Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A

Muhammad Elnaggar, Anjud Al-Mohannadi, Dhanya Kizhakayil, Christophe Michel Raynaud, Sharefa Al-Mannai, Giusy Gentilcore, Igor Pavlovski, Abbirami Sathappan, Nicholas Van Panhuys, Chiara Borsotti, Antonia Follenzi, Jean Charles Grivel, Sara Deola

Research output: Contribution to journalArticle

Abstract

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

Original languageEnglish
Pages (from-to)1-12
Number of pages12
JournalMolecular Therapy - Methods and Clinical Development
Volume17
DOIs
Publication statusPublished - 12 Jun 2020

Fingerprint

Factor VIII
Hemophilia A
Genetic Therapy
Blood Cells
Flow Cytometry
Proteins
Phosphoglycerate Kinase
Unfolded Protein Response
Genes
Reactive Oxygen Species
Monoclonal Antibodies
Gene Expression
Cell Line

Keywords

  • Cellular stress
  • F8
  • Factor VIII
  • FVIII
  • FVIII FACS
  • FVIII flow cytometry
  • FVIII in blood cells
  • HA Gene Therapy
  • Hemophilia A
  • Lentiviral vector
  • PBMCs FVIII

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells : A New Tool to Assess Gene Therapy Efficiency for Hemophilia A. / Elnaggar, Muhammad; Al-Mohannadi, Anjud; Kizhakayil, Dhanya; Raynaud, Christophe Michel; Al-Mannai, Sharefa; Gentilcore, Giusy; Pavlovski, Igor; Sathappan, Abbirami; Van Panhuys, Nicholas; Borsotti, Chiara; Follenzi, Antonia; Grivel, Jean Charles; Deola, Sara.

In: Molecular Therapy - Methods and Clinical Development, Vol. 17, 12.06.2020, p. 1-12.

Research output: Contribution to journalArticle

Elnaggar, M, Al-Mohannadi, A, Kizhakayil, D, Raynaud, CM, Al-Mannai, S, Gentilcore, G, Pavlovski, I, Sathappan, A, Van Panhuys, N, Borsotti, C, Follenzi, A, Grivel, JC & Deola, S 2020, 'Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A', Molecular Therapy - Methods and Clinical Development, vol. 17, pp. 1-12. https://doi.org/10.1016/j.omtm.2019.11.003
Elnaggar, Muhammad ; Al-Mohannadi, Anjud ; Kizhakayil, Dhanya ; Raynaud, Christophe Michel ; Al-Mannai, Sharefa ; Gentilcore, Giusy ; Pavlovski, Igor ; Sathappan, Abbirami ; Van Panhuys, Nicholas ; Borsotti, Chiara ; Follenzi, Antonia ; Grivel, Jean Charles ; Deola, Sara. / Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells : A New Tool to Assess Gene Therapy Efficiency for Hemophilia A. In: Molecular Therapy - Methods and Clinical Development. 2020 ; Vol. 17. pp. 1-12.
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