Hilal Gul-Uludag

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2kDa PEI (~2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.

CXCR4 transfection of cord blood mesenchymal stromal cells with the use of cationic liposome enhances their migration toward stromal cell-derived factor-1.

BACKGROUND AIMS The interaction between stromal cell-derived factor (SDF)-1 and its receptor CXCR4 is one of the mechanisms by which mesenchymal stromal cells (MSCs) are recruited to sites of injury. SDF-1 is upregulated in damaged tissues, but because the surface expression of CXCR4 on cultured MSCs is low, we investigated whether the delivery of CXCR4 into MSCs with the use of the cationic liposomal reagent IBAfect would increase their migration toward SDF-1. METHODS We examined (i) the effect of MSC confluency, passage number, duration of transfection and amount of IBAfect and plasmid on transfection efficiency as determined by flow cytometric analysis of CXCR4 and (ii) whether IBAfect-mediated CXCR4 transfection affected the viability, proliferation and differentiation of MSCs as well as their response toward an SDF-1 gradient in a trans-Matrigel migration assay. RESULTS We found that transfection efficiency of up to 40% was achieved after 24-h transfection of 50% confluent MSCs (at passage 4) with an IBAfect:plasmid ratio of 3.6 μL:0.6 μg, and CXCR4 transcript expression in transfected MSCs was 10(5)-fold higher than in non-transfected cells. Transfected MSCs retained their ability to differentiate to osteocytes and chondrocytes but had lower proliferation. Importantly, overexpression of surface CXCR4 with the use of IBAfect significantly increased (>3-fold) the number of cells migrating toward an SDF-1 gradient relative to cells migrating to media alone, compared with non-transfected cells (1.3-fold). CONCLUSIONS Our results suggest that IBAfect-mediated delivery of CXCR4 into MSCs is a highly efficient technique that may be useful for enhancing the recruitment of systemically infused MSCs for tissue repair.

Cationic Liposome-Mediated CXCR4 Gene Delivery into Hematopoietic Stem/Progenitor Cells: Implications for Clinical Transplantation and Gene Therapy

The chemokine stromal cell-derived factor (SDF)-1α/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34(+) HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome. Therefore, a method for CXCR4 gene delivery into HSPC that is safe, nonviral, and highly efficient is needed to improve clinical transplantation and gene therapies. In this work, we investigated the nonviral CXCR4 gene delivery into HSPC using the cationic liposome agent IBAfect. We used CD34(+) cells from cord blood and the models of immature hematopoietic cells expressing CD34 antigen, namely, leukemic cell lines KG-1a and KG-1. Transfection efficiency was determined by flow cytometric analysis 12, 24, 48, and 72 h after transfection, and the viability of cells analyzed by trypan blue exclusion and MTS assays. The functional response of CXCR4-transfected HSPC toward an SDF-1α gradient was determined by chemotaxis assay. We found that ~25% transfection is achieved for KG-1a and KG-1 cells and 20% for HSPC, and that the viability of CXCR4-transfected HSPC is not significantly altered. More importantly, overexpression of CXCR4 using IBAfect significantly increased the chemotaxis of KG-1 cells and HSPC toward SDF-1α. However, we tested 2 other commercially available cationic liposomes (Lipofectamine 2000 and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) in parallel, and we found that they failed to deliver the CXCR4 gene into cells under the same conditions. These results suggest that IBAfect-mediated in vitro gene delivery to overexpress CXCR4 on HSPC is a safe and efficient technique with great potential for improving the efficacy of HSPC transplantation and gene therapy protocols.

Effective non-viral delivery of siRNA to acute myeloid leukemia cells with lipid-substituted polyethylenimines.

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ∼0.5 and led to siRNA/polymer complexes of ∼100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.

Progress in RNAi-mediated Molecular Therapy of Acute and Chronic Myeloid Leukemia

Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia. We first present unique aspects of RNAi-mediated therapy, followed by a brief background on the delivery technology of RNAi reagents. The need for leukemia-specific delivery of siRNA is discussed by describing approaches that targeted agents to leukemic cells. Pharmacokinetics and biodistribution of RNAi agents are then presented, highlighting the critical issues pertinent to emerging siRNA therapy. Efforts to deliver specific RNAi therapies are then summarized in the context of expected clinical outcomes, focusing on limiting leukemic cell survival, sensitizing malignant cells to chemotherapy, mobilization of leukemic cells, and eradication of leukemic stem cells. We conclude with a perspective on the future of RNAi therapy, emphasizing the technological requirements and mechanistic challenges for clinical entry.

Targeting CXCR4/SDF-1 axis by lipopolymer complexes of siRNA in acute myeloid leukemia

In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.

siRNA therapy in cutaneous T-cell lymphoma cells using polymeric carriers

Cutaneous T-cell lymphomas (CTCLs) arise from specific molecular aberrations that lead to uncontrolled cell proliferation. RNA interference (RNAi) with short interfering RNAs (siRNAs) is a feasible approach to interrupt aberrant signal processing in CTCL cells, but functional biomaterial carriers are needed to effectively deliver siRNAs intracellularly. Towards this goal, we explored the utility of lipid-substituted polyethylenimines (PEI) carriers in a cell model of CTCL. Using caprylic and linoleic acid substituted 2 kDa PEI (PEI-CA and PEI-LA, respectively), we showed effective delivery of siRNA to T-lymphocyte Hut78 and Jurkat cells, but silencing of a model protein (Green Fluorescent Protein, GFP) was possible only in the Hut78 cells. To enhance siRNA delivery to Hut78 cells, a high siRNA: carrier ratio used to assemble the complexes and centrifugation of cells in the presence of complexes were found effective. The toxicities of PEI-CA and PEI-LA were significantly lower than other commercial carriers, 25 kDa PEI and Lipofectamine(®) RNAiMAX. This might have contributed to reduced siRNA delivery efficiency of the latter carriers. Screening several endogenous targets led us to identify phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and cyclin-dependent kinase 18 (CDK18) as viable targets to induce siRNA-mediated cell growth inhibition. The results of this study identified promising polymeric carriers and molecular targets that could control proliferation of CTCL cells based on RNAi therapy.

Ultrasound-mediated gene delivery into hard-to-transfect KG-1 cells

In this paper, we explore the possibility of increasing the transfection rate of hard-to-transfect cells (such as KG-1 cells) using ultrasound-mediated gene delivery (USD). Single application ultrasound induced as well as the synergistic properties of ultrasound with commonly used transfection reagents and repeated sessions USD were investigated and evaluated using GFP plasmid. KG-1 cells were not transfected by combining a commonly used transfection reagent and USD. However, repeated ultrasound-mediated treatments were able to increase transfection rate to 15% with a viability of 97%, as compared to 2% with the transfection reagent, Lipofectamine, alone.