Using Retro Viral Vector for HAX1 Gene Delivery in Severe Congenital Neutropenia

Background and Aim: Neutrophils are considered as one abundantinnate immune cells in peripheral blood that quickly act in bacterial andfungal infections. Decrease and malfunction of these cells cause severeinfectious diseases. Severe neutropenia is recognized with the Absoluteneutrophil count less than 500 per microliter that many of the patientshave a mutation in ELA2, HAX1, G6PC3, GFI1, VPS45, and JAGN1. In2007 HAX1 gene is defined as one of the genes cause severe neutropeniawith autosomal recessive inheritance and the affected patients haverepeated infections.Methods: In the past years, gene therapy is introduced as a therapeuticapproach for some of the monogenic disorders that some of the clinicaltrials have confirmed that. In this current project, the retroviral vectorcontains HAX1 gene isotype A was produced and using Plat-A cells aspackaging cell, viral particles were produced and HEK293T as targetedcells were transduced. To confirm HAX1 protein expression, indirectintracellular staining and flow cytometry was applied and double positivecells – GFP and HAX1 protein – confirmed HAX1 protein expression.Results: Retroviral vector contains HAX1 was confirmed by EcoRIand BgLII double digest and sequencing. Using TurboFect transfectionreagent, Plat-A cell as retroviral packaging cell was infected andfluorescent microscopy confirmed the infection. To quantify infected cell,flow cytometry for GFP was done and it was about 20 %. HEK293T cellwas chosen as a target cell to analysis transduction efficiency. This cellwas transduced with 70 % confluency with help of polybrene. To analysistransduction efficiency, indirect intracellular staining flow cytometry forHAX1 protein was done. Double positive cells for HAX1 protein and GFPwere about 40 %.Conclusion: In the next step, we hope by transduction hematopoieticstem cell of HAX1 deficient patient; we want to analysis in vitro resolvingmaturation arrest and myeloid differentiation series.

کلیدواژه ها:

HAX1 ، Gene therapy ، Severe neutropenia