Methods and compositions for single-stranded rna-initiated risc assembly

{
  "publication_number": "US2026103706A1",
  "country": "US",
  "kind": "A1",
  "title": "Methods and compositions for single-stranded rna-initiated risc assembly",
  "abstract": "The present disclosure provides compositions comprising ss-guides with superior binding and degradation profiles, along with methods to design compositions comprising ss-guides with superior binding and degradation profiles.",
  "claims": [
    "1. A synthetic nucleic acid composition comprising a ss-guide RNA, wherein the ss-guide RNA comprises at least 14 nucleotides and fewer than 20 nucleotides, and wherein the ss-guide RNA comprises a binding site for a PAZ domain of an RNA-induced silencing complex (RISC). 2. The synthetic nucleic acid composition of claim 1, wherein the ss-guide RNA comprises 19 nucleotides. 3. The synthetic nucleic acid composition of claim 1, wherein the RISC is completely assembled or partially assembled. 4. The synthetic nucleic acid composition of claim 1, wherein the ss-guide RNA comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity to a binding region of a target nucleic acid, or any amount less than or in-between these values. 5. The synthetic nucleic acid composition of claim 1, wherein the synthetic nucleic acid composition further comprises a pharmaceutically acceptable carrier. 6. The synthetic nucleic acid composition of claim 5 wherein the pharmaceutically acceptable carrier comprises an excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or nanoparticle. 7. A synthetic RISC composition comprising the synthetic nucleic acid composition of claim 1. 8. The synthetic RISC composition of claim 7, wherein the synthetic RISC composition further comprises a pharmaceutically acceptable carrier. 9. The synthetic RISC composition of claim 8, wherein the pharmaceutically acceptable carrier comprises an excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or nanoparticle. 10. A method of designing a guide RNA to be used with a RISC assembly, the method comprising: a) identifying a target nucleic acid, and b) sequencing together a plurality of nucleotides to generate a single stranded (ss)-guide RNA, wherein the ss-guide RNA comprises at least 14 nucleotides and less than about 20 nucleotides. 11. The method of claim 10, wherein the ss-guide RNA comprises 19 nucleotides. 12. The method of claim 10 - e -11-, wherein the ss-guide RNA comprises a tinyRNA(tyRNA), siRNA, shRNA, or a miRNA. 13. The method of claim 10, wherein the ss-guide RNA comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity to a binding region of the target nucleic acid, or any amount less than or in-between these values. 14. The method of claim 10, wherein the ss-guide RNA comprises a binding site for a PAZ domain of an RNA-induced silencing complex (RISC). 15. The method of claim 10, wherein the target nucleic acid comprises DNA or RNA. 16. The method of claim 15, wherein the RNA comprises mRNA. 17. The method of claim 10, wherein the RISC assembly comprises an AGO molecule. 18. The method of claim 17, wherein the AGO comprises AGO1, AGO2, AGO3, or AGO4. 19. A method of regulating expression of a target nucleic acid using an AGO molecule, the method comprising exposing the target nucleic acid to the AGO molecule loaded with a guide RNA comprising at least 14 nucleotides and less than about 20 nucleotides. 20. The method of claim 19, wherein the target nucleic acid is silenced by the AGO. 21 - 45. (canceled)"
  ],
  "description_excerpt": "The present disclosure relates synthetic guide RNAs and RISC compositions, and methods thereof for regulating gene expression impacting a disease and/or disorder. BACKGROUND Four Argonaute proteins (AGO1-4) are expressed in humans. The four human Argonaute proteins are structurally very similar but nevertheless contain few non-conserved amino acids in their functional domains. Four conserved domains have been elucidated: the N-terminal domain (N), the PIWI/Argonaute/Zwille (PAZ) domain, the MID domain, and the P-element-induced whimpy tested (PIWI) domain. The PAZ domain is required for anchoring the 3′ end of guide RNAs.\n\nMicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are both loaded into AGOs efficiently as duplexes with the aid of seven chaperones. This duplex-initiated RISC assembly has been heavily studied and exploited in basic research and clinical therapeutics to silence the expression of target genes. Previous studies have reported that single-stranded (ss) RNA-derived guides (ss-guides) were incorporated into RNA-induced silencing complexes (RISCs). However, the molecular mechanisms and requirements for the single stranded (ss)-RNA-initiated RISC assembly have been poorly understood. What is needed in the art are ss-guide RNAs which are useful in the RISC assembly.\n\nMiRNAs were defined to be about 22 nt because they are generated by Dicer, a molecular ruler. In contrast, there has been no clear definition of tinyRNAs (tyRNAs) to distinguish them from miRNAs and siRNAs.",
  "cpc": [
    "C12N 15/113",
    "C12N 15/10",
    "C12N 2310/141",
    "C12N 2330/30"
  ],
  "assignees": [
    "Ohio State Innovation Foundation"
  ],
  "inventors": [
    "Kotaro NAKANISHI"
  ],
  "filing_date": "2024-04-01",
  "publication_date": "2026-04-16",
  "priority_date": "2023-03-30",
  "application_number": "US-202419470312-A",
  "cited_by_count": 0
}