Search

Melanoma, a malignant form of skin cancer, has been swiftly increasing in recent years. Although there have been significant advancements in clinical treatment underlying a well-understanding of melanoma-susceptible genes and the molecular basis of melanoma pathogenesis, the permanency of response to therapy is frequently constrained by the emergence of acquired resistance and systemic toxicity. Conventional therapies, including surgical resection, chemotherapy, radiotherapy, and immunotherapy, have already been used to treat melanoma and are dependent on the cancer stage. Nevertheless, ineffective side effects and the heterogeneity of tumors pose major obstacles to the therapeutic treatment of malignant melanoma through such strategies. In light of this, advanced therapies including nucleic acid therapies (ncRNA, aptamers), suicide gene therapies, and gene therapy using tumor suppressor genes, have lately gained immense attention in the field of cancer treatment. Furthermore, nanomedicine and targeted therapy based on gene editing tools have been applied to the treatment of melanoma as potential cancer treatment approaches nowadays. Indeed, nanovectors enable delivery of the therapeutic agents into the tumor sites by passive or active targeting, improving therapeutic efficiency and minimizing adverse effects. Accordingly, in this review, we summarized the recent findings related to novel targeted therapy methods as well as nanotechnology-based gene systems in melanoma. We also discussed current issues along with potential directions for future research, paving the way for the next-generation of melanoma treatments.

Inflammation is a vital process in response to injury and infection. However, hyper-inflammation have been linked to the pathophysiology of diseases such as atherosclerosis, cancers, neuroinflammat...

Dysregulated mRNA–miRNA profiles might have the prospective to be used for early diagnosis of gastrointestinal cancers, estimating survival, and predicting response to treatment. Here, a novel biomarker based on miRNAs binding to mRNAs in single nucleotide polymorphism (SNP) sites related to gastrointestinal cancers is introduced that could act as an early diagnosis. The electronic databases used for the recruiting published articles included EMBASE, SCOPUS, Web of Science, and PubMed, based on MESH keywords and PRISMA methodology. Based on the considered criteria, different experimental articles were reviewed, during which 15 studies with the desired criteria were collected. Accordingly, novel biomarkers in prediction, early prognosis, and diagnosis of gastrointestinal cancers were highlighted. Moreover, it was found that 20 SNP sites and 16 miRNAs were involved in gastrointestinal cancers, with altered expression patterns associated with clinicopathological and demographic data. The results of this systematic study revealed that SNPs could affect the binding of miRNAs in the SNP sites that might play a principal role in the progression, invasion, and susceptibility of gastrointestinal cancers. In addition, it was found that the profiles of SNPs and miRNAs could serve as a convenient approach for the prognosis and diagnosis of gastric and colorectal cancers.

Simple Summary Cancer-associated fibroblasts (CAFs; as components of cancer stroma) use different signaling pathways to promote tumor progression and growth, invasion, and metastasis in diverse cancers. They have crosstalk with cancer cells during tumor progression. To investigate this crosstalk, in this study, a coculture system of CAF cells isolated from breast cancer patients with breast cancer cells was used to investigate leukemia inhibitory factor (LIF) production from CAFs. LIF is a signaling molecule that activates distinct signaling pathways through which cell cycle progression, cell death, adhesion, migration, and tumorigenesis are regulated. Abstract Leukemia inhibitory factor (LIF), as a member of the interleukin-6 cytokine family, plays a complex role in solid tumors. However, the effect of LIF as a tumor microenvironment factor on plasticity control in breast cancer remains largely unknown. In this study, an in vitro investigation is conducted to determine the crosstalk between breast cancer cells and fibroblasts. Based on the results, cancer-associated fibroblasts are producers of LIF in the cocultivation system with breast cancer cells. Treatment with the CAF-CM and human LIF protein significantly promoted stemness through the dedifferentiation process and regaining of stem-cell-like properties. In addition, the results indicate that activation of LIFR signaling in breast cancer cells in the existence of CAF-secreted LIF can induce Nanog and Oct4 expression and increase breast cancer stem cell markers CD24−/CD44+. In contrast, suppression of the LIF receptor by human LIF receptor inhibition antibody decreased the cancer stem cell markers. We found that LIF was frequently overexpressed by CAFs and that LIF expression is necessary for dedifferentiation of breast cancer cell phenotype and regaining of cancer stem cell properties. Our results suggest that targeting LIF/LIFR signaling might be a potent therapeutic strategy for breast cancer and the prevention of tumor recurrence.

