Periodic Reporting for period 1 - 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: 2022-10-01 do 2023-09-30
X-PAND's main objectives are the following: i) Leverage novel and emerging ex vivo HSC expansion protocols to support next-generation genetic engineering of HSC, improving the safety and efficiency of gene editing and allowing to select correctly-engineered HSC and guarantee a polyclonal long-term graft. ii) Leverage single cell multiomics approaches to faithfully identify, track and molecularly characterize HSC in ex vivo culture, to accelerate optimization of HSC engineering protocols while reducing and, eventually, replacing the need for in vivo xenograft experiments. iii) Assess genome and epigenome integrity of edited & expanded HSC and modulate the cellular response to genetic engineering towards abrogating its negative impact on HSC functionality. iv) Apply the novel expansion-based and single-cell multiomics-driven HSC engineering protocols to optimize ex vivo gene therapy approaches for genetic model diseases and cancer immunotherapy. v) Collect, integrate and reanalyze high-content multidimensional data by machine-learning to develop an Open Source tool for deep and reproducible characterization of genetically-engineered ATMPs. vi) Enable clinical development of new HSC gene therapy approaches through Start-Ups/SMEs.
Overall X-PAND aims to revolutionize genetic editing in hematopoietic stem cells (HSC) through a high-profile interdisciplinary team. This team combines expertise in gene editing, HSC expansion, multiomics data analysis, and clinical translation for monogenic disorders and cancer treatment.
The challenge is designing protocols for HSC culture that balance productive expansion with efficient genetic manipulation while preserving long-term repopulating potential. X-PAND 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.
This approach has the potential to accelerate the development of safer and more effective therapies based on genetically-engineered HSCs for genetic diseases and beyond.
Overall X-PAND aims to revolutionize genetic editing in hematopoietic stem cells (HSC) through a high-profile interdisciplinary team. This team combines expertise in gene editing, HSC expansion, multiomics data analysis, and clinical translation for monogenic disorders and cancer treatment.
The challenge is designing protocols for HSC culture that balance productive expansion with efficient genetic manipulation while preserving long-term repopulating potential. X-PAND 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.
This approach has the potential to accelerate the development of safer and more effective therapies based on genetically-engineered HSCs for genetic diseases and beyond.
Objective 1: The OSR team has defined the conditions of an optimised protocol for ex vivo expansion of mobilised peripheral blood CD34+ cells, including a GMP-compatible sorting strategy that allows enriching for HSC by a factor of 5-10 fold with respect to the total CD34+ cell population. To this end, a GMP-compatible cell sorter has been implemented. Moreover, lentiviral transduction, as well as HDR-based gene editing as well as base editing have been successfully integrated into the ex vivo expansion context at the bulk CD34+ cell level, achieving superior results compared with previously used standard procedures.
Objective 2: The UNAV team is creating a combined scATACseq + scRNAseq reference of BM and mPB CD34+ cells from healthy donors, together with the integrated analysis of the transcriptomic profile and the expression of 6 surface proteins using CITE-seq technology. A long-term signature score has been developed for the identification of the most primitive HSC subpopulation. Moreover, they set up the experimental conditions to evaluate gene transfer/editing efficiency across HSC subpopulations by using single cell multiomic assessments.
Objective 3: The UKLFR team has started to establishing a robust pipeline for unbiased, genome-wide identification of ON and OFF-target effects triggered by the various genome editing strategies. In particular, UKLFR has adapted CAST-Seq to detect on- and off-target aberrations induced by various base editors in hematopoietic cells. Data obtained are the first-time reports of the genome-wide detection of chromosomal rearrangements and OFF-target effects prompted by both adenine and cytosine base editors.
In parallel, the OSR team investigating cellular pathway activated by DSBs, NHEJ, HR, base and prime editing discover that proliferative stress is a driver of human HSPC dysfunction with fundamental implications for clinical applications.
Objective 4: The CIEMAT team positively tested the efficiency of adenine base editing in correcting the phenotype in lymphoblastic cell lines from a Fanconi Anaemia patient carrying this novel FancA protein. Secondly, they selected as the most efficient the use of a second sgRNA that promotes the generation of a nick in the non-edited strand to correct the stop codon mutation in FANCA gene confirming that the efficiency of prime editing is not affected in comparison with healthy donor cells.
In the context of Diamond Blackfan anemia, the CIEMAT team tested the efficacy and safety of a lentiviral vector-based strategy for the RPS19 defect developed by single cell RNA sequences and in parallel, they tested a knock-in gene editing approach based on AAV to target RPL5 locus in BM CD34+ from healthy donors.
Objective 5: The NBM team progressed in the first objective "Generation of a Gene Therapy DB/Atlas to organize, annotate and share multi-omics data". The core progress can be summarized in: the selection of the meta-data and data formats to be considered in the DB, and the generation of a XPAND repository within the European Genome-Phenome Archive. Additionally, different repositories have been created, such as GitHub, to ensure reproducibility, keep track of the process and provide open-source resources.
