SOW - Novel mouse models for prion disease_9.11.pdf

Statement of Work (SOW)

Project: Development of novel gene-targeted mouse models for studying livestock prion disease of cattle, sheep and goats, cervids, and camel

GENERAL INFORMATION

1.0 Scope of Work:

Prion disease is a group of always-fatal transmissible neurodegenerative diseases of humans and animals with devastating impact on human public health, food safety, wildlife welfare, and agriculture. There are historic outbreaks of prion diseases in livestock including scrapie in sheep and goats, BSE in cattle, TME in mink, camel prion disease (CPD) in camelids, and chronic wasting disease (CWD) in captive and free-raging cervids. Studying prion disease requires transmission of the prion materials to natural host animals, rodents, genetically engineered mice, and in vitro cultured cells. USDA Virus and Prion Research Unit (VPRU) is now the largest international research institution with the capacity of studying prion diseases using various large animals, a position that is and will remain unchallenged. The advantages of using large animals are that they can accurately reflect the clinical manifestations, neuropathological features, and epidemiological patterns of prion diseases, as well as being irreplaceable for future testing of anti-prion therapies.

However, large-animal studies are very expensive, moreover, complex Prion Protein (PrP) polymorphisms/variants make it extremely difficult to breed enough homozygous experimental cohort for transmission studies, upfront experiment planning being very hard, and prion incubation times in large animals are often up to years. To accelerate and strengthen the research power of VPRU, we urgently need to add cutting-edge gene-targeted mouse, cellular and organoid cultural models in our research platform. In this project, we are developing novel mouse models based on our previous research, combining the advantages of traditional transgenic mouse and gene-targeted mouse strategies to create gene-targeted normal and overexpressors. The success of this project will force reconsideration of current strategies to develop authentic and timely gene-engineered rodent models, it will have a significant impact on the basic and application prion research. We will develop 16 gene-targeted mouse models using CRISP-Cas9 genome editing, or embryo stem cell (ESC) based homologous recommendation strategies.

2.0 Background:

The USDA research scientist has extensive experience in mouse model development. Working with three of the top seven mice model companies (https://www.verifiedmarketresearch.com/blog/top-mice-model-companies/), he has designed, generated, and characterized over 60 PrP transgenic mouse (random integration) and gene-targeted mouse models using embryo stem cell homologous recombination and CRSPR-Cas9 genome editing strategies. This project will generate (below Table): 1) 15 gene-targeted mice expressing various species prion proteins by replacing the endogenous FVB mouse PrP coding sequence using CRISPR/Cas9- or ESC-based homologous recombination strategies; and 2) 1

Rosa26 KI mice containing gRNA-dCas9-VPR transcription activator cassette (~12,000 bps).

CONTRACTOR REQUIREMENTS

3.0 Technical Requirements/Tasks:

1) Contractor must have experience with and be able to offer comprehensive services covering the entire process of novel mouse model generation using transgenic and gene-targeted strategies including model design, development, breeding, long-term cryopreservation, and phenotype analysis; with extensive collaborations with scientists from the United States federal laboratories and other research institutes, leading to publications in prestigious journals. Previous service experience with prion protein transgenic or gene-targeted mouse development is preferred. Contractor will provide references of this work with quote.

2) All project design plans must be approved by the USDA principal investigator prior to starting work on any project. The company will provide timely project milestone reports. The investigator will be notified should the company encounter issues during the project.

3) Project milestones and deliverables

Milestone 1: A detailed project design packet.

Milestone 2: Guide RNA (gRNA) design and verification, and DNA donor vector design and sequence verification.

Milestone 3: Microinjection of reagents into single-cell fertilized FVB embryos, transfer of embryos into pseudo pregnant females.

Milestone 4: Identification of founders, tissue biopsy, genotyping assay

(PCR/Sequencing based), the interest gene and the up- and down-stream junction regions (up to 1000 bp each) will be sequence verified.

Milestone 5: Off-target analysis, top 10 high-risk off-target sites verification.

Milestone 6: Generation of F1 heterozygous mice, up to 4 founders will be bred to wildtype FVB/NJ mice to generate F1, F1 will be genotyped, at least 4 F1 mice (both male and female) will be shipped.

4) The proposed models must be generated using Mouse line: FVB/NJ, using the same FVB/NJ strain.

Expected Turnaround timeframes:

Milestone 1: 4 weeks from the date of award Milestone 2: 14 weeks from the date of award Milestone 3: 24 weeks from the date of award Milestone 4: 34 weeks from the date of award Milestone 5: 42 weeks from the date of award Milestone 6: 52 weeks from the date of award

5) Turnaround time: the estimate delivery timelines of F1 heterozygous mice are 52 weeks from the date of award.

6) Model developmental strategies: utilize the CRISPR/Cas9, or embryo stem (ES) cell homologous recombination, to precisely replace the endogenous mouse Prnp coding sequence (from ATG to TGA in exon 3 of NM_011170.3) with other species prion protein coding sequences (Table, electronic sequences will be provided by the USDA Research Scientist) without introducing any unwanted DNA sequences/variations including indels, synonymous and nonsynonymous mutations in the interest gene and the flanking regions. No scar sequence in mouse genome if ESC homologous recommendation strategy used.

7) Thorough off-target analysis of the top 10 high risk off-target sites to confirm there is no off-target editing.

