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US-20260124233-A1 - COMPOSITIONS FOR AQUACULTURING

US20260124233A1US 20260124233 A1US20260124233 A1US 20260124233A1US-20260124233-A1

Abstract

Particulate compositions comprising low molecular weight chitosan and ss or dsRNA partially complementary to, binding to or at least 90% identical to mRNA targets of viruses pathogenic in farmed crustaceans, compositions and farmed aquatic crustaceans comprising the same, and methods for their use in treating or preventing viral infection in aquaculture are provided.

Inventors

  • Shai Ufaz
  • Ami Schlesinger
  • Ori KOSHET
  • Margarita KANTEEV

Assignees

  • VIAQUA THERAPEUTICS LTD.

Dates

Publication Date
20260507
Application Date
20251229

Claims (18)

  1. 1 . A particulate composition comprising: particles comprising chitosan and at least one type of RNA molecule comprising a nucleic acid sequence at least 50 bases in length, wherein said chitosan is 50-100% deacetylated low molecular weight chitosan, wherein the molecular weight of said chitosan is in the range of 5-100 kDa, wherein the chitosan:RNA mass ratio (w/w) of the particles is in the range of 0.3-0.9, wherein said RNA molecule is capable of silencing expression of a gene when administered to an organism expressing said gene, and wherein the particulate composition comprises >50% particles in the range of 10-300 μm.
  2. 2 . The particulate composition of claim 1 , wherein said chitosan is 80-95% deacetylated low molecular weight chitosan.
  3. 3 . The particulate composition of claim 1 , wherein the molecular weight of the chitosan is in the range of 10-50 kDa.
  4. 4 . The particulate composition of claim 1 , wherein the molecular weight of the chitosan is in the range of 20-30 kDa.
  5. 5 . The particulate composition of claim 1 , wherein the molecular weight of said chitosan is about 30 kDa.
  6. 6 . The particulate composition of claim 1 , wherein the chitosan:RNA mass ratio is in the range of 0.45-0.55 and the molecular weight of said chitosan is in the range of 20-30 kDa.
  7. 7 . The particulate composition of claim 1 , and/or a nutraceutical composition comprising farmed crustacean food and the particulate composition of claim 1 , wherein said at least one type of RNA molecule comprises one or more of the following: (a) at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a virus pathogenic in farmed crustaceans; (b) at least one sequence having at least 90% sequence identity to a target mRNA molecule of a virus pathogenic in farmed crustaceans; (c) at least one sequence at least partially complementary to a target mRNA molecule of a virus pathogenic in farmed crustaceans; (d) at least one sequence identical to at least 21 contiguous bases of a target mRNA molecule of a virus pathogenic in farmed crustaceans; (e) at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a farmed crustacean; (f) at least one sequence identical to at least 21 contiguous bases of a target mRNA molecule of a farmed crustacean; (g) at least one sequence at least partially complementary to a target mRNA molecule of a farmed crustacean.
  8. 8 . The particulate composition and/or nutraceutical composition of claim 7 , wherein said RNA molecule comprising at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a virus pathogenic in farmed crustaceans or comprising at least one sequence at least partially complementary to a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises: (a) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 5.
  9. 9 . The particulate composition and/or nutraceutical composition of claim 7 , wherein said RNA molecule comprising at least one sequence having at least 90% sequence identity to a target mRNA molecule of a virus pathogenic in farmed crustaceans or at least one sequence identical to at least 21 contiguous bases of a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises: (a) a nucleic acid sequence identical to at least 21 contiguous bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 59, 64, 77, 78 and 79, or (b) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59, 64, 77, 78 and 79.
  10. 10 . The particulate composition and/or nutraceutical composition of claim 7 , comprising at least one additional RNA sequence capable of directing cleavage of a target mRNA molecule of a virus pathogenic in farmed crustaceans.
  11. 11 . A nutraceutical composition comprising farmed crustacean food and the particulate composition of claim 1 .
  12. 12 . The nutraceutical composition of claim 11 , wherein the RNA content (w/w) of said composition is in the range of 0.1-3.0%.
  13. 13 . The nutraceutical composition of claim 11 , wherein the particulate composition is prepared by: (i) mixing an RNA solution to a volume of 50-100% deacetylated low molecular weight chitosan solution, wherein the molecular weight of said chitosan is in the range of 5-100 kDa, wherein said chitosan solution comprises 2-20 mg/ml low molecular weight chitosan in 0.01-0.1 M citric acid, (ii) agitating the mixture chitosan-RNA solution for 1-10 minutes, (iii) desalting and concentrating the chitosan-RNA complex solution, and (iv) drying the desalted, concentrated chitosan-RNA complex solution.
  14. 14 . The nutraceutical composition of claim 11 , wherein the molecular weight of the chitosan is in the range of 10-50 kDa.
  15. 15 . The nutraceutical composition of claim 11 , wherein the molecular weight of the chitosan is in the range of 20-30 kDa.
  16. 16 . A farmed crustacean comprising the particulate composition of claim 1 .
  17. 17 . The farmed crustacean of claim 16 , wherein said farmed crustacean is selected from the group consisting of Shrimp, Prawns, Crabs, Lobsters and Crayfishes.
  18. 18 . The farmed crustacean of claim 16 , wherein said farmed crustacean is a shrimp or a prawn.

