Periodic Reporting for period 3 - iPSpine (Induced pluripotent stem cell-based therapy for spinal regeneration)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
Low back pain (LBP) is a leading cause of disability worldwide. Degeneration of the spine’s intervertebral discs (IVDs) is a major contributor to LBP. It accounts for at least 40% (~200 million) of all LBP cases, leading to an annual economic burden of ~€240 billion for the EU. To date, there are no treatments that can stop or reverse disc degeneration. The aim of iPSpine is to investigate and develop a new advanced therapy medicinal product (ATMP), based on a game-changing therapeutic approach involving induced pluripotent stem cells (iPSC) and smart biomaterials.
1. A strong evidence base for the effective and safe use of iPSC-based strategies for regenerative treatment of IDD, restoring the IVD to a functional unit.
We characterized the differentiated iPS-notochordal-like cells (iPS-NLCs) at the single cell level to identify unique surface markers assisting in enrichment methods for iPS-NLCs. Two biomaterials were shortlisted based on their physicochemical, cytotoxicity, biomechanical and biocompatibility testing. Both were shown to be safe and have been tested with the progenitors of iPS-NLCs. An advanced platform (e.g. the dynamic loading bioreactor for disc tissue) was used to evaluate their performance: the biomaterials supported the iPS-NLC progenitors after injection into the degenerate disc and seem to also support their maturation towards NLCs. Furthermore, we confirmed the capacity of these cells to survive inside degenerated discs at 30 days upon injection in sheep, whereafter we continued with their evaluation at 3 months post-injection. We achieved full evaluation of the sheep spines, including biomechanical analysis using the portable spine biomechanics tester prior analysis at the macro- and microscopic, and biochemical level. Safety biodistribution/toxicity and tumorigenicity studies in mice are ongoing. The final study will employ the iPSpine aggrecan reporter iPSC line to study the iPS-NLC capacity to obtain a healthy matrix-producing phenotype. The latter is essential for biomechanical and functional restoration of the disc in the long term.
2. Enabling technologies for the development, characterisation, and validation of the iPSC-based strategy.
While RNA-based iPSC reprogramming is successful in disc-derived cells, we continue to pursue GMP-compliant RNA reprogramming of cells derived from blood to foster broad applicability beyond the iPSpine therapy. We confirmed the validity of a new method for depletion of undifferentiated iPSC from the iPSpine therapy, an essential safety step.
A new multi-omics approach enabled single-cell characterization of the transcriptomic and epigenetic landscapes of the heterogenous cell populations that reside within the NP of the healthy and degenerated IVDs, using dog disc cells. This work will feed into new strategies to achieve maturation of the iPS-NLCs.
DigiWest technology enabled targeted proteomic analysis of iPS-NLC phenotypic identity, studying their anti-catabolic effects and the newly developed biomaterials as well as characterizing challenging samples with very limited quantities of protein, e.g. extracellular vesicles.
The consortium successfully generated cloaked/FailSafe cells with a dual fluorescence reporter system, a marker for notochord lineage and a marker of slow-proliferating cells, to facilitate the in vitro and in vivo tracing of iPS-NLC differentiation. In vitro testing showed that the cloaked/FailSafe cells are immune privileged.
The iTOP intracellular delivery technology was demonstrated to be feasible for iPSC. CRISPR-Cas9 proteins are being produced for the purpose of (epi)genetic reprogramming of iPSCs to enable lineage commitment using iTOP. In parallel, we continued CRISPRα targeting of multiple transcription factors essential in iPS-NLC differentiation that will be transferred to an iTOP-based approach.
The in vitro and ex vivo models from the cell-/tissue-/organ-scale together with culture media capable of mimicking the healthy and degenerate disc environment have been finalized and enabled objective 1. Collaborative within and beyond the iPSpine consortium led to two seminal papers that will aid the scientific community in increased harmonization of methodology. Finally, the prototype of the open digital platform has been beta-tested and is ready to accept OMICs data across the consortium and provides interfaces to the smart iPSpine digital platform for ATMP development.
3. A recommendation on ethical and regulatory affairs to enhance and accelerate further development and implementation of iPSC-based therapeutic strategies.
The consortium continues to analyse the complex ethical, policy-related, and regulatory aspects involved in ATMPs. We have worked to understand how responsible innovation in stem cell research could be improved and on how moral imagination can be used as a teaching strategy for biomedical research students. Policy studies have been conducted on the topics of stem cell policy in Europe and stem-cell based embryo models. In addition, we developed a framework for responsibilization.
Regulatory advice is continuously being embedded to guide the consortium through the regulatory field of ATMP . One of the main activities was preparation and conduct of the first National Scientific Advice for the iPSpine therapy focusing on the design of safety and efficacy studies in animals, as well as feedback on iPSC line development and biomaterial classification. The feedback was used to modify the iPSpine studies in such a way that studies and processes will more likely be acceptable to agencies when e.g. a clinical trial application or application for marketing authorisation is filed.
Project management is conducted in different dimensions (scientific, financial, risk, ethical, IP aspects). The website (www.iPSpine.eu) is kept up to date. iPSpine authored various scientific publications, whereby lay versions have been disseminated. An educational webinar series on a broad range of topics has been presented to the consortium and shared online. Patient experts of iPSpine's Patient Advisory Board (PAB) shared their stories on social media and will also be the focus of iPSpine café sessions, to raise awareness of the needs the iPSpine project aims to meet.
