Periodic Reporting for period 2 - X-PAND (Exploiting ex vivo expansion and deep multiomics profiling to bring novel, efficient and safer hematopoietic stem cell gene therapies to clinical application)
Okres sprawozdawczy: 2023-10-01 do 2025-03-31
X-PAND aims to revolutionize hematopoietic stem cell (HSC) genetic engineering. The challenge is designing protocols for HSC culture that balance productive expansion with efficient genetic manipulation while preserving long-term repopulating potential. It seeks to develop a novel ex vivo genetic engineering approach for HSC expansion, focusing on symmetric self-renewal of HSCs and single-cell multiomics analysis.
Its objectives are 1) Leverage novel and emerging ex vivo HSCs expansion protocols to support next-generation genetic engineering of HSCs, improving safety and efficiency of gene editing and allowing to select correctly-engineered HSCs, ensuring a polyclonal long-term graft. 2) Use single-cell multiomic approaches to identify, track and characterize HSCs in ex vivo culture, accelerating optimization of HSC engineering protocols while reducing and eventually replacing the need for in vivo xenograft experiments. 3) Assess genome and epigenome integrity of edited and expanded HSCs and modulate cellular response to engineering towards abrogating its negative impact on HSC functionality. 4) Apply novel expansion-based, single-cell multiomics-driven HSC engineering protocols to optimize ex vivo gene therapy approaches for genetic diseases and cancer. 5) Collect, integrate and reanalyze high-content multidimensional data by machine-learning to create an open source tool for deep and reproducible characterization of genetically-engineered ATMPs. 6) Enable clinical development of new HSC gene therapy approaches.
Its objectives are 1) Leverage novel and emerging ex vivo HSCs expansion protocols to support next-generation genetic engineering of HSCs, improving safety and efficiency of gene editing and allowing to select correctly-engineered HSCs, ensuring a polyclonal long-term graft. 2) Use single-cell multiomic approaches to identify, track and characterize HSCs in ex vivo culture, accelerating optimization of HSC engineering protocols while reducing and eventually replacing the need for in vivo xenograft experiments. 3) Assess genome and epigenome integrity of edited and expanded HSCs and modulate cellular response to engineering towards abrogating its negative impact on HSC functionality. 4) Apply novel expansion-based, single-cell multiomics-driven HSC engineering protocols to optimize ex vivo gene therapy approaches for genetic diseases and cancer. 5) Collect, integrate and reanalyze high-content multidimensional data by machine-learning to create an open source tool for deep and reproducible characterization of genetically-engineered ATMPs. 6) Enable clinical development of new HSC gene therapy approaches.
1) OSR refined an HSC expansion protocol supporting a net HSCs maintenance during ex vivo culture, though lentiviral transduction adds stress that may impact HSC function if non-optimised protocols are used. Long-term grafts deriving from ex vivo expanded HSCs have a highly polyclonal composition, which is encouraging for clinical translation.
Homology-driven repair (HDR) has been successfully established for gene editing in the expansion culture setup and lipid nanoparticles (LNPs) resulted beneficial in improving HDR efficiency and stress response.
Integrating base (BE) and prime editing (PE) techniques in the HSC expansion protocol resulted promising with a slightly higher editing efficiency using LNPs.
2) UNAV developed a comprehensive multiome dataset to explore HSC subpopulations and novel regulatory features within CD34+ cells isolated from bone marrow and mobilized peripheral blood. UNAV and CIEMAT established standardized protocols for isolating, culturing, and modifying CD34+ cells, ensuring consistent multiomic assessments, with further experiments planned. OSR contributed multiome data from ex vivo expanded cells, which are being integrated with the data from UNAV. Gene editing experiments targeting the AAVS1 locus and initial single-cell sequencing studies are currently underway.
