated tissue engineered intestine (TEI) implantation in a mouse model.
We have begun pre-clinical work in a large animal model, developing an immunosupression protocol which was demonstrated to improve growth of the TEI. Further work is now needed to demonstrate safety in a pig model of the engineered graft – this has been delayed primarily due to the impact of COVID-19 pandemic. Early engagement with the EMA Taskforce has been established to determined feasible next steps to progress towards the clinical trial. Patient have also been engaged through various opportunities at festivals, workshops for schools, open research days. We also published outputs of a health economic study which demonstrated that patient-level cost of care analysis is feasible applied to SBS.
WP 1
Across this work package a number of consortium members have made seminal contributions. This has enabled the development of scaffolds in the subsequent work packages. In WP1, this includes the development and optimisation of protocols for the derivation of different cell types found in the small intestine from both patient biopsies and pluripotent stem cells. To ascertain the correct cellular identities and the appropriateness of these cell types in downstream implementations, we have developed tests to prove the functional properties of the different cell derivatives and also aspects relevant for their safety.
WP 2
We designed a chemically-defined low-defect thiol-Michael addition (LDTM) hydrogels that can be formed at low polymer content without loss of mechanical integrity. These hydrogels promote the development of patterned mouse and human intestinal organoids, the latter in the absence of any animal-derived components, and thus provide a substitute for ill-defined Matrigel for successful translation of organoid technology from the lab to the clinic.
WP3
We have successfully generated mucosal grafts, with organoids alone and fibroblasts and organoids, using RAFT® scaffolds. This is advantageous as, unlike decellularized tissue scaffold, there is an unlimited supply and both seeding density (of organoids and fibroblasts generated in WP1) and thickness of RAFT can be adjusted.
Organoids on grafts organised into a columnar epithelium and, most importantly, we have optimised a protocol to turn epithelial layer to form crypt-villus structures and differentiate mucosal graft to give rise to all intestinal epithelial cell lineages. We plan to go on to assess the digestive and absorptive function of these grafts in vitro and utilise them for orthotopic transplantation in vivo.
A protocol, detailing work utilising decellularized native intestine to generate jejunal mucosal grafts, in vitro, has been published in Nature Protocols “Bio-engineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds”. The teams will carry on working on further developments to eventually translate the use of engineered mucosa for first-in-human trial.
WP4
The INTENS program has resulted in the development of culture conditions allowing the production of intestinal cells in vitro in conditions compatible with clinical application including cell-based therapy. These conditions can be used with a diversity of human induced Pluripotent Stem Cell lines including cells lines generated in GMP conditions. Finally, we have generated hiPSC line lacking key immune molecule using genome engineering. These immune silent cells will avoid the use of heavy immune suppressive treatments since they will not be recognised by the immune system.
The possibility to generate human intestinal organoids, mini-organs in a dish, has opened tremendous opportunities for regenerative medicine. However, the growth of these organoids relies on the use of animal-derived (growth) factors and poorly defined 3D Matrices, excluding clinical application. In WP4, we developed and tested the ideal culturing conditions allowing optimal, safe and good manufacturing practice (cGMP)-level expansion of human adult stem cell derived intestinal organoids.
WP5
This work package has generated several major advances towards potential tissue-engineered solutions to Short Bowel Syndrome:
• Development of scaffolding materials that will serve as the essential substrate and support for engineered intestine.
• Demonstration that human tissue-engineered intestine can readily be implanted in immunocompromised or immunosuppressed mouse and pig models in order to study potential translational use.
• Identification of a key challenge (transport of intestinal human organoids to distant sites) that will need to be addressed for successful translation of the technology.
WP6
Dynamic public-facing website developed, updated and integrated with eurostemcell.org.
- Development and dissemination of digital engagement tools. Ongoing dissemination secured through integration with eurogct.org.
- Built capacity within the INTENS consortia for public engagement through establishing a network of communicators, the production of tools to communicate digitally and through face-to-face engagement.
- Two online Q&As with patient families
- INTENS researchers received training at Hydra Summer School on Stem Cell Biology and Regenerative Medicine.
WP7 and WP8
WP7 activities covered all aspects of project monitoring, reporting, financial and contractual administration in accordance with the Commission’s rules, ensuring proper communication within the consortium and implementing the project governance’s decisions.
Copies of ethics approvals and renewals were collected and checked by Ethic External Experts. First submission to EMA Innovation Task Force has been achieved and the consortium is waiting for feedback.
While challenges have been encountered in delivery of this proposed project, the consortium have made great progress in demonstrating potential of this technology for clinical use. In particular, the develop-ment of a protocol utilising human-derived cells presents an important step that would enable future scalability to production of the therapy in readiness for the clinical trial.