Tailoring FPOX enzymes for enhanced stability and expanded substrate recognition.

Fructosyl peptide oxidases (FPOX) are deglycating enzymes that find application as key enzymatic components in diabetes monitoring devices. Indeed, their use with blood samples can provide a measurement of the concentration of glycated hemoglobin and glycated albumin, two well-known diabetes markers. However, the FPOX currently employed in enzymatic assays cannot directly detect whole glycated proteins, making it necessary to perform a preliminary proteolytic treatment of the target protein to generate small glycated peptides that can act as viable substrates for the enzyme. This is a costly and time consuming step. In this work, we used an in silico protein engineering approach to enhance the overall thermal stability of the enzyme and to improve its catalytic activity toward large substrates. The final design shows a marked improvement in thermal stability relative to the wild type enzyme, a distinct widening of its access tunnel and significant enzymatic activity towards a range of glycated substrates.

Biosimilar Gene Therapy: Investigational Assessment of Secukinumab Gene Therapy.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-α), checkpoint inhibitors, and interleukin-17 (IL-17) are critical targets in inflammation and autoimmune diseases. Monoclonal antibodies (mAbs) have a successful portfolio in the treatment of chronic diseases. With the current progress in stem cells and gene therapy technologies, there is the promise of replacing costly mAbs production in bioreactors with a more direct and cost-effective production method inside the patient's cells. In this paper we examine the results of an investigational assessment of secukinumab gene therapy. MATERIALS AND METHODS: In this experimental study, the DNA sequence of the heavy and light chains of secukinumab antibodies were cloned in a lentiviral vector. Human chorionic villous mesenchymal stem cells (CMSCs) were isolated and characterized. After lentiviral packaging and titration, part of the recombinant viruses was used for transduction of the CMSCs and the other part were applied for systemic gene therapy. The engineered stem cells and recombinant viruses were applied for ex vivo and in vivo gene therapy, respectively, in different groups of rat models. In vitro and in vivo secukinumab expression was confirmed with quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and ELISA by considering the approved secukinumab as the standard reference. RESULTS: Cell differentiation assays and flow cytometry of standard biomarkers confirmed the multipotency of the CMSCs. Western blot and qRT-PCR confirmed in vitro gene expression of secukinumab at both the mRNA and protein level. ELISA testing of serum from treated rat models confirmed mAb overexpression for both in vivo and ex vivo gene therapies. CONCLUSION: In this study, a lentiviral-mediated ex vivo and in vivo gene therapy was developed to provide a moderate dose of secukinumab in rat models. Biosimilar gene therapy is an attractive approach for the treatment of autoimmune disorders, cancers and other chronic diseases.

Stable Knockdown of Adenosine Kinase by Lentiviral Anti-ADK miR-shRNAs in Wharton's Jelly Stem Cells.

OBJECTIVES: 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 miRshRNAs 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 miRshRNA 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. CONCLUSIONS: 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 with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model.

OBJECTIVES: 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 2×105 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 2-3×107 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. CONCLUSIONS: 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.

BACKGROUND: 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. METHODS: 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: Results showed that MSCs were efficiently transduced with lentiviral vector resulting in efficient tumor penetration. CONCLUSION: 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.

OBJECTIVE: 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. MATERIALS AND METHODS: 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-IRES- EBI3-EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. RESULTS: 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. CONCLUSION: 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.

Selective ß2 adrenergic agonist increases Cx43 and miR-451 expression via cAMP-Epac.

It has been demonstrated that connexin 43 (Cx43) and microRNAs have significant roles in glioma. Cyclic adenosine monophosphate (cAMP) is suggested to be a regulator of connexins and microRNAs. However, it remains elusive whether cAMP and exchange protein directly activated by cAMP (Epac2), have a regulatory effect on Cx43 and microRNA-451 (miR-451) in astrocytoma cells. We treated 1321N1 astrocytoma cells with a selective ß2 adrenergic agonist and a selective Epac activator with and without adenyl cyclase and protein kinase A inhibition. Cx43 and miR-451 expression were measured. Next, we evaluated the effect of miR-451 overexpression on Cx43 expression. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that cAMP-Epac2 increased Cx43 and miR-451 expression. However, the alteration of miR-451 expression required a higher dose of drugs. Overexpression of miR-451 had no significant effect on Cx43 expression. The MTT assay showed that cAMP-Epac stimulation and miR-451 overexpression had a synergic inhibitory effect on cell proliferation. These findings may expand our understanding of the molecular biology of glioma and provide new potential therapeutic targets.