Periodic Reporting for period 1 - HEAL (HLA-homozygous iPSC-cardiomyocytE Aggregate manufacturing technoLogies for allogenic cell therapy to the heart)
Reporting period: 2022-09-01 to 2024-02-29
Background
The project “HLA-homozygous iPSC-cardiomyocyte Aggregate manufacturing technologies for allogenic cell therapy to the heart” (HEAL) is focused on overcoming numerus hurdles to human induced pluripotent stem cell (iPSC)-based therapies to heart failure, which remains a major global cause of morbidity and mortality.
Aims of the project
The overarching goal is to establish new tools, technologies and expertise for accelerating the development of advanced iPSC-based therapies. The project also aims at overcoming scientific, regulatory and in particular safety hurdles, necessary to initiate a first in-man (FIM) clinical trial for the therapeutic administration of allogeneic, human leukocyte antigen (HLA)-homozygous iPSC-CMAs, for improving the function of damaged hearts in heart failure patient for whom very few alternative therapies currently exist. Project outcome will facilitate decision-making by authorities and clinicians in a secure and ethical manner, for respecting individual integrity and underpinned with public acceptance and trust.
The project “HLA-homozygous iPSC-cardiomyocyte Aggregate manufacturing technologies for allogenic cell therapy to the heart” (HEAL) is focused on overcoming numerus hurdles to human induced pluripotent stem cell (iPSC)-based therapies to heart failure, which remains a major global cause of morbidity and mortality.
Aims of the project
The overarching goal is to establish new tools, technologies and expertise for accelerating the development of advanced iPSC-based therapies. The project also aims at overcoming scientific, regulatory and in particular safety hurdles, necessary to initiate a first in-man (FIM) clinical trial for the therapeutic administration of allogeneic, human leukocyte antigen (HLA)-homozygous iPSC-CMAs, for improving the function of damaged hearts in heart failure patient for whom very few alternative therapies currently exist. Project outcome will facilitate decision-making by authorities and clinicians in a secure and ethical manner, for respecting individual integrity and underpinned with public acceptance and trust.
WP 1
• Calculable process upscaling strategy and validation by wet lab experiments established.
• Modulated protocol for the efficient iPSC cardiomyocyte aggregates (iPS-CMA's) differentiation was established by applying the R&D-grade iPSC line R26_6 (from CAT) in combination with an ACF expansion and advanced CM differentiation formulation (from BI).
• Production of R26-derived iPS-CMA's for their transplantation and validation in respective animal safety experiments in WP 3.
• Substantial progress regarding the upscaling of iPS-CMA production by closed-loop bioprocessing; this has led to substantial 4fold upscaling from 500 mL to 2000 mL scale.
• SOPs development for optimized iPSC expansion and differentiation has resulted into a SOP-like structured publication entitled “Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks”; Kriedemann et al. and Martin, Zweigerdt; Nat. Prot. in print (2024).
• Preparation of R&D-grade cell bank of the iPSC line R26_6 and respective QC testing.
• Cell distribution consortium.
• Contribution to the optimization of the GMP compliant differentiation protocol.
• Development of ACF expansion medium in accordance with established GMP SOPs.
• Performance of the iPSC expansion medium demonstrated through successful testing with the R26_6 line.
• Cultured R26_6 aggregates in the ACF expansion media underwent CM differentiation in CDM3 medium and an advanced differentiation formulation currently undergoing optimization and stability studies.
• Despite alternative strategies, poor recovery after iPS-CMAs freezing was found; further extensive testing ongoing.
• Different option: single CMs freezing of dissociated iPS-CMAs; applicability to large scale is tested.
WP2
• Single-cell immune-phenotyping protocol established.
• Immune-modulation assay prototype format tested.
• Animal license and work protocol finalized and accepted.
• Arranging logistics for cell transfer between Hannover and Utrecht.
• Tumorigenicity assay key to the quality and sensitivity of the HEAL CM aggregate safety under validation; broadly applicable for safety of other iPSC- products.
WP 3
• Determining the status of cancer-related mutations in differentiated cells derived from human pluripotent stem cells.
• Establishing a bioinformatic tool “EpiTyping” to identify epigenetic aberrations in human pluripotent stem cells.
• Adaption of established, gene editing iCasp9 suicide construct to implement proliferation-specificity in two different strategies by instructed gene synthesis and standard cloning techniques.
• Adaption of culture conditions to the HLAh iPSC line R26_6
• Testing of two genetic engineering strategies under GMP-compliant culture conditions: i) iCasp9 suicide safety switch under control of the proliferation-specific promotor human CyclE; ii) knock-in of iCasp9 suicide safety switch into the proliferation-related gene locus CDK1.
• Generation and preliminary analysis of transgenic clones.