3) UKLFR developed a comprehensive safety pipeline to assess CRISPR-based editing in HSC expansion, highlighting the persistence of chromosomal risks despite double-nickase use. OSR found that HDR-based engineering induces a senescence-like response in HSPCs, involving p53 and inflammation, necessitating mitigation strategies. Ex vivo culture triggers p38 MAPK activation, increasing ROS and proliferative stress, which can be alleviated by p38 inhibition to enhance self-renewal. The team aims to test DNA damage response (DDR) and senescence modulators, optimize genome editing base and prime editors, and evaluate genotoxicity risks.
4) CIEMAT significantly advanced studies on editing approaches in Fanconi Anemia (FA) and Diamond Blackfan Anemia (DBA). They applied PE and BE to identify the most suitable approaches for future treatments. Ongoing research aims to develop precise and effective therapies to address their underlying genetic causes. CHUV has tested novel, potentially less toxic strategies for engraftment of genetically-engineered HSCs, showing promising potential for further refinement. Progress was also made in HSC-based gene therapies for cancer, focusing on tumor infiltrating macrophage-specific loci driving the expression of antitumor genes. These advances will guide further development in the coming year.
5) NBM-FMS established a long-term gene therapy data repository within EGA. To support education and engagement within X-PAND, video resources have been expanded to cover key topics like Gene Therapy for Multiple Myeloma and Deep Learning. The team made significant progress in refining the HSC fingerprint, with consistent findings across hematopoietic compartments. Multiomic profiling confirmed the ability to distinguish cells carrying transferred genes, replicating in vitro expression patterns observed in vivo. Efforts to map HSC gene regulatory networks are ongoing, with a strong focus on CellOracle-based analyses to identify key transcriptional elements involved in haematopoiesis. The team continues to enhance open-source tools and guidelines. (i) The GitHub repository for single-cell multiome and CITE-seq pipelines is regularly updated to improve usability and reproducibility. (ii) The newly improved GeneSetCluster2.0 tool has been designed to facilitate functional analysis in single-cell data.
Homology-driven repair (HDR) has been successfully established for gene editing in the expansion culture setup and lipid nanoparticles (LNPs) resulted beneficial in improving HDR efficiency and stress response.
Integrating base (BE) and prime editing (PE) techniques in the HSC expansion protocol resulted promising with a slightly higher editing efficiency using LNPs.
2) UNAV developed a comprehensive multiome dataset to explore HSC subpopulations and novel regulatory features within CD34+ cells isolated from bone marrow and mobilized peripheral blood. UNAV and CIEMAT established standardized protocols for isolating, culturing, and modifying CD34+ cells, ensuring consistent multiomic assessments, with further experiments planned. OSR contributed multiome data from ex vivo expanded cells, which are being integrated with the data from UNAV. Gene editing experiments targeting the AAVS1 locus and initial single-cell sequencing studies are currently underway.
3) UKLFR developed a comprehensive safety pipeline to assess CRISPR-based editing in HSC expansion, highlighting the persistence of chromosomal risks despite double-nickase use. OSR found that HDR-based engineering induces a senescence-like response in HSPCs, involving p53 and inflammation, necessitating mitigation strategies. Ex vivo culture triggers p38 MAPK activation, increasing ROS and proliferative stress, which can be alleviated by p38 inhibition to enhance self-renewal. The team aims to test DNA damage response (DDR) and senescence modulators, optimize genome editing base and prime editors, and evaluate genotoxicity risks.
4) CIEMAT significantly advanced studies on editing approaches in Fanconi Anemia (FA) and Diamond Blackfan Anemia (DBA). They applied PE and BE to identify the most suitable approaches for future treatments. Ongoing research aims to develop precise and effective therapies to address their underlying genetic causes. CHUV has tested novel, potentially less toxic strategies for engraftment of genetically-engineered HSCs, showing promising potential for further refinement. Progress was also made in HSC-based gene therapies for cancer, focusing on tumor infiltrating macrophage-specific loci driving the expression of antitumor genes. These advances will guide further development in the coming year.
