ERKUT BAHCECI, MD
Osteopathic Medicine in New Haven, CT

2008026, Oct 23, 2008

Feb 4, 2008

12/025700

Peter M. Rabinovich - Madison CT, US
Sherman M. Weissman - Milford CT, US
Marina E. Komarovskaya - Milford CT, US
Erkut Bahceci - Hamden CT, US

A61K 35/12, C12N 15/11, C12P 19/34, C12N 5/06, A61P 43/00, C12N 15/87, A61K 31/70

424 9321, 536 231, 435 912, 435440, 514 44, 424 932, 435377, 435455

A method of mRNA production for use in transfection is provided, that involves in vitro transcription of PCR generated templates with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), the gene to be expressed, and a polyA tail, typically - bases in length. This RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less time consuming because it does not require cloning, and also consequently eliminates the unwanted errors and effects related to RNA made on DNA templates obtained with cloning techniques. The results of transfection of RNAs demonstrate that RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with DNA constructs. Further, the levels of gene expression following mRNA transfection are consistent from cell to cell in an experiment and these levels can be controlled over a wide range simply by changing the amount of mRNA that is transfected, and without obvious cytotoxic effects due to the levels of RNA per se. Due to high efficiency the cells can be simultaneously transfected with multiple genetic constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for DNA, in vitro and in vivo.