Source: UNIVERSITY OF ARIZONA submitted to
A VIRAL VECTOR FOR AN ORAL DELIVERY OF RNAI-BASED THERAPEUTICS IN SHRIMP
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1027875
Grant No.
2022-67015-36332
Project No.
ARZW-2021-06960
Proposal No.
2021-06960
Multistate No.
(N/A)
Program Code
A1221
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2024
Grant Year
2022
Project Director
Dhar, A.
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Animal&Biomedical Sciences
Non Technical Summary
White Spot Disease (WSD), caused by the White Spot Syndrome Virus (WSSV) is a threat to crustacean farming (e.g., shrimp & crawfish) in the US and elsewhere in the world causing billions of dollars of losses, and there are no commercially available therapeutics against the disease, as of today. Our goal is to develop a therapeutic against WSD using an RNA interference (RNAi) approach. The objective is to develop a replication deficient viral vector to deliver RNAi molecules via oral route using commercial feed used in shrimp farming. We expect to develop an oral vaccine against WSD in shrimp and other crustaceans. Availability of an oral vaccine against WSD delivered by mixing it with a commercial diet, used in shrimp farming, can have an enormous impact in mitigating losses due to WSD, in the US and other shrimp producing countries in Asia and Latin America.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31137211101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3721 - Marine shrimp;

Field Of Science
1101 - Virology;
Goals / Objectives
White Spot Disease (WSD), caused by the White Spot Syndrome Virus (WSSV) is a threat to crustacean (e.g., shrimp & crawfish) farming in the US and globally. There is no measure to control WSD except prevention. RNA interference (RNAi)-based therapeutics delivered via injection to live shrimp has shown promise. However, due to the lack of an oral delivery method, there is no RNAi-based therapeutic for a commercial application, because injection of shrimp is not feasible.We propose to use a freshwater prawn, Macrobrachium rosenbergii Nodavirus (MrNV)-based viral vector to deliver RNAi molecules via oral route to control WSD.The objectives of the project are:Construction of a shrimp viral vector containing hairpin-RNA (hRNA) targeted against WSSV structural and non-structural genes. Confirm expression of hRNA molecules delivered using a shrimp viral vector in vitro (using Sf9 cells and shrimp primary hemocytes) and in vivo (live shrimp).Determine the efficacy of shrimp viral vector in delivering hRNA via diet and protecting shrimp against WSD.
Project Methods
1. Construction of a shrimp viral vector containing hairpin RNA (hRNA) targeted against WSSV structural and non-structural genes.2. Expression of hRNA delivered using a shrimp viral vector in vitro (using Sf9 cells and shrimp primary hemocytes) and in vivo (live shrimp).3.Determiningthe efficacy of shrimp viral vector in delivering hRNA via diet and protecting shrimp against WSD.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience: Shrimp and crayfish industry in the US and elsewhere in the world. Aquafeed industry working to develop functional feed to control white spot disease in crustaceans. Animal health professionals working in government, academia, and industry working to develop therapeutics against viral diseases in crustaceans. Changes/Problems:We encountered a technical challenge in cloning a hairpin RNA in the shrimp viral vector. Initially, to expedite the project, the cloning ofhairpin RNA in a shrimp viral vectorwas outsourced to a vendor who routinely provide services of viral vector cloning. Unfortunately, the vendor failed to make stable clone containing therapeutic RNA molecule targeting white spot syndrome virus. Then apostdoctoral fellow working on the project had to clone the gene in hpuse through iterations and improvizing cloning technique. This delayed the project outcomes. So far, have made one out of three viral clones originally proposed in the project proposal. This clone was tested in an injection bioassay to determine the efficacy in providing protection against WSD. We got succesful result in the injection bioassay and we are now cloning the remaining two therapeutic RNA in the shrimp viral vector. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The research findings of this project was communicated through a peer-reviewed publication in the journal Proceedings of National Academy of Science-Nexus and through oral presentations in several national and international conferences/ meetings worldwide. The audience of these conferences and meetings included professionals from academic institutions, industry, government, and non-governmental organizations working on shrimp and VMOs, aquatic animal health professionals. A complete list of publications is provided under "Publication" Section. In addition, the findings of this project was also disseminated during a "Shrimp Pathology Short Course", a summer course held in the Aquaculture Pathology Laboratory, School of Animal & Comparative Biomedical Sciences, University of Arizona in June 12-17, 2023. The training was attended by 12 participants from five countries including Brazil, Colombia, Mexico, Venezuela, and USA . What do you plan to do during the next reporting period to accomplish the goals?1. So far we have made recombinant shrimpnodaviral vector carrying hairpin RNA for one of the three targets of WSSV, VP28 gene. We plan to complete the cloning and production ofrecombinant shrimpnodaviral vectorcarrying hairpin RNA for the remaining two target genes of WSSV. 2. An injection bioassay using purified recombinant shrimpnodavirus carrying hairpin RNA targeting WSSV VP28 gene provided control against white spot disease. We will now conduct a bioassay by delivering the recombinant shrimpnodaviruscarrying hairpin RNA targeting WSSV VP28 gene via commercial diet and determine the efficacy in protecting shrimp against WSD. 3. Based on the finding of this bioasay, as decribed in #2, that involves delivering WSSV VP28 RNAi therapeutic via commercial diet, we will design experimental bioasay to deliver the remaining two RNAi constructs via oral route and determine which of three RNAi construct provides highest level of protection against WSD.