8) Mice will be Specific and Opportunistic Pathogen-Free (SOPF) and arrive from a maximum barrier facility. If an additional quarantine facility other than NADC animal facility is needed, the cost will be covered by the company.

9) USDA will not be charged for the service if provider is unable to generate and deliver the model(s) as agreed in the project contract.

4.0 Government Furnished: N/A

5.0 Deliverables / Schedule:

The principal investigator at Virus and Prion Research Unit, National Animal Disease Center, USDA-ARS will be responsible for reviewing and approving milestone reports and final products generated under the contract.

The service company will deliver timely progress reports, and the mice within approximately 12 months from the date of award (The timeline is approximate.

Any changes to the timeline must be approved by the USDA.)

1) Project milestone reports and deliverables

Milestone report 1: A detailed project design packet. 4 weeks from the date of award.

Milestone report 2: Guide RNA (gRNA) design and verification, and DNA donor vector design and sequence verification. 14 weeks from the date of award.

1 KI-Rosa26-dCas9-VPR ~12,000 bps 2 KI-chimeric mouse prion protein gene ~3000 bps 3 Prion protein CDS 1 ~800 bps 4 Prion protein CDS 2 ~800 bps 5 Prion protein CDS 3 ~800 bps 6 Prion protein CDS 4 ~800 bps 7 Prion protein CDS 5 ~800 bps 8 Prion protein CDS 6 ~800 bps 9 Prion protein CDS 7 ~800 bps 10 Prion protein CDS 8 ~800 bps 11 Prion protein CDS 9 ~800 bps 12 Prion protein CDS 10 ~800 bps 13 Prion protein CDS 11 ~800 bps 14 Prion protein CDS 12 ~800 bps 15 Prion protein CDS 13 ~800 bps 16 Prion protein CDS 14 ~800 bps

Gene‐targeted mouse models

Milestone report 3: Microinjection of reagents into single-cell fertilized FVB embryos, transfer of embryos into pseudo pregnant females. 24 weeks from the date of award.

Milestone report 4: Identification of founders, tissue biopsy, genotyping assay (PCR/Sequencing based), the interest gene and the up- and down-stream junction regions (up to 1000 bp each) will be sequence verified. 34 weeks from the date of award.

Milestone report 5: Off-target analysis, top 10 high-risk off-target sites verification. 42 weeks from the date of award.

Milestone report 6: Generation of F1 heterozygous mice, up to 4 founders will be bred to wildtype FVB/NJ mice to generate F1, F1 will be genotyped, at least 4 F1 mice (both male and female) will be shipped. 52 weeks from the date of award.

2) Mouse delivery

6.0 Data Rights: N/A

7.0 Place of Performance: Work detailed above will be conducted and completed at

Contractor’s laboratory facility. Mice will be delivered to USDA following at USDA, 1920 Dayton Avenue, Ames, IA 50010.

8.0 Period of Performance:

Milestone report 1: A detailed project design packet. 4 weeks from the date of award.

Milestone report 2: Guide RNA (gRNA) design and verification, and DNA donor vector design and sequence verification. 14 weeks from the date of award.

Progress report Mice

1 KI-Rosa26-dCas9-VPR ~12,000 bps Milestone reports 1 ‐ 6

2 KI-chimeric mouse prion protein gene ~3000 bps Milestone reports 1 ‐ 6

3 Prion protein CDS 1 ~800 bps Milestone reports 1 ‐ 6

4 Prion protein CDS 2 ~800 bps Milestone reports 1 ‐ 6

5 Prion protein CDS 3 ~800 bps Milestone reports 1 ‐ 6

6 Prion protein CDS 4 ~800 bps Milestone reports 1 ‐ 6

7 Prion protein CDS 5 ~800 bps Milestone reports 1 ‐ 6

8 Prion protein CDS 6 ~800 bps Milestone reports 1 ‐ 6

9 Prion protein CDS 7 ~800 bps Milestone reports 1 ‐ 6

10 Prion protein CDS 8 ~800 bps Milestone reports 1 ‐ 6

11 Prion protein CDS 9 ~800 bps Milestone reports 1 ‐ 6

12 Prion protein CDS 10 ~800 bps Milestone reports 1 ‐ 6

13 Prion protein CDS 11 ~800 bps Milestone reports 1 ‐ 6

14 Prion protein CDS 12 ~800 bps Milestone reports 1 ‐ 6

15 Prion protein CDS 13 ~800 bps Milestone reports 1 ‐ 6

16 Prion protein CDS 14 ~800 bps Milestone reports 1 ‐ 6

Deliverable

A minimum four heterozygous F1/N1 mice, including both male and female, will be delivered in 9 ‐ 12 months after initiation for each project.

Gene‐targeted mice and insertions

Milestone report 3: Microinjection of reagents into single-cell fertilized FVB embryos, transfer of embryos into pseudo pregnant females. 24 weeks from the date of award.

Milestone report 4: Identification of founders, tissue biopsy, genotyping assay (PCR/Sequencing based), the interest gene and the up- and down-stream junction regions (up to 1000 bp each) will be sequence verified. 34 weeks from the date of award.

Milestone report 5: Off-target analysis, top 10 high-risk off-target sites verification. 42 weeks from the date of award.

Milestone report 6: Generation of F1 heterozygous mice, up to 4 founders will be bred to wildtype FVB/NJ mice to generate F1, F1 will be genotyped, at least 4 F1 mice (both male and female) will be shipped. 52 weeks from the date of award.

9.0 Technical Point of Contact:

N/A