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In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia Volume 29, numéro 4, October-November-December 2018

Illustrations

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Tableaux

Auteurs
1 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of microbiology and Immunology, faculty of Veterinary medical, University of Tehran, Tehran, Iran
3 Blood Transfusion Research Centre, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
4 Virology Department, Pasteur Institute of Iran, Tehran, Iran
5 Division of Haematology and Oncology, Children's Medical Center, Pediatrics Center of Excellence, Tehran university of medical sciences, Tehran, Iran
6 Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
7 Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran
* Correspondence: Nima Rezaei, Children's Medical Center Hospital, 62 Qarib St, Keshavarz Blvd, Tehran 14194, Iran
  • Mots-clés : severe congenital neutropenia, HAX1, vector, gene therapy
  • DOI : 10.1684/ecn.2018.0419
  • Page(s) : 146-52
  • Année de parution : 2018

Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.