Stable knockdown of adenosine kinase by lentiviral anti-ADK miR-shRNAs in wharton's jelly stem cells

Objective: In this study, we describe an efficient approach for stable knockdown of adenosine kinase [ADK] using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells [hWJMSCs]. These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy

Materials and Methods: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA [shRNAs]. First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction [qRT-PCR] was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker [CD44+] and lack of expression of hematopoietic lineage marker [CD45-]. Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs

Results: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level

Conclusion: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors [AAV] might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy

Multiple sclerosis gene therapy using recombinant viral vectors: overexpression of IL-4, IL-10 and leukemia inhibitory factor in wharton's jelly stem cells in the EAE mice model

Objective: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 [IL-4], IL-10 and leukemia inhibitory factor [LIF] in Wharton's jelly stem cells [WJSCs] in the experimental autoimmune encephalomyelitis [EAE] mice model

Materials and Methods: In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector [as the transfer vector]. Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 [HEK-293T] cells with helper plasmids which produced recombinant lentiviral viruses [rLV]. WJSCs were transduced with rLV to make recombinant WJSC [rWJSC]. In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction [qPCR], enzyme-linked immunosorbent assay [ELISA] and western blot [WB] analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×10[5] rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions

Results: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×10[7] infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group

Conclusion: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis [MS]. In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Efficient lentiviral transduction of adipose tissue-derived mouse mesenchymal stem cells and assessment of their penetration in female mice cervical tumor model

Although the incidence of cervical cancer has reduced during last years, but it causes mortality among women. Many efforts have performed to develop new drugs and strategy for treatment of cervical cancer. Adipose Tissue-Derived mouse Mesenchymal Stem Cells [MSCs] has many advantages which make them a suitable choice as a cell therapeutic agent in cancer treatment. In this study, we aimed to develop an improved protocol for Mouse MSCs transduction as well as assess the homing capacity and incorporation of MSCs in cervical cancer model. MScs were isolated from the mouse adipose tissue and characterized by differentiation and flow cytometry. In our study, lentiviral vector transductions of MSCs performed. Their penetrations were detected in tissue sections after injection of transduced MSCs to female C57BL/6 mice as a cervical cancer model. Results showed that MSCs were efficiently transduced with lentiviral vector resulting in efficient tumor penetration. The results provide evidence that MSCs were able to penetrate into the tumor mass of cervical tumor model and are good vehicles for gene transfer to cervical cancer

Lentiviral mediating genetic engineered mesenchymal stem cells for releasing IL-27 as a gene therapy approach for autoimmune diseases

Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 [Th17] and interleukin [IL]-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells [MSCs] that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases. In this experimental study, we isolated adipose-derived MSCs [AD-MSCs] from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 [p28 and EBI3] were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 [Epstein-Barr virus induced gene 3] were determined by real time polymerase chain reaction [PCR]. MSCs were transduced by a pCDH-CMV-p28-IRESEBI3- EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27. The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics