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
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9822361-B2
Publication Date
2017-11-21
Expiration Date
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 invention provides hydrolase-resistant, non-enveloped MS2-derived virus-like particle (VLP) capsids used as nanocontainers. These capsids are used for encapsidating and purifying heterologous cargo molecules, with particular emphasis on oligoribonucleotides including siRNA, shRNA, sshRNA, lshRNA, miRNA, and mRNA.
In some embodiments, the VLPs include ribozymes flanked by capsid packing sequences to release defined short RNA products. The disclosed ribozyme-mediated processing supports the construction of RNA cargo that includes ribozyme elements and capsid packing sequences, with ribozyme types including Hammerhead and Hepatitis Delta Virus (HDV) ribozymes.
The document further describes isolating and purifying target cargo from whole cell lysate by selectively hydrolyzing unenclosed proteins while keeping intact the hydrolase-resistant capsids. The encapsidating and purifying system is supported by compositions and nucleic acid constructs for producing ribozyme-packaging systems, and by examples indicating purification performance for MS2 capsids/phage and validation of RNA packaging and ribozyme-mediated processing in vitro and in vivo.
Claims Coverage
The independent claims are directed to oligoribonucleotides that contain a capsid specific packaging sequence and multiple contiguously arrayed complementary regions arranged as siRNA sense/loop/antisense structures, including variants where the sense and antisense regions are catenated. The claims define how the complementary regions are constructed, with core inventive features focused on the contiguously arrayed packaging-complement architecture, the sense/loop/antisense composition of each complementary region, and optional ribozyme-based connectivity or ribozyme incorporation within the loop-forming segment.
Capsid specific packaging sequence with contiguously arrayed complementary regions as sense-loop-antisense blocks
An oligoribonucleotide comprising a capsid specific packaging sequence and multiple complementary regions, wherein each complementary region comprises a sense strand siRNA sequence, immediately followed by a non-homologous RNA sequence capable of forming a loop, immediately followed by an antisense strand siRNA sequence complementary to the sense strand siRNA sequence, wherein the multiple complementary regions are contiguously arrayed.
Ribozyme connectivity between contiguously arrayed complementary regions
An oligoribonucleotide of the capsid specific packaging sequence and multiple complementary regions wherein the complementary regions are connected by one or more ribozymes.
Loop-forming RNA sequence includes one or more ribozymes
An oligoribonucleotide of the capsid specific packaging sequence and multiple complementary regions wherein the RNA sequence capable of forming a loop includes one or more ribozymes.
Capsid specific packaging sequence with contiguously arrayed complementary regions using catenated sense and antisense strands
An oligoribonucleotide comprising a capsid specific packaging sequence and multiple complementary regions, wherein each complementary region comprises multiple catenated sense strand siRNA sequences immediately followed by a non-homologous RNA sequence capable of forming a loop, immediately followed by multiple catenated antisense strand siRNA sequences complementary to the multiple catenated sense strand siRNA sequences, wherein the multiple complementary regions are contiguously arrayed.
Bulged RNA by linking catenated sense strand siRNA sequences with 1 to 3 non-complementary nucleotides
An oligoribonucleotide of the capsid specific packaging sequence and multiple complementary regions wherein a bulged RNA is formed by linking multiple catenated sense strand siRNA sequences with 1 to 3 non-complementary nucleotides.
Loop-forming RNA sequence includes one or more ribozymes (catenated complementary regions variant)
An oligoribonucleotide of the capsid specific packaging sequence and multiple complementary regions wherein the loop-forming RNA sequence comprises one or more ribozymes.
Across the independent claims, the document covers oligoribonucleotides with a capsid specific packaging sequence and multiple contiguously arrayed complementary regions constructed as sense/loop/antisense siRNA blocks, including a variant where each sense and antisense portion is formed by multiple catenated strands. Dependent claim variants further specify ribozyme connectivity between complementary regions and/or ribozyme inclusion within the loop-forming RNA segment, and additionally define a bulged RNA formation using a constrained 1 to 3 non-complementary nucleotides linkage.
Stated Advantages
Capsids resistant to peptide-bond hydrolases EC 3.4 enable selective hydrolysis of unenclosed proteins while keeping intact capsids.
Enables encapsidating and purifying heterologous cargo molecules using hydrolase-resistant, non-enveloped MS2-derived VLP capsids.
Supports RNA packaging and ribozyme-mediated processing, enabling defined short RNA products.
Documented Applications
Use of VLP capsids as nanocontainers for encapsidating and purifying heterologous oligoribonucleotide cargo, including siRNA, shRNA, sshRNA, lshRNA, miRNA, and mRNA.
Release of defined short RNA products via ribozyme-mediated processing associated with capsid packing sequences.
Purification of RNA cargo from whole cell lysate using selective hydrolysis of unenclosed proteins while keeping intact hydrolase-resistant capsids.
mRNA delivery using VLPs is described.
Long RNA/dsRNA strategies, including bulged or mismatched constructs, are described.
Small-molecule loading using aptameric bifunctional polynucleotides targeting herbicide/pesticide targets is described.
Interested in licensing this patent?