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Improved method for fermentative large scale production of dsRNA Disclosure Number: IPCOM000247387D
Publication Date: 2016-Aug-31
Document File: 38 page(s) / 1M

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Described are methods for cost efficient dsRNA production, isolation and purification that is applicable in large scale and in versatile multiparallel production of diverse dsRNA molecules.

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Improved method for fermentative large scale production of dsRNA

Field of the Invention

The present invention relates to a method for fermentative production and isolation of

dsRNA molecules.

Description of the Invention
dsRNA is known to regulate gene expression in various organisms e.g. plants and animals. Recently it has been discovered that dsRNA molecules may be applied topically to organ-

isms in order to regulate gene expression (e.g. WO2006070227; WO2011112570). In addi- tion, dsRNA may potentially be useful as medicament to regulate gene expressions of genes causing diseases such as cancer (e.g. US20070104688).

A limitation to the methods of application of dsRNA molecules in large scale is the lack of cost effective production of dsRNA molecules. dsRNA molecules are mostly produced by

means of chemical synthesis or in vitro transcription which are expensive and do not allow for large scale production.

There is a need in the art for the provision of methods for cost efficient dsRNA production, isolation and purification that allows versatile multiparallel production of diverse dsRNA mol- ecules as well as large scale production of such molecules.

Production of dsRNA in E. coli has been described previously (US2003/0051263) but the dsRNA molecules were isolated by gel purification which would not be applicable in large scale.

In another publication, fermentative production of dsRNA in E.coli is disclosed (US2014/0271559), however, the dsRNA molecules are not isolated but rather the entire

cell lysate comprising total RNA including for example single stranded RNA, rRNA, tRNA and dsRNA is used for subsequent applications.

Especially the method for isolation of the dsRNA molecule from the bacteria and growth medium as described in the prior art is time consuming and cost intensive. The method of

the present invention established an easy and inexpensive isolation and purification method using differential precipitation, for example (NH4)2SO4 precipitation as single precipitant of dsRNA after cell lysis.

Other methods for dsRNA purification are known from literature, e.g., via phenolic com-

pounds like TRIzol [Posiri et al. (2013), J Vir Meth 188:64] or via fractionation with LiCl [Diaz-Ruiz & Kaper (1978), Prep Biochem 8:1]. However, purification via the described (NH4)2SO4 method of this invention is
- Safer, since ((NH4)2SO4 is neither toxic like phenol nor corrosive like LiCl. In addition, phe- nolic purifications mostly include an ethanol or isopropanol step, demanding explosion-re-

sistant equipment for a production plant. Moreover, processes using (NH4)2SO4 are well es- tablished also at large scales, e.g., for protein purifications.










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- Cheaper, since raw material costs for (NH4)2SO4 are much lower than, e.g., for LiCl. Addi- tionally, no explosion-resistant equipment is needed like for a phenolic process.

- Not the least, the method of the invention is yi...