Source: UNIV OF MARYLAND submitted to
GENERATION OF ZOONOTIC INFLUENZA RESISTANT RECOMBINANT PIGS VIA SITE-DIRECTED TECHNOLOGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1004964
Grant No.
2015-67015-22845
Project No.
MD-ANSC-6377
Proposal No.
2014-09126
Multistate No.
(N/A)
Program Code
A1241
Project Start Date
Jan 1, 2015
Project End Date
Dec 31, 2019
Grant Year
2015
Project Director
Telugu, B.
Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Animal & Avian Sciences
Non Technical Summary
Influenza or flu is one of top 3 economic diseases affecting the pork industry. In humans, it causes up to 41,000 human fatalities in United States and upwards of 500,000 casualties worldwide. Pigs serve as reservoirs for swine, avian, and human viruses and produce novel high potency strains, similar to the "Swine flu" pandemic H1N1 strain. Our hypothesis is that eliminating receptors for viral entry and interfering with viral replication will serves as a dual mechanism for protecting the pigs from viral infection, and transmission of flu to human and pig hosts. There are three objectives in the proposal: 1) assemble and validate genome editing tools to delete the receptor for flu virus entry, and prevent viral propagation; 2) generate recombinant pigs; and 3) test the recombinant pigs for susceptibility to infection by swine and human type adapted viruses to infection and dissemination. We expect to develop a pig model of influenza research and identify druggable targets. From agricultural stand point, we anticipate elimination of flu from commercial swine herds.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31135101040100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The major goals of the project are:Aim-1 Assemble and validate novel shRNA and defective interfering particle based attenuation vectors targeting IAV replication, assembly, and dissemination of swine, avian and human IAV strains ex vivo.Aim-2 Create genome edited pigs resistant to IAV infection and transfer. Two strategies will be employed.Inject TALEN constructs into 1- cell embryos (zygotes) to induce site-specific double strand breaks and knockout human (ST6GAL1) and/or avian (ST3GAL1) IAV receptors Using TALENs, knock-in shRNA DI- decoys into IAV receptor loci thereby effectively knocking out the receptors. Using the modified cells generate recombinant pigs via nuclear transfer (cloning).Aim-3 Challenge ST6GAL1 KO/shRNA decoy pigs with swine IAV subtypes and assess viral production and transmission rates.
Project Methods
The project employs advanced genome editing tools such as:1) Transcriptional activators like effector nucleases (TALENS)2) Gene decoys that mimic viral replication3) Genome editing by injections of TALENs and CRISPRs into the early embryos.

Progress 01/01/18 to 12/31/18

Outputs
Target Audience:Results from the research were presented to the scientific community, students and academicians at several national and international meetings acknowledging the funding source. Changes/Problems:The defective interfering particles/decoys that were proposed in the proposal and since been tested at the Roslin Institute and have been found not be adequately expressed and/or ineffective in mounting resilience against viral infections. In this regard, we have altered the strategy and have since used interferon inducible MX transgene as a second layer of defense, in addition to the receptor ablation. What opportunities for training and professional development has the project provided?A postdoctoral student and a graduate student have received training in generating targeting constructs, nucleofection and stable selection of cells and generating stable selected cells. How have the results been disseminated to communities of interest?The results have been presented at various national and international conferences (E.g. LAGE, Large Animal Genome Editing Conference) to target audiences. What do you plan to do during the next reporting period to accomplish the goals?Theknockout/knockin cells were used in somatic cell nuclear transfer/cloning to generate a pregnancy. Cell lines from the piglets will be tested for IAV resilience. The validated lines will be used to generate a cohort of pigs for whole animal viral challenge studies to be performed by co-PI, Dr. Amy Vincent at NADC, Iowa.

Impacts
What was accomplished under these goals? CRISPR/Cas knockout of ST3 and ST6GAL1 knockout animals have been generated. Cell lines from the tracheal epithelium were established for invitro viral challenge experiments. A mouse MX transgene under the regulation of IFNB promoter has been knockin into the ST6GAL1 knockout fibroblasts.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: 1) Sheets TP, Park KE, Park CH, Swift SM, Powell A, Donovan DM, Telugu BP*. CRISPR/Cas9 Ablation of NEUROGENIN 3 (NGN3) in Domestic Pigs Impairs Pancreatic Endocrine but not Exocrine Development. Scientific Reports. 2018 Feb 26;8(1):3582. PMID: 29483633. 2) Zhou Y, Shen B, Jiang J, Padhi A, Park KE, Oswalt A, Sattler CG, Telugu BP, Chen H, Cole JB, Liu GE, Ma L. Construction of PRDM9 allele-specific recombination maps in cattle using large-scale pedigree analysis and genome-wide single sperm genomics. DNA Research. 2017 Nov 27. doi: 10.1093. PMID: 29186399


Progress 01/01/17 to 12/31/17

Outputs
Target Audience:Target audiences reached by the project include academians, researchers, and animal husbandry industry. The target audiences are reached by laboratory instruction, or practicum experiences; development of innovative research methodologies; workshops; and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: The grant serves to train a postdoctoral fellow, a graduate student and a technician in advanced CRISPR/Cas genome editing tools. Professional development: The technical knowledge and skill level of the trainees was enhanced by encouraging participation in workshops, conferences, seminars, study groups, and individual study. How have the results been disseminated to communities of interest?The results were communicated by research articles, review articles and at seminars at national and international meetings (Large animal genetic engineering summing; CRISPR AG Bio conference; 6th Swine in Biomedical Research Conference). What do you plan to do during the next reporting period to accomplish the goals?We plan to validate that the ablation of ST3 and ST6GAL1 results in reduced infection from IAV. Once confirmed, the fibroblast cells will be used for cloning to generate a cohort of animals for challenge studies at NADC, IA.