Objective 2: The UNAV team is creating a combined scATACseq + scRNAseq reference of BM and mPB CD34+ cells from healthy donors, together with the integrated analysis of the transcriptomic profile and the expression of 6 surface proteins using CITE-seq technology. A long-term signature score has been developed for the identification of the most primitive HSC subpopulation. Moreover, they set up the experimental conditions to evaluate gene transfer/editing efficiency across HSC subpopulations by using single cell multiomic assessments.
Objective 3: The UKLFR team has started to establishing a robust pipeline for unbiased, genome-wide identification of ON and OFF-target effects triggered by the various genome editing strategies. In particular, UKLFR has adapted CAST-Seq to detect on- and off-target aberrations induced by various base editors in hematopoietic cells. Data obtained are the first-time reports of the genome-wide detection of chromosomal rearrangements and OFF-target effects prompted by both adenine and cytosine base editors.
In parallel, the OSR team investigating cellular pathway activated by DSBs, NHEJ, HR, base and prime editing discover that proliferative stress is a driver of human HSPC dysfunction with fundamental implications for clinical applications.
Objective 4: The CIEMAT team positively tested the efficiency of adenine base editing in correcting the phenotype in lymphoblastic cell lines from a Fanconi Anaemia patient carrying this novel FancA protein. Secondly, they selected as the most efficient the use of a second sgRNA that promotes the generation of a nick in the non-edited strand to correct the stop codon mutation in FANCA gene confirming that the efficiency of prime editing is not affected in comparison with healthy donor cells.
In the context of Diamond Blackfan anemia, the CIEMAT team tested the efficacy and safety of a lentiviral vector-based strategy for the RPS19 defect developed by single cell RNA sequences and in parallel, they tested a knock-in gene editing approach based on AAV to target RPL5 locus in BM CD34+ from healthy donors.
Objective 5: The NBM team progressed in the first objective "Generation of a Gene Therapy DB/Atlas to organize, annotate and share multi-omics data". The core progress can be summarized in: the selection of the meta-data and data formats to be considered in the DB, and the generation of a XPAND repository within the European Genome-Phenome Archive. Additionally, different repositories have been created, such as GitHub, to ensure reproducibility, keep track of the process and provide open-source resources.
The X-PAND scientific project has the potential to significantly elevate the development of new Advanced Therapy Medicinal Products (ATMPs) through genetic Hematopoietic Stem Cell (HSC) engineering. This platform promises to expedite the optimization, validation, and characterization of products. This has the potential to create a substantial impact on various industries involved in the cell and gene therapy arena, ultimately enhancing Europe's technological leadership in this field.
Broadly, X-PAND's scientific advancements in the cell and gene therapy research field revolve around refining the HSC platform and delving into the dynamics of clonal HSCs. The project encompasses the utilization of editing technologies and the analysis of patient cells carrying mutations in DNA repair genes, which will provide valuable insights into cellular biology questions related to DNA Damage Response (DDR). The bioinformatics tools developed during the project will be accessible to the broader scientific community, offering applications that extend well beyond the initial project scope. Furthermore, the discoveries related to off-target effects induced by base editors will contribute to our understanding of the safety of gene editing approaches on HSCs.
X-PAND is poised to effectively harness and strengthen the European Union's scientific talent pool. This is achieved through the involvement of leading investigators with complementary expertise within the consortium and a strategic portfolio of activities aimed at building a network of researchers focused on cell and gene therapy across Europe.
In conclusion, the successful completion of this project will yield tangible benefits for an increasing number of patient categories affected by genetic diseases impacting hematopoietic cells and oncological patients. It will help transform the promise of cell and gene therapy into a clinical reality.
Broadly, X-PAND's scientific advancements in the cell and gene therapy research field revolve around refining the HSC platform and delving into the dynamics of clonal HSCs. The project encompasses the utilization of editing technologies and the analysis of patient cells carrying mutations in DNA repair genes, which will provide valuable insights into cellular biology questions related to DNA Damage Response (DDR). The bioinformatics tools developed during the project will be accessible to the broader scientific community, offering applications that extend well beyond the initial project scope. Furthermore, the discoveries related to off-target effects induced by base editors will contribute to our understanding of the safety of gene editing approaches on HSCs.
X-PAND is poised to effectively harness and strengthen the European Union's scientific talent pool. This is achieved through the involvement of leading investigators with complementary expertise within the consortium and a strategic portfolio of activities aimed at building a network of researchers focused on cell and gene therapy across Europe.
In conclusion, the successful completion of this project will yield tangible benefits for an increasing number of patient categories affected by genetic diseases impacting hematopoietic cells and oncological patients. It will help transform the promise of cell and gene therapy into a clinical reality.