Background and purpose: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene ( ctxA ) for inhibiting CT toxin production in Vibrio cholera (V. cholera) . Experimental approach: An engineered ZFN was designed to target the catalytic site of the ctxA gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and E2-crimson plasmid and transformed to Escherichia coli (E. coli) Top10 and V. cholera . The efficiency of ZFN was evaluated by colony counting. Findings/Results: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed E. coli . The ctxA gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of E. coli Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of V. cholera with E2-crimson vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay. Conclusions and implications: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.

Coronavirus disease-19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 virus strains has geographical diversity associated with diverse severity, mortality rate, and response to treatment that were characterized using phylogenetic network analysis of SARS-CoV-2 genomes. Although, there is no explicit and integrative explanation for these variations, the genetic arrangement, and stability of SARS-CoV-2 are basic contributing factors to its virulence and pathogenesis. Hence, understanding these features can be used to predict the future transmission dynamics of SARS-CoV-2 infection, drug development, and vaccine. In this review, we discuss the most recent findings on the mutations in the SARS-CoV-2, which provide valuable information on the genetic diversity of SARS-CoV-2, especially for DNA-based diagnosis, antivirals, and vaccine development for COVID-19.

Cancer patients, as a highly vulnerable population, are receiving a great deal of attention in the current crisis of coronavirus 2019 (COVID-19). To date, shreds of evidence are not sufficient to the description of COVID-19 outcomes in patients with cancer. This study was performed to evaluate the demographic and clinical characteristics and subsequent outcomes of COVID-19 in cancer patients.A hospital-based study was conducted involving 66 cancer patients with a confirmed diagnosis of COVID-19 from January 15, 2020, to December 21, 2020, in Isfahan, Iran. The clinical information was collected by interview and medical records. The statistical analyses were performed to describe categorical variables as well as mean, standard deviation, median, and the interquartile range for quantitative variables.In our study, 66 cancer patients with confirmed COVID-19 (age: 17-97 years; 50% female) were included. Leukemia and bone marrow cancer with a frequency of 25.7% were the most common types of cancer among them. Cancer patients mostly complained of fever, cough and fatigue, and shortness of breath. Among 76.9% of patients discharged from the hospital with relative recovery, 23% died; the most common cause of death was acute respiratory distress syndrome. Age, gender, and type of cancer did not affect cancer mortality. COVID-19 had no potential effect to increase the risk of side effects of anticancer therapies.The results of our studies revealed that cancer is an important risk factor for the higher rate of mortality in patients with COVID-19. These findings could help physicians for the management, treatment, and supportive care of COVID-19 cancer patients.

In late 2019, a new member of the Coronaviridae family, officially designated as “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth.

Human respiratory viral infections are the leading cause of morbidity and mortality around the world. Among the various respiratory viruses, coronaviruses (e.g., SARS-CoV-2) have created the greatest challenge and most frightening health threat worldwide. Human coronaviruses typically infect the upper respiratory tract, causing illnesses that range from common cold-like symptoms to severe acute respiratory infections. Several promising vaccine formulations have become available since the beginning of 2021. Nevertheless, achievement of herd immunity is still far from being realized. Social distancing remains the only effective measure against SARS-CoV-2 infection. Nanobiotechnology enables the design of nanobiosensors. These nanomedical diagnostic devices have opened new vistas for early detection of viral infections. The present review outlines recent research on the effectiveness of nanoplatforms as diagnostic and antiviral tools against coronaviruses. The biological properties of coronavirus and infected host organs are discussed. The challenges and limitations encountered in combating SARS-CoV-2 are highlighted. Potential nanodevices such as nanosensors, nanobased vaccines, and smart nanomedicines are subsequently presented for combating current and future mutated versions of coronaviruses.

Coronavirus disease-19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 virus strains has geographical diversity associated with diverse severity, mortality rate, and response to treatment that were characterized using phylogenetic network analysis of SARS-CoV-2 genomes. Although, there is no explicit and integrative explanation for these variations, the genetic arrangement, and stability of SARS-CoV-2 are basic contributing factors to its virulence and pathogenesis. Hence, understanding these features can be used to predict the future transmission dynamics of SARS-CoV-2 infection, drug development, and vaccine. In this review, we discuss the most recent findings on the mutations in the SARS-CoV-2, which provide valuable information on the genetic diversity of SARS-CoV-2, especially for DNA-based diagnosis, antivirals, and vaccine development for COVID-19.

