Optimized Method for Manufacturing and Purifying Nkx2.2 Modified-Mrna for Therapeutic Applications

Background: Nkx2.2 is a homeodomain-containing transcrip-tion factor, robustly expressed in pancreatic islets and central nervous system (CNS). It is required for final differentiation and maturation of pancreatic β-cells to produce insulin and oligodendrocyte precursor cells to remyelinate demyelinating lesions. The necessity of Nkx2.2 overexpression with the aim of clinical applications, needs an efficient and safe system to produce this factor. Synthetic modified-mRNAs have consid-ered as an effective method for delivering genetic information and inducing transient expression without genetic modification. Modified nucleotides reduce the immunogenicity of therapeutic RNAs likewise increase their stability. The goal of this study was to optimize the best condition for producing large amounts of Nkx2.2 modified-mRNAs with less immunogenicity.Materials and Methods: Linearized pMRNA-Nkx2.2 and T7-Nkx2.2-PolyA PCR product were served as template for in vitro Transcription (IVT) using NEB HiScribeT7-Kit. Transcribed mRNAs were purified by phenol-chloroform, LiCl and Qiagen-RNeasy column. To analyze the mRNA concentration, purity and integrity, spectrophotometer as well as agarose gel were used.Results: Our result demonstrated that the yield of produced modified-mRNAs was the same in two different template con-ditions; equal number of molecules and equal concentration. However, the amount of unintended double-stranded RNAs (dsRNAs) was lower using linearized vector. Additionally, all aforementioned characteristics of transcribed modified-mRNAs was significantly higher in LiCl purification method. Moreover, increased centrifugation time, enhanced the purification effi-ciency.Conclusion: In conclusion, using linearized vector as IVT tem-plate produces less dsRNA, which ultimately resulting in less immune system stimulation. Furthermore, LiCl method with higher centrifugation time is the best condition for purifying modified-mRNAs.

کلیدواژه ها:

Nkx2.2 ، Pancreatic Islets ، Central Nervous System ، Modified-Mrna ، in vitro Transcription