We characterized the differentiated iPS-notochordal-like cells (iPS-NLCs) at the single cell level to identify unique surface markers assisting in enrichment methods for iPS-NLCs. Two biomaterials were shortlisted based on their physicochemical, cytotoxicity, biomechanical and biocompatibility testing. Both were shown to be safe and have been tested with the progenitors of iPS-NLCs. An advanced platform (e.g. the dynamic loading bioreactor for disc tissue) was used to evaluate their performance: the biomaterials supported the iPS-NLC progenitors after injection into the degenerate disc and seem to also support their maturation towards NLCs. Furthermore, we confirmed the capacity of these cells to survive inside degenerated discs at 30 days upon injection in sheep, whereafter we continued with their evaluation at 3 months post-injection. We achieved full evaluation of the sheep spines, including biomechanical analysis using the portable spine biomechanics tester prior analysis at the macro- and microscopic, and biochemical level. Safety biodistribution/toxicity and tumorigenicity studies in mice are ongoing. The final study will employ the iPSpine aggrecan reporter iPSC line to study the iPS-NLC capacity to obtain a healthy matrix-producing phenotype. The latter is essential for biomechanical and functional restoration of the disc in the long term.
2. Enabling technologies for the development, characterisation, and validation of the iPSC-based strategy.
While RNA-based iPSC reprogramming is successful in disc-derived cells, we continue to pursue GMP-compliant RNA reprogramming of cells derived from blood to foster broad applicability beyond the iPSpine therapy. We confirmed the validity of a new method for depletion of undifferentiated iPSC from the iPSpine therapy, an essential safety step.
A new multi-omics approach enabled single-cell characterization of the transcriptomic and epigenetic landscapes of the heterogenous cell populations that reside within the NP of the healthy and degenerated IVDs, using dog disc cells. This work will feed into new strategies to achieve maturation of the iPS-NLCs.
DigiWest technology enabled targeted proteomic analysis of iPS-NLC phenotypic identity, studying their anti-catabolic effects and the newly developed biomaterials as well as characterizing challenging samples with very limited quantities of protein, e.g. extracellular vesicles.
The consortium successfully generated cloaked/FailSafe cells with a dual fluorescence reporter system, a marker for notochord lineage and a marker of slow-proliferating cells, to facilitate the in vitro and in vivo tracing of iPS-NLC differentiation. In vitro testing showed that the cloaked/FailSafe cells are immune privileged.
The iTOP intracellular delivery technology was demonstrated to be feasible for iPSC. CRISPR-Cas9 proteins are being produced for the purpose of (epi)genetic reprogramming of iPSCs to enable lineage commitment using iTOP. In parallel, we continued CRISPRα targeting of multiple transcription factors essential in iPS-NLC differentiation that will be transferred to an iTOP-based approach.
The in vitro and ex vivo models from the cell-/tissue-/organ-scale together with culture media capable of mimicking the healthy and degenerate disc environment have been finalized and enabled objective 1. Collaborative within and beyond the iPSpine consortium led to two seminal papers that will aid the scientific community in increased harmonization of methodology. Finally, the prototype of the open digital platform has been beta-tested and is ready to accept OMICs data across the consortium and provides interfaces to the smart iPSpine digital platform for ATMP development.
3. A recommendation on ethical and regulatory affairs to enhance and accelerate further development and implementation of iPSC-based therapeutic strategies.
The consortium continues to analyse the complex ethical, policy-related, and regulatory aspects involved in ATMPs. We have worked to understand how responsible innovation in stem cell research could be improved and on how moral imagination can be used as a teaching strategy for biomedical research students. Policy studies have been conducted on the topics of stem cell policy in Europe and stem-cell based embryo models. In addition, we developed a framework for responsibilization.
Regulatory advice is continuously being embedded to guide the consortium through the regulatory field of ATMP . One of the main activities was preparation and conduct of the first National Scientific Advice for the iPSpine therapy focusing on the design of safety and efficacy studies in animals, as well as feedback on iPSC line development and biomaterial classification. The feedback was used to modify the iPSpine studies in such a way that studies and processes will more likely be acceptable to agencies when e.g. a clinical trial application or application for marketing authorisation is filed.
Project management is conducted in different dimensions (scientific, financial, risk, ethical, IP aspects). The website (www.iPSpine.eu) is kept up to date. iPSpine authored various scientific publications, whereby lay versions have been disseminated. An educational webinar series on a broad range of topics has been presented to the consortium and shared online. Patient experts of iPSpine's Patient Advisory Board (PAB) shared their stories on social media and will also be the focus of iPSpine café sessions, to raise awareness of the needs the iPSpine project aims to meet.
Upon successful implementation of the iPSpine therapy, we envision improved outcome and QoL for patients with IVD degeneration-related LBP, through effective regeneration of the degenerated tissue with restored biomechanical function of the disc, and long-lasting reduction of LBP. Altogether, this will result in reduced LBP-related premature retirement and improved socio-economic contribution.
The novel and extended knowledge, tools and technologies that will be the outcome of the iPSpine project, will provide unique platforms of knowledge, testing, and exploitation with broad applicability. These platforms will contribute to acceleration, innovation, and implementation of novel technologies and ATMPs for treatment of large patient groups. Hereby, iPSpine realizes its ambition to make a significant contribution by reducing translational bottlenecks through open innovation and taking European leadership in the development of ATMPs.
The novel and extended knowledge, tools and technologies that will be the outcome of the iPSpine project, will provide unique platforms of knowledge, testing, and exploitation with broad applicability. These platforms will contribute to acceleration, innovation, and implementation of novel technologies and ATMPs for treatment of large patient groups. Hereby, iPSpine realizes its ambition to make a significant contribution by reducing translational bottlenecks through open innovation and taking European leadership in the development of ATMPs.