WP 4
• The current regulatory background of the HEAL project outputs and guidance for the IMP class has now been carried out with a comprehensive regulatory roadmap for the project developed.
• A first draft of a target product profile has been completed with gaps identified to be addressed in the 2nd part of the project.
• IMPD development has begun with input being gathered by all relevant partners and assessed (nonclinical and quality) to identify critical areas to be discussed in a scientific advice at the PEI in the second part of the project.
• Calculable process upscaling strategy and validation by wet lab experiments established.
• Modulated protocol for the efficient iPSC cardiomyocyte aggregates (iPS-CMA's) differentiation was established by applying the R&D-grade iPSC line R26_6 (from CAT) in combination with an ACF expansion and advanced CM differentiation formulation (from BI).
• Production of R26-derived iPS-CMA's for their transplantation and validation in respective animal safety experiments in WP 3.
• Substantial progress regarding the upscaling of iPS-CMA production by closed-loop bioprocessing; this has led to substantial 4fold upscaling from 500 mL to 2000 mL scale.
• SOPs development for optimized iPSC expansion and differentiation has resulted into a SOP-like structured publication entitled “Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks”; Kriedemann et al. and Martin, Zweigerdt; Nat. Prot. in print (2024).
• Preparation of R&D-grade cell bank of the iPSC line R26_6 and respective QC testing.
• Cell distribution consortium.
• Contribution to the optimization of the GMP compliant differentiation protocol.
• Development of ACF expansion medium in accordance with established GMP SOPs.
• Performance of the iPSC expansion medium demonstrated through successful testing with the R26_6 line.
• Cultured R26_6 aggregates in the ACF expansion media underwent CM differentiation in CDM3 medium and an advanced differentiation formulation currently undergoing optimization and stability studies.
• Despite alternative strategies, poor recovery after iPS-CMAs freezing was found; further extensive testing ongoing.
• Different option: single CMs freezing of dissociated iPS-CMAs; applicability to large scale is tested.
WP2
• Single-cell immune-phenotyping protocol established.
• Immune-modulation assay prototype format tested.
• Animal license and work protocol finalized and accepted.
• Arranging logistics for cell transfer between Hannover and Utrecht.
• Tumorigenicity assay key to the quality and sensitivity of the HEAL CM aggregate safety under validation; broadly applicable for safety of other iPSC- products.
WP 3
• Determining the status of cancer-related mutations in differentiated cells derived from human pluripotent stem cells.
• Establishing a bioinformatic tool “EpiTyping” to identify epigenetic aberrations in human pluripotent stem cells.
• Adaption of established, gene editing iCasp9 suicide construct to implement proliferation-specificity in two different strategies by instructed gene synthesis and standard cloning techniques.
• Adaption of culture conditions to the HLAh iPSC line R26_6
• Testing of two genetic engineering strategies under GMP-compliant culture conditions: i) iCasp9 suicide safety switch under control of the proliferation-specific promotor human CyclE; ii) knock-in of iCasp9 suicide safety switch into the proliferation-related gene locus CDK1.
• Generation and preliminary analysis of transgenic clones.
WP 4
• The current regulatory background of the HEAL project outputs and guidance for the IMP class has now been carried out with a comprehensive regulatory roadmap for the project developed.
• A first draft of a target product profile has been completed with gaps identified to be addressed in the 2nd part of the project.
• IMPD development has begun with input being gathered by all relevant partners and assessed (nonclinical and quality) to identify critical areas to be discussed in a scientific advice at the PEI in the second part of the project.
PoC for the newly designed ACF CM differentiation formulation demonstrated for R26_6 cells
Substantial process upscaling for iPS-CMA production in STBRs achieved.
SOPs development resulted into publication of “Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks”; Kriedemann et al. Nat. Prot. (in print 2024).
• Central tumorigenicity assay established and validated.
• Novel 60- and 354-marker single-cell level deep immunophenotyping prototype established.
• Defining high rate of cancer mutations in progenies of hPSC published in Nat. Biotech. doi: 10.1038/s41587-023-02090-2.
• Unique tool demonstrating the epigenetic status of hPSC published in Nat. Prot. doi: 10.1038/s41596-023-00898-5).
Substantial process upscaling for iPS-CMA production in STBRs achieved.
SOPs development resulted into publication of “Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks”; Kriedemann et al. Nat. Prot. (in print 2024).
• Central tumorigenicity assay established and validated.
• Novel 60- and 354-marker single-cell level deep immunophenotyping prototype established.
• Defining high rate of cancer mutations in progenies of hPSC published in Nat. Biotech. doi: 10.1038/s41587-023-02090-2.
• Unique tool demonstrating the epigenetic status of hPSC published in Nat. Prot. doi: 10.1038/s41596-023-00898-5).