Biology subjects

Bozhilov, Y.

Publications and source records attributed to Bozhilov, Y..

6 recordsLinked to original sources

bioRxiv2025

Genome editing for treatment of JAK2 V617F-driven myeloproliferative neoplasms

JAK2 V617F is a common haematological driver mutation and underlies most cases of myeloproliferative neoplasms (MPNs). Reducing variant allele frequency (VAF) is an important treatment goal, but no current treatment modalities fully and specifically inhibit mutant signalling. Thus, the consequences of V617F inactivation are unclear, including whether selective inhibition of V617F signalling will eradicate mutant cells due to oncogene addiction. Here, we describe an allele-selective CRISPR-Cas genome editing strategy that achieves selective and efficient JAK2 V617F inactivation in patient stem and progenitor cells. We show that JAK2 V617F heterozygous cells are not oncogene addicted and maintain viability and differentiation potential upon loss of their mutant allele. In contrast, homozygous mutant cells are eradicated upon deletion of both mutant alleles. Across in vitro, organoid, xenotransplantation and single-cell assays, selective deletion of V617F alleles reverts MPN hallmarks including erythroid clonogenicity, inflammatory gene expression signatures, as well as myelofibrosis and splenomegaly phenotypes in an in vivo xenograft model. Collectively, our results show that is it possible to revert heterozygous JAK2 V617F mutant cells to a normal phenotype and suggest that ex vivo genome editing of stem and progenitor cells may be a viable treatment option to achieve rapid and deep reductions in VAF.

cancer biology↗

bioRxiv2025

Polymer-based culture system enables expansion of human haematopoietic stem and progenitor cells while preserving ageing-associated transcriptional programs

The global rise in ageing populations is driving an increase in age-associated diseases, including haematological malignancies such as myelodysplastic syndromes and acute myeloid leukaemia. A major challenge in addressing this burden is the lack of experimental systems that enable mechanistic studies and therapeutic screening in ageing haematopoiesis. Here, we evaluate a polymer-based ex vivo culture platform for studying human haematopoietic stem and progenitor cells (HSPCs) in the context of ageing. Using CD34+ cells from cord blood, bone marrow and peripheral blood from different age groups, we show that this culture system enables robust expansion of HSPCs while preserving age-associated transcriptional programs. Single-cell RNA sequencing revealed enrichment of key progenitor populations with consistent transcriptional identities across donors. Importantly, expanded HSPCs from older individuals retained ageing-associated signatures, such as upregulation of inflammatory pathways. These findings demonstrate that our polymer-based expansion system enables robust modelling of ageing haematopoiesis and provide a single cell transcriptional landscape of this ageing model. We envision that this system could provide a versatile platform for mechanistic studies and pharmacological testing, addressing a critical gap in experimental frameworks for age-related haematological disease research.

cell biology↗

bioRxiv2025

Inhibition of MLLT1 limits growth of MLL-AF4 leukaemias without killing healthy haematopoietic stem cells

A major challenge in cancer therapeutics has been the identification of targets that are selectively toxic to cancer cells while displaying limited effects on healthy counterparts. Toxicities related to blood production from haematopoietic stem and progenitor cells (HSPCs) can be particularly problematic and result in patient morbidity and mortality. Within haematological malignancies, therapy response rates and patient survival vary widely between cancer subtypes, with leukaemias driven by the MLL-AF4 fusion protein associated with poor prognosis. MLLT1 has been recently identified as a key potential target in acute myeloid leukaemia. Here we evaluated a panel of leukaemia cell lines and healthy HSPCs for their sensitivity to the MLLT1 inhibitor SGC-iMLLT. We found that SGC-iMLLT strongly inhibited MLL-AF4-driven leukaemia growth in vitro and in vivo. By contrast, SGC-iMLLT did not alter in vitro colony forming potential of human HSPCs or affect long-term in vivo function of mouse HSPCs. These results suggest that SGC-iMLLT may have a promising therapeutic window in the treatment of MLL-AF4-driven leukaemias, and that further clinical development is warranted.

cancer biology↗

bioRxiv2024

Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing

Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to [~]70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.

cell biology↗

bioRxiv2022

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to mutations in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, it remains elusive how this seemingly ubiquitous factor is specifically upregulated in neural crest cells. Here, we use epigenomic profiling of chick neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7. To our knowledge, this is the first report of tissue-specific regulation of a chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Collectively our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

developmental biology↗

bioRxiv2019

An integrated platform to systematically identify causal variants and genes for polygenic human traits.

Genome-wide association studies (GWAS) have identified over 150,000 links between common genetic variants and human traits or complex diseases. Over 80% of these associations map to polymorphisms in non-coding DNA. Therefore, the challenge is to identify disease-causing variants, the genes they affect, and the cells in which these effects occur. We have developed a platform using ATAC-seq, DNaseI footprints, NG Capture-C and machine learning to address this challenge. Applying this approach to red blood cell traits identifies a significant proportion of known causative variants and their effector genes, which we show can be validated by direct in vivo modelling.

genetics↗