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Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes.

ABSTRACT: Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34⁺ cells/μL required to initiate apheresis. A median of 21.8 CD34⁺ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34⁺ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34⁺ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34⁺ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34⁺ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.

SUBMITTER: Sevilla J

PROVIDER: S-EPMC8390450 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes.

Sevilla Julián J Navarro Susana S Rio Paula P Sánchez-Domínguez Rebeca R Zubicaray Josune J Gálvez Eva E Merino Eva E Sebastián Elena E Azqueta Carmen C Casado José A JA Segovia José C JC Alberquilla Omaira O Bogliolo Massimo M Román-Rodríguez Francisco J FJ Giménez Yari Y Larcher Lise L Salgado Rocío R Pujol Roser M RM Hladun Raquel R Castillo Ana A Soulier Jean J Querol Sergi S Fernández Jesús J Schwartz Jonathan J García de Andoín Nagore N López Ricardo R Catalá Albert A Surralles Jordi J Díaz-de-Heredia Cristina C Bueren Juan A JA

Molecular therapy. Methods & clinical development 20210612

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34<sup>+</sup> cells/μL required to initiate apheresis. A median of 21.8 CD34< ...[more]

PMID: 34485595

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Project description:We used Fancd2-/- mice to understand its mechanism of action. Transcriptome analysis of cKit+ Sca1+ Lin- (KSL) cells discovered that only four genes changed their expression levels significantly after chronic OXM administration in both Fancd2−/− and wild-type mice: mKi67 and Cenpf were up-regualted by 1.4 fold; Spp1 and Oasl2 were significantly down-regulated by 10.5 and 1.5 fold, respectively. Both mKi67 and Cenpf genes are cell cycle-regulated genes and proliferation markers. Their up-regulation was consistent with our observation in flow cytometry analysis that oxymetholone stimulated the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis showed no effects on mTert mRNA expression with chronic androgen therapy, but instead suggested down-regulation of Spp1 and Oasl2 as an important mechanism for the drug’s action. Our RNAseq analysis also revealed that Fancd2−/− KSL cells showed clear changes in mRNA expression profiles compared to wild-type controls: 430 genes were down-regulated by more than 1.5 fold, whereas 159 genes were up-regulated. Gene ontology analysis revealed key pathways to be significantly altered in Fancd2−/− KSL cells. Besides the abnormal cell cycle status expected from our earlier flow cytometry analysis, surprisingly we noticed that a group of genes involved in immune responses and inflammation, comprising Cfp (Properdin), Socs2, Ccr1, Ccr2, Ccr5, Chga (Chromogranin A), Ifi30 (Interferon Gamma-Inducible Protein 30), Lgmn, Txn, and Sell (selectin L), were up-regulated in Fancd2−/− KSL cells. We therefore hypothesize that some genes up-regulated in FA HSPCs may be part of an innate immune response to DNA damage. In addition, whole bone marrow cells were also analyzed in parallel with KSL cells. As compared to whole bone marrow cells, the genes enriched in KSL cells in wild-type mice were listed in details in the corresponding publication. This information can be a good resource for the future gene expression analysis of HSPCs. Finally, we compared the gene expression profiles of early progenitors between OXM-treated and placebo-treated mice. There were no significant differences at all in gene expression between OXM-treated wild-type erythroid progenitors and their placebo-treated wild-type counterparts, with no genes displaying an expression change higher than 1.2 fold. Importantly, no up-regulation of EPO-inducible genes such as Socs1, Socs2, Socs3, and Cish was seen in wild-type mice treated with OXM. Furthermore, there was no differential expression of the well-known EPO target transferrin receptor or any other major players of the Epo-R signaling network such as Bcl2l1, Cdc25a, Btg3, Ccnd2, Lyl1, Pim3, and Tnfrsf13c. These results indicate that EPO might not play a role in the action of OXM in the erythroid lineage. The goal of this study is to investigate gene expression changes in Fancd2 knockout mice in response to oxymetholone treatment. The study focuses on two bone marrow cell populations: cKit+ Sca1+ Lin- cells (representing hematopoietic stem and progenitor cells) and Ter119+/CD71high/FSChigh cells (representing proerythroblasts and basophilic erythroblasts). Both populations were sorted twice by FACS to ensure the purity. Cells of interest were collected in Trizol and RNA was isolated using RNAeasy mini prep kit. mRNAs were positively selected using oligo(dT)- Dynobeads and treated with DNase I. RNAseq libraries were then constructed using Illumina TruSeq RNA Sample Prep Kit and sequenced as 51 base-length reads on an Illumina HiSeq 2000 genome analyzer. For KSL libraries, each sample represented total mRNA isolated from pooled KSL cells of 5 individual mice; for basophilic erythroblast libraries, each library represented total mRNA isolated from basophilic erythroblasts of one individual mouse; for whole bone marrow libraries, each sample represented a combined library originally from 5 individual mice. All reads were mapped to the mouse reference genome (version mm9) using Bowtie short read aligner software (http://bowtie-bio.sf.net). Most of the data analysis was performed using EdgeR GLM algorithms. For the comparison of oxymetholone KSL libraries vs placebo KSL libraries, more stringent pair-wise comparisons were used to keep a consistent flow cytometric setting among each pair. The common gene list was the one shared by all three comparisons: COM17 vs HSC_101b, HSC_13 vs HSC_18, and HSC_23 vs QZ_35 for Fancd2−/− KSL cells; HSC_3 vs QZ_36, HSC_22 vs HSC_24, and COM15 vs COM16 for wild-type KSL cells. Data-mining and pathway analysis were carried out with the MetaCore integrated software suite (Thomson Reuters, New York, USA).

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Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes.

Publications

Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes.

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