Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells
Introduction
Efficient gene transfer techniques offer the possibility to manage both malignant and non-malignant human diseases. Since hematopoietic stem cells (HSCs) are capable of differentiating into specialized blood cells as well as undergoing self-renewal, these cells are important targets for gene delivery. Several gene therapy trials have been conducted to investigate the use of HSCs in the treatment of cancers, immunodeficiencies, and AIDS [1], [2], [3]. Virus-mediated gene transfer is essential to stably integrate the gene of interest into the genome of target cells and to sustain long-term expression [4]. Retrovirus-mediated gene transfer has been widely used since this method is safe and allows for stable vector integration and expression with simple manipulation [5], [6], [7]. However, this gene transfer method is problematic due to low gene transfer efficiency into hematopoietic cells [8]. The human immunodeficiency-based lentivirus vector (LV) was developed to improve gene transfer efficiency into HSCs, including both dividing and non-dividing cells [9], [10], [11], [12]. Multiple trials have been conducted with this vector to investigate newly discovered cytokines, highly expressed alternative viral receptors, and a modified vector.
Previous gene delivery studies have shown that fibronectin promotes colocalization between stem cells and vector particles and increases gene delivery efficiency [13], [14]. In addition, fibronectin fragments containing the RGDS in segment-1 (CS-1) and heparin-binding domains induce colocalization of virus particles and HSCs, promoting efficient gene transduction into HSCs [15]. The biological activity of fibronectin significantly enhances the phenotypic and functional improvement of murine bone marrow hematopoietic cells. In fact, in vitro culture and in vivo transplantation tests have shown that fibronectin can increase expansion of primitive HSCs/HPCs by 800-fold [16]. Fibronectin is abundantly expressed in the bone marrow microenvironment and supports cell-to-cell adhesion as well as cell-to-matrix adhesion to influence homing, cell survival, and proliferation [17]. The CS-1 domain binds to HSCs/hematopoietic progenitor cells (HPCs) via VLA-4 integrin, and this binding is lost upon differentiation. The RGDS domain binds to HSCs/HPCs as well as many differentiated cells via VLA-5 integrin [18], [19]. On the other hand, the heparin domain binds to virus particles. RN is a fibronectin fragment containing all three of these domains (i.e., RGDS, CS-1, and heparin domains). This fragment not only supports colocalization of target cells expressing integrins and virions, but also enhances retroviral gene delivery into murine and human HSCs [5], [20].
This study was conducted to investigate whether HSCs can be genetically modified through intra-bone marrow (IBM) injection of mice with a combination of RN and LV encoding a VEGF transgene. Our data show that co-injection of lentivirus and RN increases the efficiency of gene delivery into HSCs. Our data also reveal that RN enhances gene delivery in cultured bone marrow-derived c-Kit+ Lin− HPCs and augments the colony-forming ability of these cells. These findings suggest that RN supports in vivo lentiviral gene transfer into HPCs in bone marrow.
Section snippets
LV preparation
The LV contained the packaging construct pMDLg/pRRE, which deleted all accessory genes (vif, vpr, vpu, and nef) and regulatory genes (tat and rev) [21], [22]. Human immunodeficiency virus type 1 expressing the VENUS gene under the control of the human polypeptide chain elongation factor-1α (EF-1α) promoter was produced by transiently transfecting 293T cells with envelop plasmid [pRSV-REV harboring the vesicular stomatitis virus G glycoprotein (VSV-G) gene], packaging plasmid (pCAG-HIVgp), and
Effect of RN on gene delivery in cultured HPCs
We used VSV-G pseudotyped LV encoding the VENUS transgene (summarized in Fig. 1A) to determine the effect of RN on gene transfer into HPCs. The c-Kit+ (stem cell factor receptor/CD117) murine HPCs were isolated from bone marrow by MACS. The purity of isolated c-Kit+ cells showed up to 93%, as determined by FACS analysis. These cells were grown on RN-coated dishes or PBS control dishes and then transduced with LV encoding VENUS under the control of the EF-1α promoter. Gene transfer efficiency
Discussion
HSCs hold great potential for treatment of several human genetic diseases and therefore, are very important targets for genetic modification. In this study, we have shown that RN significantly enhances lentivirus-mediated gene transfer into HPCs and mature hematopoietic cells both in vitro and in vivo. We have found that RN increases transduction efficiency in cultured c-Kit+ Lin− HPCs by 3-fold. The gene delivery with lentivirus in the absence of RN via intra-bone marrow injection increases
Acknowledgements
This work was supported by a grant (Code 200807010340010080500) from the BioGreen 21 Program, Rural Development Administration, and by the Korea Research Foundation Grant (KRF-2008-313-C00674), Republic of Korea.
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