BackgroundThe most common malignancy is breast cancer, among women in the world. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Cancer associated fibroblasts (CAFs) play a critical role to support tumor cells in all aspect of cancer development such as cell proliferation, migration and angiogenesis. MiRNAs are one of the regulatory molecules that regulate the genes contributing to cell growth, differentiation, migration and apoptosis. Based on other studies, miR-200c, as a tumor suppressor, has low expression levels in cancer associated fibroblasts. In this investigation, effect of miR-200c overexpression was evaluated on proliferation, migration and angiogenesis of TNBC cells. MethodsThe fibroblasts were isolated from normal and cancer breast tissue. MiR-200c expression was assessed using RT PCR in cancer associated fibroblasts (CAFs) and normal fibrobalasts (NFs) and then, were transfected using miR-200c. Finally, its effect on proliferation, migration and angiogenesis of TNBC cells were evaluated. ResultsOur results confirm that in presence of miR-200c transfected fibroblasts, the proliferation, migration and angiogenesis of cancer cells significantly decreased. This effect may be due to the reduction of growth factors provided by CAFs after miRNAs dysregulation. ConclusionThese results suggest miR-200c act as an effective tumor suppressor in many aspects of TNBC cancer development and can be regarded as a potential therapeutic tool for breast cancer in the future.

Breast cancer is the second leading cause of cancer-related death among females, worldwide. The cytokines are proteins that have a significant role in the development of tumor growth. Leukemia inhibitory factor (LIF) of the interleukin-6 cytokines superfamily plays a significant role, by the modulation of many signaling pathways. This study summarizes some current works in breast cancer, in which LIF intervention is being discussed. LIF promotes tumorigenesis, invasion, migration of breast cancer cells in vitro, metastasis of breast cancer in vivo, epithelial-mesenchymal transition, and mediates pro-invasive activation of stromal fibroblast. LIF contributes to inducing growth, tumorigenesis, and metastasis of breast cancer and is a significant biomarker for breast tumors and can be a therapeutic target for clinical intervention.

Organoids are powerful systems to facilitate the study of individuals' disorders and personalized treatments. This emerging technology has improved the chance of translatability of drugs for preclinical therapies and mimicking of the complexity of organs, proposing numerous approaches for human disease modeling, tissue engineering, drug development, diagnosis, and regenerative medicine. In this review, we outline the history of organoid technology and summarize its faithful applications, and then we discuss the challenges and limitations encountered by three-dimensional organoids. Finally, we propose that human organoids offer a basic mechanistic infrastructure for “human modeling” systems to prescribe personalized medicines.

BackgroundOn December 2019, Novel coronavirus disease (COVID-19) was detected in Wuhan, China, and then spread around the world. There is little information about effects of COVID-19 on Pregnant women and newborns as a sensitive population. The current study is a systemic review and Meta-analysis to measure the risks and determine the presentations of COVID-19 in pregnant women and newborn.Methodsonline data bases were searched on march 20. Heterogeneity of the included studies was assessed using the Cochran Q test and Higgins I2 statistic and expressed as percentage. All data were analyzed with 95% confidence intervals.ResultsA total of 7 studies involving 50 participants with Positive test of COVID-19 were enrolled. Mean age of pregnant women was 30.57 years old and the Mean Gestational age was 36.9 weeks. Other variables such as Apgar score, birth weight, Sign and symptoms, Complications and Laboratory data were Analyzed.ConclusionOur findings showed same clinical characteristics in pregnant women as in non-pregnant adults, with the main symptoms being cough and fever. No vertical transmission was seen and all patients delivered healthy neonates. Our findings would be of great help to the decision making process, regarding the management of pregnant women diagnosed with COVID-19.

The emerging new Coronaviridae member, nCoV 19, outbreak announced a pandemic by WHO with an increased morbidity and mortality rate worldwide. nCoV 19 known as the third highly pathogen coronavirus in the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), the nCoV 19. The renin-angiotensin (RAS) signaling pathway, oxidative stress and cell death, cytokines storm and endothelial dysfunction are four major pathways involved in the pathogenesis of nCoV 19. Acute respiratory distress syndrome (ARDS) generally develops with a massive oxidative/nitrosative stress following virus entry and RAS activation. The DNA damage subsequent to oxidative burst activates poly-ADP ribose polymerase-1 (PARP-1), viral macrodomain (NSP3) poly (ADP-ribose) glycohydrolase (PARG) and transient receptor potential channel, melastatin 2 (TRPM2) in a sequential manner ultimately leading to apoptosis and necrosis due to NAD and ATP depletion. Regarding the molecular mechanisms involved in nCoV 19 pathogenesis, angiotensin II receptor blockers and/or PARP, PARG and TRPM2 blockers could be engaged as therapeutic candidates for inhibition of RAS and quenching oxidative stress, respectively. In this review, the molecular aspects of nCoV 19 pathogenesis would be studied precisely and possible therapeutic targets would be proposed. It is recommended to evaluate the proposed drugs and supplements via registered clinical trials along with conventional guideline-based multi-drug regimen.

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.

