Lentiviral Based Gene Transduction and Promoter Studies in Human Hematopoietic Stem Cells (hHSCs)

Nadimpalli Ravi S Varma ,Asm Iskander ,Meser M Ali ,Branislava Janic ,Ali S Arbab

doi:10.46582/jsrm.0701005

Nadimpalli Ravi S Varma , Asm Iskander + Show 3 more

Open Access

https://doi.org/10.46582/jsrm.0701005

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Abstract

Human hematopoietic stem cells (hHSCs) have enormous potential for clinical use in cell-based therapies, especially as a gene delivery system. Moreover, lentiviral transduction in stem cells is very often associated with low transduction efficiency and low levels of foreign gene expression. Therefore, it is important to analyze vector and promoter systems that can generate robust foreign gene expression in these cells. In this study, we evaluated and compared the ability of different commercially available promoters to drive the expression of exogenous reporter genes in hHSCs and evaluated the effect of different doses of stem cell growth factors on the expression of transgenes. We used lentivirus based vector system carrying the following promoters: 1) Human cytomegalovirus (CMV) promoter, 2) Simian virus 40 (SV40) promoter, 3) mammalian Ubiquitin C (UBC) promoter and 4) cellular polypeptide chain elongation factor 1 alpha (EF1) promoter. EF1 and CMV promoters robustly drove the expression of green fluorescence protein (GFP) reporter gene, while SV40 and UBC promoters induced very low level of GFP expression. Lentivectors containing EF1 and CMV promoters showed high-level stable GFP expression in human cord blood stem cells for 6 weeks period after post transduction. CD133+ hHSCs stimulated with higher concentration of growth factors exhibited enhancement of transduction rate. Cord blood derived CD133+ hHSCs could be effectively transduced with lentivectors under CMV or EF-1 promoters for the expression of foreign gene.

Highlights

Human cord blood derived hematopoietic stem cell can renew it-self and can differentiate to a variety of specialized cells [1, 2]
To confirm the purity of the culture isolated from cord blood, flow cytometry was done at day 1 to assess the levels of progenitor markers CD133, CD34, hematopoietic CD45, CD117 and CD14 markers, and CD20 and CD3 that are commonly expressed on B and T cells, respectively
The lentiviral particles were produced by transiently transfecting the plasmid constructs (CMV-Green fluorescent protein (GFP), elongation factor 1 (EF1)-GFP, Ubiquitin C (UBC)-GFP and SV40-GFP) along with the pHDMH Gagpol, VSVG, REV plasmids into 293TN cells

Summary

Introduction

Human cord blood derived hematopoietic stem cell (hHSCs) can renew it-self and can differentiate to a variety of specialized cells [1, 2]. The promoters in the lentiviral vector system are critical for maximizing and stabilizing the foreign gene expression in the HSCs. For robust transgene expression in transduced cells, a variety of cellular and viral promoters have been used [14, 15]. The human Ubiquitin C (UBC) promoter was used to generate stable transgenic cell lines in situations where the use of CMV promoter was impeded by different silencing mechanism of host cells [21,22,23]. There is limited data available on the effectiveness of different promoters in human cord blood derived CD34+/AC133+ (CD34+/CD133+) HSCs. it is important to analyze the promoter strength in the hHSCs before using these cells as a gene delivery vehicle for therapeutic purposes. We attempted to determine optimal promoter for transgene expression in hHSCs and to enhance the viral transduction rate in hHSCs using cell growth factors

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