Introduction: Neural stem cells (NSCs) are multipotent stem cells residing in the central nervous system that is capable of self-renewal to support ongoing requirements for neurogenesis in the adult brain. Since NSCs are considered potential candidate cells for neuro-regenerative medicine, applying safe induction methods for them is very important. Synthetic Modified-mRNA (mmRNA) as an alternative to traditional DNA-or protein-based methods, is regarded as a powerful tool for inducing short-term gene expression in cells with no genetic manipulation. Methods: Here, we aimed to develop an optimized condition for mmRNA transfection in primary NSCs. In vitro-transcribed EGFP mmRNA (mmRNAEGFP) was delivered to human embryonic kidney cells (HEK293T) and mouse NSCs by using two commercial agents, Lipofectamine-2000 (LF2000) and TransIT. Also, a plasmid DNA was used to transfect cells considered EGFP-expressing positive control. In addition, the poly(A) tail (poly adenosine tail) elongation and chloroquine (CQ) treatment were performed to improve transfection efficiency. Finally, flow cytometry, fluorescence microscopy, and MTT assays were performed to assess the cells. Results: In comparison with HEK293T, NSCs were very sensitive to transfection, the efficacy of transfection using DNA/LF2000 was higher in HEK293T cells, but mmRNAEGFP/ TransIT showed better transfection efficacy in NSCs. Poly(A) tail elongation,also, treating the cells with CQ before transfection significantly improved its efficacy. Conclusion: The mmRNA poly(A) tail elongation and the use of specific transfection agents in combination with TLR inhibitors can lead to a more effective transfection in NSCs.

Vaccination is one of the important approaches in the prevention and control of diseases. Although the capacity to present antigens other than the disease-specific antigen in the traditional vaccine composition provides a potential benefit by increasing its protective efficacy, many components that are not needed for the related disease are also transferred. These components can reduce vaccine activity by lowering immunity against protective antigens. The reasons such as the low effectiveness of traditional vaccines and the high cost of production and time-consuming reasons show that it is necessary to develop a new vaccine method for our world, which is struggling with epidemics almost every year. Among nucleic acids, mRNA has many advantages, such as genomic integration, induction of anti-DNA autoantibodies, and immune tolerance induced by long-term antigen expression. mRNA vaccines have become a therapeutic target for reasons such as efficacy, safety, fast and non-expensive production. The fact that mRNA triggers both humoral and cellular immunity and goes only to the cytoplasm, not to the nucleus, makes it highly efficient. The mRNA must cross the lipid bilayer barrier and entry to the cytoplasm where it is translated into protein. There are two main ways of mRNA vaccine delivery for this: ex vivo loading of mRNA into dendritic cells (DCs) and direct injection of mRNA with or without a carrier. Studies continue to understand which delivery system is therapeutically more efficient. Preclinical and clinical trials showed that mRNA vaccines trigger a long-lasting and safe immune response.

Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) -Modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated.Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG), DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-Modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy.Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-Modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P<0.05).Conclusions: Hydroxyapatite NPs conjugated with DOPE are a good choice for gene delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be addressed more in future studies.

Introduction: Hepatitis C virus (HCV) is a blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5' cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality. Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of 5ʹ and 3ʹ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the Modified mRNA, the 5' cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay. Results: Cell transfection results showed that despite the absence of 5' cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of GFP and Core genes were achieved. Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.