Background and purpose: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene (ctxA) for inhibiting CT toxin production in Vibrio cholera (V. cholera). Experimental approach: An engineered ZFN was designed to target the catalytic site of the ctxA gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and E2-crimson plasmid and transformed to Escherichia coli (E. coli) Top10 and V. cholera. The efficiency of ZFN was evaluated by colony counting. Findings/Results: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed E. coli. The ctxA gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of E. coli Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of V. cholera with E2-crimson vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay. Conclusions and implications: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4β1 and lymphocyte Peyer’s patch adhesion molecule-1 or α4β7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 (ITGA4) is overexpressed in some immune disorders and cancers. Thus, inhibition of ITGA4 could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress ITGA4 expression. The ITGA4 3' UTR was amplified, cloned in the Z2827-M67-(ITGA4) plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the ITGA4 expression analysis. The findings of this study showed that miR-30a downregulated ITGA4 expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the ITGA4 gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy.

Prostate cancer usually develops to a hormone-refractory state that is irresponsive to conventional therapeutic approaches. Therefore, new methods for treating aggressive prostate cancer are under development. Because of the importance of androgen receptors (ARs) in the development of the hormone-refractory state and AR mechanism of action, this study was designed. A single-stranded DNA as an aptamer was designed that could mimic the hormone response element (HRE). The LNCaP cells as an AR-rich model were divided into three sets of triplicate groups: the test group was transfected with Aptamer Mimicking HRE (AMH), Mock received only transfection reagents (mock) and a negative control. All three sets received 0, 10 and 100 nM of dehydroepiandrosterone (DHEA) separately. Data analysis showed hormone dependency of LNCaP cells in the negative control group upon treatment with 10 and 100 nM DHEA (compared with cells left untreated (P=0.001)). Transfection of AMH resulted in significant reduction of proliferation in the test group when compared with the negative control group with 10 (P=0.001) or 100 nM DHEA (P=0.02). AMH can form a hairpin structure at 37 °C and mimic the genomic HRE. Hence, it is capable of effectively competing with genomic HRE and interrupting the androgen signaling pathway in a prostate cancer cell line (LNCaP).

Background: Familial hypercholesterolemia (FH) leads to elevated low-density lipoprotein cholesterol (LDL-C) levels in plasma. Mutations of its related gene; apolipoprotein B (APOB) is seen in about two percent of the patient with FH. Thyroid disease is usually part of the exclusion criteria for the detection of FH which alters the lipid profile. We evaluated mutations in the APOB gene in patients with high LDL-C levels. Materials and Methods: Patients aged between 2 and 80 years with at least one LDL-C level of more than 190 mg/dl were selected (120 patients) from Isfahan Laboratories. Blood samples were obtained from all patients. Genomic DNA was extracted. Primer sequences were designed by Oligo 7.60 to amplify the desired 844 bp region of exon 26 of the APOB gene containing R3500Q and R3500W variants associated with FH. Results: Overall, two patients showed a heterozygous form of a common pathogenic variant in exon 26 named c. 10579 C > T (R3500W, cDNA.10707), and one patient was hypothyroidism. We also recognized another nonpathognomonic variant c. 10913G > A (rs1801701, cDNA.11041) in 13 patients, two of them were hypothyroidism. Conclusion: This study for the first time shows the coexistence of APOB mutation in hypothyroidism, which emphasis screening of patients with hypothyroid for FH detection.

Aims Breast cancer is the most frequently occurring cancer in women. Lumpectomy followed by radiotherapy is suggested to be as effective as a total mastectomy. Radiation-induced dermatitis often occurs as a result of breast radiotherapy. Recent studies suggest that doxepin has promising anti-inflammatory properties. This study was undertaken to evaluate the effects of doxepin therapy on radiation dermatitis. Methods A double-blind randomized clinical trial was launched from 2016 to 2017, with a total of 48 patients who had undergone breast-conserving surgery and received postoperative radiation therapy. Radiotherapy was applied 5 days per week for 5 weeks. Adverse dermatological effects were evaluated by a physician at the beginning of the fifth week of radiotherapy and the patients were then randomly assigned (1:1 ratio) to receive either doxepin (5%) or placebo cream for 7 days. Results There were no significant differences in the dermatitis grade between doxepin and placebo groups at baseline (P > .5). The occurrence of acute dermatitis (grade 2 or higher) was significantly lower with the use of doxepin than with placebo (P ≤ .0001, Zα = 1.96 at 95% confidence interval). Conclusion Doxepin cream prevents dermatitis grade 2 or higher during post-operative breast irradiation. Doxepin cream is easy to use, affordable and prevents pain and irritation.

Elevation of hemoglobin F (HbF) ameliorates symptoms of β-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to β hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in β-thalassemia patients.

Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.