Genetic Reprogramming Of Cells And Tissue With mRNA Services

At Seattle Genov, we have a broad range of experience in supplying clients with mRNA products at the appropriate quality level for each program stage. Our proprietary mRNA synthesis technology achieves highly efficient and economical co-transcriptional capping, and we offer numerous post-transcriptional modifications, including Dnase and phosphatase treatments, enzymatic capping, and polyadenylation. Additionally, you can select from a variety of purification options, including silica gel purification, liquid chromatography isolation, and high-performance liquid chromatography.

mRNA reprogramming is the most unambiguously “footprint-free,” largely productive, and possibly the best fitted for the clinical generation of stem cells.

The emergence of the mRNA reprogramming system in 2010 took the stem cell area by surprise. Synthetic mRNA did not feature in the definitive toolkit of cell biologists at the time, in contrast with retroviruses, lentiviruses, plasmids, small interfering RNAs (siRNAs), soluble proteins, and small molecules. Synthetic mRNA has been generated and applied in niche research applications going as far back as the 1980s.

The published “modified mRNA” reprogramming protocol integrates various optimizations notified by earlier iPSC research, containing an mRNA cocktail stoichiometrically weighted in favour of the most potent reprogramming factor, Oct4, and the aim of 5% oxygen culture.

Direct cell reprogramming, also named transdifferentiation, enables for the reprogramming of one somatic cell type rapidly into another, without the need to transition through an induced pluripotent state. Therefore, it is an impressive approach to formulate novel tissue engineering applications to treat disorders and injuries where there is a shortage of proliferating cells for tissue repair. In specific tissue damage, terminally differentiated somatic cells lose their ability to proliferate, as an outcome, damaged tissues cannot heal by themselves. 

Examples of these scenarios contain myocardial infarctions, neurodegenerative diseases, and cartilage injuries. Transdifferentiation is able of reprogramming cells that are sufficient in the body into desired cell phenotypes that are able to restore tissue function in damaged areas. Accordingly, direct cell reprogramming is a favorable direction in the cell and tissue engineering and regenerative medicine fields.

In conclusion, the mRNA reprogramming system gives an attractive path around one of the main stumbling blocks to future iPSC-based therapeutics and, accordingly, continues to deserve and receive the attention of scientists working to bring that dream to reality.

Step 1. Plasmid Manufacturing 

◆ mRNA synthesis begins with plasmid design and production. 

◆ Plasmids are generated in bacterial cultures, then harvested and purified.

Step 2. Transcrption 

In-vitro transcription (IVT)

◆ In vitro transcription is a method that facilitates for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of various kilobases in μg to mg quantities. 

◆ It is based on the engineering of a template that contains a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest fulfilled by transcription utilizing the corresponding RNA polymerase.

Step 3. mRNA purification

◆ After certain manufacturing steps it is significant to purify the mRNA. 

◆ mRNA purification eliminates enzymes, remaining nucleotides, plasmid DNA, and defective mRNA. New emerging technologies like Fibro chromatography, currently accessible for mAb purification, are in development for molecules such as DNA plasmids and mRNA.

Step 4. mRNA encapsulation and polishing

◆ The purified mRNA-based therapeutic is formulated in lipid nanoparticles (LNPs) as a drug delivery vehicle.

◆ Core chromatography can be used to further eliminate impurities.

Step 5. QC release and stability testing

Relying on your final mRNA application and clinical stage, the quality control testing requirements may vary.

◆ RNA content by UV-Vis

◆ Purity by IRRP HPLC

◆ Residual DNA by RT-qPCR

◆ Residual protein by MS

◆ Potency by cell-free translation

◆ Endotoxin and residuals measurements

◆ Sequencing

SERVICE HIGHLIGHTS

◆ High quality products and services at competitive prices

◆ Custom tailored assistance to meet specific application or program needs

◆ Vast variety of modification, treatment, and purification options

◆ Accessible custom synthesis up to gram scales of mRNA and long RNA (multiple   kilobases)

◆ In-house plasmid manufacturing optimized for therapeutic mRNA generation

DELIVERABLES

We provide high throughput evaluations along with faster results. In addition to that, we provide many different mRNA formulation services to meet your various end-point in vaccine delivery. These formulations come with specific functionality to improve the efficiency of vaccines in the physiological environment.

References

1.Takahashi K., Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. 

2.Normile D. First-of-its-kind clinical trial will use reprogrammed adult stem cells to treat Parkinson’s. Science. 2018 Published online July 30, 2018. 

3.Kim D., Kim C.H., Moon J.I., Chung Y.G., Chang M.Y., Han B.S., Ko S., Yang E., Cha K.Y., Lanza R., Kim K.S. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009;4:472–476.