Compositions and methods using capsids resistant to hydrolases

Inventors

Arhancet, Juan Pedro Humberto • Arhancet, Juan P. • Delaney, Kimberly • Hall, Kathleen B. • Summers, Neena • OATES, Edward

Assignees

RNAissance Ag LLC

Abstract

Novel processes and compositions are described which use viral capsid proteins resistant to hydrolases to prepare virus-like particles to enclose and subsequently isolate and purify target cargo molecules of interest including nucleic acids such as siRNAs and shRNAs, miRNAs, messenger RNAs, small peptides and bioactive molecules.

Core Innovation

The disclosed invention relates to virus-like particles (VLPs) that include protease resistant capsid proteins enclosing a heterologous cargo molecule. The cargo molecule comprises nucleic acids comprising long single stranded sense RNA and long single stranded antisense RNA, each having more than 30 nucleotides, and each RNA further includes a capsid specific packing sequence.

In one aspect, the method purifies first and second compositions from a bacterial lysate, where each composition comprises a VLP with protease resistant capsid proteins enclosing complementary long single stranded RNA with capsid specific packing sequences. Equal amounts of the two compositions are mixed, the encapsulated heterologous cargo molecules are isolated, and the sense and antisense RNAs are annealed to form a double stranded RNA molecule that is subsequently isolated.

In another aspect, the method is used to produce siRNA, shRNA, sshRNA, lshRNA and miRNA by using long single stranded sense RNA and long single stranded antisense RNA that are homologous to one another, encapsulated in protease resistant capsid proteins with capsid specific packing sequences. After mixing equal amounts, isolating the encapsulated RNAs, annealing to form double stranded RNA, and isolating the double stranded RNA, the method transforms a target cell with the double stranded RNA.

Claims Coverage

The document provides two independent method claims. Across both claims, the same core inventive elements appear: protease resistant capsid proteins in VLPs that encapsulate complementary long single stranded RNAs with capsid specific packing sequences, followed by equal-amount mixing, isolation of encapsulated cargo RNAs, annealing to form double stranded RNA, and isolation of the resulting double stranded RNA; the second independent claim additionally includes transforming a target cell with the double stranded RNA.

Both independent claims are directed to producing double stranded RNA by purifying two bacterial-lysate-derived VLP-containing compositions that use protease resistant capsid proteins and capsid specific packing sequences to encapsulate complementary long single stranded sense and antisense RNAs, followed by equal-amount mixing, isolation of encapsulated cargo, annealing, and isolation of double stranded RNA; the second independent claim further requires transforming a target cell with the resulting double stranded RNA to produce siRNA/shRNA/sshRNA/lshRNA/miRNA.

Stated Advantages

Documented Applications