Impacts
What was accomplished under these goals? Objective #1 : Construction of a shrimp viral vector containing hairpin-RNA (hRNA) targeted against WSSV structural and nonstructural genes. Major activities completed / experiments conducted Bivalent hairpin RNA was constructed to investigate the combined strength of dual-targeted hairpin RNAs; VP9 (non-structural) and VP28 + VP9 DNA templates. These need to be clones in BV-MrNV backbone. Also ordered custom synthesis of VP9 (non-structural) and VP28 + VP9 RNA that could be used as positive control for injection purpose. RNA detection, Viral particle isolation and transmission electron microscopy (TEM) confirming the assembly of viral vector and hairpin cargo for lhVP28 construct. Data collected: TEM micrograph showed the successful assembly of the viral vector particles. RNA isolation and visualization displayed a full-length hairpin RNA. Summary statistics and discussion of results Detection of the intact hairpin RNA and TEM micrograph showed the successful production of the viral vector and hairpin cargo complex. Cloning of hairpin RNA targeting VP 19 and Vp9 are on-going. Objective #2 : Confirm expression of hRNA molecules delivered using a shrimp viral vector in vitro (using Sf9 cells and shrimp primary hemocytes) and in vivo (live shrimp). Major activities completed / experiments conducted Analysis of full-length expression of therapeutic RNA from the viral vector construct. (Completed for VP28 hRNA). Data collected: MrNV and VP28 RNA were also detected by RTqPCR in Sf9 cells confirming the production of the therapeutic RNA molecule. RNA extracted from the viral vector and SF9 cells showed the intact hairpin RNA for VP28-hRNA but not for VP28-dsRNA. Objective #3 : Determining the efficacy of shrimp viral vector in delivering hRNA via diet and protecting shrimp against WSD. Major activities completed / experiments conducted Feed preparation and palatability test Trial bioassay run with diet treatments containing viral vector VP28-hRNA and VP28-dsRNA challenged with white spot syndrome virus. Survival assay was performed by direct administration by injecting the purified viral vector carrying lhVP28 and purified RNA from the produced viral vector; followed by a viral challenge test with white spot syndrome virus. Control groups were saline-treated (positive control), GFP RNA-treated (negative control) and naïve groups. Confirmation of the effectiveness of treatment through molecular analysis. Quantification of viral load and knockdown of target viral gene (VP28) after treatment and viral challenge. Data collected: Optimum feed preparation with 1:0.5 ratio (grams to mL) of food pellet to viral vector solution. Food pellets are fully taken up by animals within 2-5 mins. A slight delay (1-2 days) in cumulative mortality was seen in the treatment groups as compared to the control groups. All groups reached 100% mortality. 100% survival for all treatment groups observed for 14 days post-infection. Negative control groups reached 100% mortality at day 6 or 7 post-infection. The viral load was reduced in both treatment groups displaying zero viral DNA copy number. Relative quantification of RNA of the targeted WSSV VP28 gene was confirmed to be downregulated. Summary statistics and discussion of results This mode of preparation retains the integrity and palatability of the food diet ensuring successful oral take-up. A slight delay in mortality may signify that the dosage of the orally delivered therapeutic agent was insufficient, resulting in little to no protection against viral infection. The survival curve shows a significant increase in survival among treatment groups as compared to the negative control groups. Zero copy numbers in treatment groups showed a significant reduction and viral suppression compared to the 2x105 average viral copy number per nanogram DNA in infected shrimp in the negative control. Reduction of viral copies quantifying VP644 gene (unrelated to knockdown gene VP28) displays complete protection from WSSV and not only the knockdown or suppression of one viral gene. Significant downregulation of VP28 RNA in treatment groups as compared to GFP RNA treated groups demonstrates the effectivity of the active component (lhVP28 RNA) of viral vector treatment. Key outcomes or other accomplishments realized. Maintaining the integrity of the food pellet ensures food uptake by the animals. There is a need to concentrate the therapeutic material before incorporating it into the diet and ensure proper dosage rendering protection. The stability of RNA should also be investigated upon incorporation into the feed. Treatment material is capable of suppressing viral infection in shrimp when administered directly by injection. Protection against WSSV resulted from gene-targeted therapy.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Alenton, Rod Russel R., Mai, Hung N. and Dhar, Arun K. 2023. Engineering a replication incompetent viral vector for delivery of therapeutic RNA in crustacean. Proc. Natl. Acad. Sci- Nexus, 2: 1-9, https://doi.org/10.1093/pnasnexus/pgad278
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Aquaculture Pathology Laboratory, University of Arizona - The World Organization for Animal Health Reference Laboratory of Crustacean Diseases Tools⿝, The Launch of a Regional Aquatic Animal Health Laboratory Network (RAAHLN) for Africa, December 05-07, 2023, Pretoria, South Africa.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dhar, Arun K. 2023. ⿿Keynote address: Expediting pathogen discovery and developing antiviral therapy in shrimp⿝, 4th International mini-Symposium on the Control of Aquatic Animal Disease 2023 (CAAD 2023) will be held at National Cheng Kung University, Taiwan (NCKU) on November 26-29, 2023, Taiwan.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dhar, Arun K. 2023. ⿿Engineering a Viral Vector Platform for the Delivery of therapeutic RNA in Crustacean & Developing Oral Vaccine in Fish⿝, Global Fisheries Conference, November 21-22, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dhar, Arun K. 2023. ⿿Major Diseases & Disease Management in Shrimp Aquaculture⿝, College of Veterinary Medicine, Mississippi State University, Starkville, MS., November 10, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 8. Dhar, Arun K. 2023. ⿿Diseases & Disease Management in Shrimp Aquaculture⿝, Bandung Institute of Technology (Intitut Teknologi Badung), Jawa Barat, Indonesia, November 01, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Alenton, R. R.R., Mai, H. N. & Dhar, A. K. 2023. An oral RNA delivery platform using reverse-engineered Nodavirus for marine shrimp Aquaculture America 2023 Conference, World Aquaculture Society, February 23-26, 2023, New Orleans, Louisiana.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Alenton, R. R.R., Mai, H. N. & Dhar, A. K. 2023. 2023. Overcoming roadblocks for RNA-targeted antiviral therapy in shrimp through a vector-based oral delivery platform. The Conference of Research Workers in Animal Diseases, January 22-24, 2023, Chicago, Illinois.
  • Type: Other Status: Published Year Published: 2023 Citation: 1. Dhar, Arun K. 2023. Reverse genetics approaches to study viral pathogenesis and developing viral vector for an oral delivery of therapeutic molecules in shrimp, Central Fisheries Education Institute, as a part of Fulbright Specialist Visit to CIFE, September 09, 2023,


