Methods and compositions for increased double stranded RNA production

Inventors

Killmer, John L. • McLaughlin, Patrick D. • Arhancet, Juan Pedro Humberto

Assignees

RNAissance Ag LLC

Abstract

The invention provides methods and compositions for improved production of large quantities of unencapsidated double strand RNA (dsRNA) in vivo. The disclosed methods and compositions, comprising co-expression of genes encoding orotate phosporibosyl transferase, bacteriophage coat protein and dsRNA produce a significant improvement over current in vivo methods of producing unencapsidated dsRNA.

Core Innovation

The invention relates to producing unencapsidated dsRNA in a microbial cell by co-expressing, in vivo, a dsRNA stem-loop RNAi precursor together with a bacteriophage Leviviridae coat protein gene. The method provides for producing unencapsidated dsRNA that is directly recoverable from a cell lysate. The unencapsidated dsRNA is characterized as exceeding the interior diameter of the Leviviridae coat protein, so that the dsRNA remains unencapsidated while being produced in the microbial cell.

The invention further links co-expression of the Leviviridae coat protein to increased accumulation of intact unencapsidated dsRNA recovered from cell lysates. The disclosed rationale states that capsid protein increases accumulation of intact unencapsidated dsRNA, while effects are not strictly tied to cognate pac-site binding and are not limited to particular loop or stem sizes or structures. Embodiments include using the capsid protein gene directly or using an amino-terminal fragment of the capsid protein.

The disclosed embodiments include promoter and genetic arrangement options for co-expression, such as expressing the coat protein gene from a constitutive promoter while expressing the dsRNA gene from an inducible promoter, and locating both the dsRNA gene and the coat protein gene on one plasmid or extrachromosomal element. The disclosure further includes capsid substitution examples and embodiments stating that the N-terminus is sufficient, including truncated N-terminal lengths of the capsid protein.

Claims Coverage

The document contains one independent claim. The independent claim includes three core inventive elements: co-expressing dsRNA and a Leviviridae coat protein or amino-terminal fragment, direct recoverability from a cell lysate, and an unencapsidated dsRNA length exceeding the interior diameter of the Leviviridae coat protein.

Overall, the claim coverage centers on producing unencapsidated dsRNA by co-expression with a Leviviridae coat protein or amino-terminal fragment in a microbial cell, enabling direct recovery from a cell lysate, while the dsRNA length exceeds the interior diameter of the coat protein. Dependent claim coverage further refines the capsid protein source, promoter arrangement, genetic co-localization, and truncated capsid length.

Stated Advantages

Documented Applications