5) NBM-FMS established a long-term gene therapy data repository within EGA. To support education and engagement within X-PAND, video resources have been expanded to cover key topics like Gene Therapy for Multiple Myeloma and Deep Learning. The team made significant progress in refining the HSC fingerprint, with consistent findings across hematopoietic compartments. Multiomic profiling confirmed the ability to distinguish cells carrying transferred genes, replicating in vitro expression patterns observed in vivo. Efforts to map HSC gene regulatory networks are ongoing, with a strong focus on CellOracle-based analyses to identify key transcriptional elements involved in haematopoiesis. The team continues to enhance open-source tools and guidelines. (i) The GitHub repository for single-cell multiome and CITE-seq pipelines is regularly updated to improve usability and reproducibility. (ii) The newly improved GeneSetCluster2.0 tool has been designed to facilitate functional analysis in single-cell data.
X-PAND is making significant strides in advancing scientific knowledge and translating cell and gene therapies into real-world applications. An HSC expansion protocol combined with lentiviral transduction and editing techniques is being designed for clinical translation. X-PAND has refined these procedures and enhanced editing technologies, paving the way for valorising its technological platform and positioning it for future funding.
By harmonizing multi-omics datasets, X-PAND accelerates the translation of research into clinical use. A key milestone is the launch of GeneSetCluster 2.0 an advanced gene-set analysis tool that has improved knowledge-sharing. Bioinformaticians use it to perform analyses and share data with clinicians via its interactive site, facilitating the collaboration and strengthening the scientific community’s collective expertise.
Scientifically, X-PAND contributes significantly to the EU’s research landscape by uniting leading experts in gene editing, HSC expansion, multiomics data analysis, and clinical translation. Its robust multi-omics dataset and refined gene editing protocols enlarge our understanding of HSC biology and the genetic mechanisms behind disorders like FA and DBA. Research into DDR and HSC biology has provided key insights into genetic manipulation’s effects on stem cell properties and genotoxicity, ensuring the development of safe, clinically applicable procedures.
X-PAND’s societal impact is profound. Optimized HSC expansion protocols and next-generation genetic engineering technologies improve treatment efficacy and access for genetic disorders and cancer. X-PAND is poised to increase the safety and effectiveness of HSC-based therapies, offering patients new hope. The optimized protocols for gene editing and cell expansion and multiomic approaches to reduce/replace animal testing aim to cut treatment costs, making innovative therapies more affordable. Improved cost-effectiveness of gene and cell therapies will help overcome financial barriers in healthcare, ensuring long-term sustainability of HSC gene therapy. Increased accessibility and affordability will reduce healthcare burden, benefiting patients, their families and the healthcare system as a whole.
By harmonizing multi-omics datasets, X-PAND accelerates the translation of research into clinical use. A key milestone is the launch of GeneSetCluster 2.0 an advanced gene-set analysis tool that has improved knowledge-sharing. Bioinformaticians use it to perform analyses and share data with clinicians via its interactive site, facilitating the collaboration and strengthening the scientific community’s collective expertise.
Scientifically, X-PAND contributes significantly to the EU’s research landscape by uniting leading experts in gene editing, HSC expansion, multiomics data analysis, and clinical translation. Its robust multi-omics dataset and refined gene editing protocols enlarge our understanding of HSC biology and the genetic mechanisms behind disorders like FA and DBA. Research into DDR and HSC biology has provided key insights into genetic manipulation’s effects on stem cell properties and genotoxicity, ensuring the development of safe, clinically applicable procedures.
X-PAND’s societal impact is profound. Optimized HSC expansion protocols and next-generation genetic engineering technologies improve treatment efficacy and access for genetic disorders and cancer. X-PAND is poised to increase the safety and effectiveness of HSC-based therapies, offering patients new hope. The optimized protocols for gene editing and cell expansion and multiomic approaches to reduce/replace animal testing aim to cut treatment costs, making innovative therapies more affordable. Improved cost-effectiveness of gene and cell therapies will help overcome financial barriers in healthcare, ensuring long-term sustainability of HSC gene therapy. Increased accessibility and affordability will reduce healthcare burden, benefiting patients, their families and the healthcare system as a whole.