In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia.

In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia. Eur Cytokine Netw. 2019 Jan 28;: Authors: Farajifard H, Zavvar M, Rajaei T, Noorbakhsh F, Nikougoftar-Zarif M, Azadmanesh K, Kompani F, Rezaei N Abstract 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...
Source: European Cytokine Network - Category: Molecular Biology Tags: Eur Cytokine Netw Source Type: research