Impacts
What was accomplished under these goals? Aim-1: The Roslin collaborators are assembling and validating decoy vectors. Aim-2: We have successfully generated ST3- and ST6-GAL1 double knockout pigs. This illustrates that the double ablation is not embryonic lethal. Primary tracheal and fetal fibroblast cells were established from the pigs. Co-investigators are currently testing the loss of infection in these lines. Aim-3: The fetal fibroblasts from ST3- and ST6GAL1 double knockout pigs will be utilized for the generation of a cohort of pigs for challenge studies in 2018 at NADC, IA

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Park KE, Powell A, Sandmaier SES, Kim C, Mileham A, Donovan DM, Telugu BP. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Scientific Reports. 2017 Feb 14; 7:42458. PMID: 28195163
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Genovese NJ, Domeier TL, Telugu BP, Roberts RM. Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells. Scientific Reports. 2017 Feb 6; 7:41833. PMID: 28165492
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Park KE, Kaucher A, Powell A, Waqas SM, Sandmaier SES, Oatley MJ, Park CH, Tibary A, Donovan DM, Blomberg L, Lillico S, Whitelaw CBA, Mileham A, Telugu BP, and Jon M. Oatley (2016). Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Scientific Reports. 2017 Jan 10; 7:40176. PMID: 28071690
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sheets TP, Park CH, Park KE, Powell A, Donovan DM, Telugu BP. Somatic cell nuclear transfer followed by CRIPSR/Cas9 microinjection results in highly efficient genome editing in cloned pigs. International Journal of Molecular Sciences. 2016 Dec 3;17(12). pii: E2031. PMID: 27918485
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Telugu BP, Park KE, Park CH. Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications. Mammalian Genome. 2017 Jul 15. doi: 10.1007/s00335-017-9709-4. [Epub ahead of print] PMID: 28712062


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:During this funding period, the PI has attended and presented at the Large animal genetic engineering summit, Annual Animal Health meeting in Greensboro, and US-EU meeting on Genome editing in Budapest, Hungary.Using these media, the PI has reachedout to academians, students, and Governmental regulators. Additionally, the PI's work has been highlighted in NY times, You tube, and presented at the National Academy of Medicine.Via these media, the PI has reached out tolay persons. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided opportunity to train a graduate student and a postdoctoral trainee in performing genome editing in livestock. How have the results been disseminated to communities of interest?Yes, the data has been disseminated as publications. What do you plan to do during the next reporting period to accomplish the goals?The ST6- and ST3GAL1 fetal fibroblast cells will be used to generate clonal pigss for viral challenge experiments

Impacts
What was accomplished under these goals? We have successfully generated and valided the CRISPRs to generate simultaneous knockout of ST6- and ST3GAL1 loci by injection of CRISPR reagents into the cytoplasm of 1-cell pig embryos. We have established a successful pregnancy and collected fetuses for establishing fetal fibroblast lines. We have screened and confirmed double knockout of the ST6- and ST3 GAL1 loci.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 1. Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs. Sheets TP, Park CH, Park KE, Powell A, Donovan DM, Telugu BP. Int J Mol Sci. 2016 Dec 3;17(12). pii: E2031. PMID: 27918485 Free Article 2. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins. Park KE, Park CH, Powell A, Martin J, Donovan DM, Telugu BP. Int J Mol Sci. 2016 May 26;17(6). pii: E810. doi: 10.3390/ijms17060810. PMID: 27240344 Free PMC Article 3.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene Park KE, Kaucher A, Powell A, Waqas MS, Sandmaier SE, Oatley MJ, Park CH,Tibary A, Donovan DM, Blomberg L, Lillico S, Whitelaw CBA, Mileham A, Telugu BP, and Oatley JM Scientific Reports (Accepted)


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Target audience are scientific community and industry groups, primarily animal genetics companies and pharamaceutical companies. These target audience are reached via publication of a manuscript and through News article published in New York Times. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training for a postdoctoral fellow to generate and validate editors for genome editing. The project also allowed the postdoctoral fellow and a technician to generate and validate a landing site into a safe harbor locus. How have the results been disseminated to communities of interest?Yes. A publication to this effect has already been published. Additionally, the PD is already engaged the public into the work that is underway to generate flu resistant animals. What do you plan to do during the next reporting period to accomplish the goals?We will generate the IAV receptor null pigs for testing resistance to IAV We also plan to knockin a interferon responsive MX cassette into the safe harbor locus.

Impacts
What was accomplished under these goals? Aim-1: Flu decoys were assembled and were introduced into porcine zygotes and modified animals are currently being generated. Aim-2: Genome editors were generated and validated for injections into zygotes to generate ST6- and ST3GAL1 double knockout pigs. The embryo transfers will be attempted in January. Aim-3: Nothing to report here

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Whitelaw CB, Sheets TP, Lillico SG, Telugu BP (2015). Engineering large animal models of human disease. J Pathol. [Epub ahead of print] Review. PubMed PMID: 26414877