Publications by authors named "Francisco J Roman-Rodriguez"

4 Publications

  • Page 1 of 1

Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia.

Genes (Basel) 2021 Apr 9;12(4). Epub 2021 Apr 9.

Biomedical Research Institute I+12, University Hospital 12 de Octubre, 28041 Madrid, Spain.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, -mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These -mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with mutations are phenocopies of FA-HNSCC cells.
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http://dx.doi.org/10.3390/genes12040548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069753PMC
April 2021

Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia.

Nat Med 2019 09 9;25(9):1396-1401. Epub 2019 Sep 9.

Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date. Mutations in FANCA account for more than 60% of FA cases worldwide. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.
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http://dx.doi.org/10.1038/s41591-019-0550-zDOI Listing
September 2019

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

Blood Adv 2017 Oct 24;1(23):2088-2104. Epub 2017 Oct 24.

International Center for Infectiology Research, Team Enveloped Viruses, Vectors and Innate Responses, INSERM, U1111, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique, Unité Mixte de Recherche (UMR) 5308, Ecole Normale Supérieure de Lyon, Lyon, France.

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34 (hCD34) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34 cells, whereas conventional vesicular stomatitis virus G (VSV-G)-LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc (NSG) mice with H/F-LV transduced prestimulated or resting hCD34 cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34 cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34 progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34 cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.
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http://dx.doi.org/10.1182/bloodadvances.2017007773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728281PMC
October 2017

Therapeutic gene editing in CD34 hematopoietic progenitors from Fanconi anemia patients.

EMBO Mol Med 2017 11;9(11):1574-1588

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 cells from healthy donors, gene targeting was confirmed not only in cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.
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http://dx.doi.org/10.15252/emmm.201707540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666315PMC
November 2017