Progress 01/01/22 to 12/31/22

Outputs
Target Audience: Shrimp industry in the US and elsewhere in the world. Researchers and professionals from academia, industry, government and non-governmental organizations working on shrimp and fish diseases, and aquatic animal health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The research efforts of the project have been communicated through invited talks in universities worldwide and scientific conferences. We have reached students and researchers internationally through invited talks at universities in Asia (i.e., India and Taiwan) and the Americas (i.e., USA and Mexico). Most target audiences of the project were reached by presenting at strategic scientific conferences, such as Aquaculture America (2022 & 2023) and Conference for Research in Animal Diseases (2023), which bring together universities, industry, government, and non-governmental organizations working on shrimp and VMOs, aquatic animal health professionals. In addition, by holding workshops and training, such as the Aquaculture Pathology Annual Short Course, we reached representatives of government agencies and private companies from different countries in the Americas. What do you plan to do during the next reporting period to accomplish the goals? The next reporting period will cover how our viral vector will be used as a therapeutic oral treatment, which we already started. MrNV viral vector carrying hRNA and dsRNA targeting VP28, VP19, or VP9 shall be incorporated in the diet of shrimp and shall be fed in several dosages; and their successful delivery in the shrimp tissues will be determined and measured. Finally, the efficacy of the treatment materials shall be determined through viral challenge using live animals. In addition to the initial plan, we constructed double-stranded RNA (dsRNA) in addition to hairpin RNA (hRNA). Both RNA forms can trigger gene silencing against viral genes, however, testing both shall give us more options for the stability of the therapeutic materials, which is important for their incorporation into the shrimp diet. During the current reporting period, we experience delays in the synthesizing of the hRNA-carrying viral vector from our designated vendor. For this, we came up with our in-house production which will be easily adapted for additional therapeutic materials that we would need to test.

Impacts
(N/A)

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Rod Russel R. Alenton., Hung N. Mai, and Arun K. Dhar. 2022. Reverse-genetics approach in developing a Macrobrachium rosenbergii Nodavirus (MrNV)-based viral vector for an oral delivery of therapeutics in shrimp. Aquaculture America 2022 Triennial Conference. February 28 - March 4, 2022, Town and Country Conference Center San Diego, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Rod Russel R. Alenton Antiviral therapies for shrimp aquaculture. Ensenada Center for Scientific Research and Higher Education (CISESE), Ensenada, Mexico. August 08, 2022
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Arun K. Dhar. 2022. Emergent Diseases and efforts toward developing antiviral therapy in shrimp. Indian Council of Agricultural Research Lecture Series on the occasion of 75th Years of Independence of India, organized by Central Institute of Brackishwater Aquaculture, Chennai, India, July 29, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Rod Russel R. Alenton, Mary Beth B. Maningas. Diagnostics and RNAi based therapeutics for the shrimp industry. National Cheng Kung University, Taipei, Taiwan, July 14, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Arun K. Dhar. 2022. Discovering Pathogens and Developing Antiviral Therapies in Marine Shrimp Using a Reverse Genetic Approach, West Bengal University of Fishery & Animal Sciences, Kolkata, West Bengal, India, July 14, 2022.