COMPOSITIONS AND METHODS USING CAPSIDS RESISTANT TO HYDROLASES

• US 20160177299A1
• Filed: 06/05/2014
• Published: 06/23/2016
• Est. Priority Date: 06/19/2013
• Status: Active Grant

• Current Assignee: RNAiSSANCE AG LLC
• Original Assignee: APSE, LLC (RNAiSSANCE AG LLC)
• Inventors: ARHANCET, Juan Pedro Humberto, ARHANCET, JUAN P., DELANEY, Kimberly, HALL, Kathleen B., SUMMERS, Neena, OATES, Edward
• Granted Patent: US 9,822,361 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/5184 Virus capsids or envelopes enclosing drugs with additional exogenous lipids A61K9/127; bacterial membranes A61K9/5068
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell
• CPC Class Codes: C12N 2795/00023 Virus like particles [VLP]
• CPC Class Codes: C12N 2795/00042 virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 2795/18122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2795/18123 Virus like particles [VLP]
• CPC Class Codes: C12N 2795/18142 virus or viral particle as vehicle, e.g. encapsulating small organic molecule

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.

Claims

1. A virus-like particle (VLP) comprising a protease resistant capsid enclosing at least one heterologous cargo molecule, the cargo molecule further comprising a capsid specific packing sequence.

2. A VLP according to claim 1, wherein the heterologous cargo molecule comprises an oligoribonucleotide.

3. A nucleic acid encoding the oligoribonucleotide of claim 2.

34. A method for producing double stranded RNA comprising;

(a) mixing equal amounts of a first VLP of claim 2 and a second VLP of claim 2;

(b) isolating the heterologous cargo molecules thereof;

(c) annealing the heterologous cargo molecules to form a double stranded RNA molecule;

(d) isolating the double stranded RNA molecule.

35. A method for producing siRNA, shRNA, sshRNA, lshRNA and miRNA comprising transforming a host cell with the double stranded RNA of claim 34.

36. The nucleic acid of claim 3, comprising one or more complementary regions, each of said complementary regions comprising;

(a) a sense strand siRNA, immediately followed by

(b) RNA capable of forming a loop, immediately followed by

(c) an antisense strand siRNA complementary to the sense strand siRNA are contiguously arrayed.

37. The nucleic acid of claim 36, wherein the complementary regions are linked by one or more ribozymes.

38. The nucleic acid of claim 3, comprising one or more complementary regions, each of said complementary regions comprising;

(a) multiple catenated sense strand siRNA, immediately followed by

(b) RNA capable of forming a loop, immediately followed by

(c) multiple catenated antisense strand siRNA complementary to the multiple catenated sense strand siRNA

are contiguously arrayed.

39. The nucleic acid of claim 38, wherein multiple catenated sense strand siRNAs are linked by 1 to 3 non-complementary nucleotides to form bulged RNA.

40. The nucleic acid of claim 3, comprising one or more complementary regions, each of said complementary regions comprising;

(a) multiple catenated sense strand siRNA, each immediately followed by a sense strand RNA sequence for host cell RNA processing, immediately followed by

(b) RNA capable of forming a loop, immediately followed by

(c) multiple catenated antisense strand siRNA complementary to the multiple catenated sense strand siRNA, each immediately followed by an antisense strand RNA complementary to the sense strand RNA for host cell RNA processing

are contiguously arrayed.

41. The nucleic acid of claim 40, wherein the RNA sequence for host cell RNA processing is CRISPER.

43. The nucleic acid of claim 36, wherein the RNA sequence capable of forming a loop comprises one or more ribozymes.

45. The nucleic acid of claim 38, wherein the RNA sequence capable of forming a loop comprises one or more ribozymes.

46. The nucleic acid of claim 40, wherein the RNA sequence capable of forming a loop comprises one or more ribozymes.

4-33. -33. (canceled)

44. (canceled)