Description

RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 18/708,618 filed on May 9, 2024, which is a National Phase of PCT Patent Application No. PCT/IL2022/051185 having International Filing Date of Nov. 8, 2022, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63/277,176 filed on Nov. 9, 2021. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety. SEQUENCE LISTING STATEMENT The XML file, entitled 106309.xml, created on Dec. 29, 2025, comprising 154,073 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application. FIELD AND BACKGROUND OF THE INVENTION The most significant diseases injurious to the aquaculture industry are caused by infectious agents. Of these, the majority are viral diseases. The most common viral diseases affecting shrimp aquaculture include White Spot Syndrome Virus (WSSV), YellowHead Disease Virus (YHV), Taura Syndrome Virus (TSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Monodon Baculovirus Disease (MBV), causing estimated billions of dollars in losses worldwide. Despite the urgent industry need for them, anti-viral methods for controlling shrimp viral pathogens are mostly unavailable due, in part, to lack of an adaptive immune response in crustaceans that renders conventional vaccination methods ineffective RNAi gene silencing methods were first described for successful protection of shrimp against white spot disease (WSD) by injecting dsRNA specific to genes of white spot syndrome virus (WSSV) into shrimp in the laboratory as early as 2005. Despite the proven efficacy of anti-viral RNAi technology, to date no method for use of dsRNA in shrimp farms has seen widespread acceptance in the industry. Most likely this is the result of the lack of simple and cost-effective delivery methods for farm-scale anti-viral RNAi methodologies. Recent studies on use and delivery of dsRNA to shrimp via injection and oral routes in hatcheries and on farms, have employed oral delivery using dsRNA-expressing bacteria as a component of dry feed pellets or use of living brine shrimp (Artemia) pre-fed with dsRNA before they are fed to shrimp. Also tested have been dsRNA enclosed in the chitosan-RNA particles including chitosan, liposomes and viral-like particles (VLP, empty, non-infective viral capsids) for direct injection or use as components of feed pellets for hatchery or pond-reared shrimp. Objective challenges to effective delivery of RNAi agents in feed include vulnerability to chemical and enzymatic degradation, leakage to the aqueous environment, and its opposite concern: limited bioavailability of encapsulated RNAi agents, as well as palatability, simplicity of operation and cost-effectiveness. US Patent Publication No. 2014/0371295 to Loy et al teaches the delivery of dsRNA targeting organisms pathogenic to shrimp and other aquatic invertebrates (specifically the Myonecrosis virus) by feeding, injection, biolistic delivery, immersion, poration, liposomes, alphavirus replicon particles and various forms of encapsulation. US Patent Publication No. 2005/0080032 to Gross et al teaches delivery of dsRNA targeting pathogenic/parasitic microorganisms of marine invertebrates by injection, ingestion, immersion, encapsulation, specifically by microbial biodelivery of genetically engineered microorganisms expressing the dsRNA. US20200032267—to Sayre et al teaches RNAi strategies for biocontrol of aquatic pathogens in aquatic organisms using paratransgenic probiotic bacteria for RNAi delivery. US20150240236—to Brown et al teaches the use of viral-derived double-stranded RNA particles for delivery of RNAi targeting aquatic pathogens, and in particular, viral pathogens of salmonid fish and penaeid shrimp. Additional relevant publications include Itsathitphaisarn et al, J. Invert. Pathol, 2017, 147:76-85, US20190175518 to Ufaz et al, US 2013/0245091 and U.S. Pat. No. 9,011,919 to Rozema et al, US 2014/0335192 to Ward et al, US 2011/0033547 to Kjems et al., PCT Publication No: WO 2008/003329 to Besenbacher et al, US Patent Publication 20080194504 to Kyle et al, EP Patent No. 2397123 to Aarhus Univ, US201020295355-A1 to Baker Shenda et al, US20110064664-A1 to Lopez-Berestein et al, US20120238735-A1 to McManus et al; Sarathi et al, Marine Biotech 2008 10.3:242-249; Feroskhan et al, Current Nanoscience 2014, 10.3:453-464. SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a particulate composition comprising chitosan and at least one type of RNA molecule comprising a nucleic acid sequence at least 50 bases in length, wherein the chitosan is 50-100% deacetylated low molecular weight chitosan, wherein the chitosan:RNA r