Source: PreGentis, Inc. submitted to
CHARACTERIZATION OF PRIMARY BOVINE CELLS FOR GENOME-EDITING AT THE PRP LOCUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0199599
Grant No.
2004-33610-14322
Project No.
PENK-2004-00163
Proposal No.
2004-00163
Multistate No.
(N/A)
Program Code
8.3
Project Start Date
May 15, 2004
Project End Date
Dec 31, 2005
Grant Year
2004
Project Director
Metz, R. A.
Recipient Organization
PreGentis, Inc.
(N/A)
Newtown,PA 18940
Performing Department
(N/A)
Non Technical Summary
Problem: With the recent development of BSE or mad cow disease, markets around the world are crying out for a safer beef product. Given that the incidence of BSE is actually quite low, why has it had such a profound impact on the public? The answer relates to the discovery in the UK during 1994 of an outbreak in young people of the fatal neurodegenerative disease Creutzfeld-Jakob Disease (CJD) that previously had been seen only in individuals in their 6th and 7th decades. BSE and sporadic CJD are both prion-mediated diseases and the brain pathology of patients dying with this new variant CJD (nvCJD) confirmed that it is also prion-mediated and of BSE origin. To date there have been approximately 130 fatal human cases of nvCJD. However since over 1 million contaminated cattle may have entered the human food chain, there is great concern that the eventual total number of CJD cases stemming from the BSE source could be much higher. Purpose: As no vaccine, antemortem diagnostic test or therapy exists for either nvCJD or BSE, protection depends on preventative measures. The development of cattle that are genetically resistant to acquiring or transmitting BSE would be a major step in this direction. We will use the genome editing technology GenEdit to: 1) Disable the bovine PrP gene so there is no target for BSE infection, or 2) introduce a single amino acid change to render the animal resistant to infection. GenEdit does not introduce foreign DNA into the genome of the cell (non-GMO) and the modified cells are indistinguishable from cells carrying a naturally occurring mutation.
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
3113399108050%
7123499108050%
Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3499 - Dairy cattle, general/other; 3399 - Beef cattle, general/other;

Field Of Science
1080 - Genetics;
Goals / Objectives
The first reported U.S. incident of Bovine spongiform encephalopathy (BSE) or Mad Cow Disease has raised significant public concern regarding the safety of beef and dairy products. The purpose of this research program is to develop and optimize procedures for generating beef and dairy cattle that are genetically resistant to acquiring or transmitting BSE by modifying the endogenous prion gene (PrP) in those cattle. The main objective of this specific proposal is to establish the optimal method of delivering GenEdit targeting molecules to primary bovine cells in tissue culture and to identify which primary cells possess the most suitable characteristics for genome editing and subsequent cloning by nuclear transfer. The successful completion of the six month phase I study will permit the initiation of Phase II studies which uses GenEdit technology to generate and isolate cell clones containing either a null or modified (PrPARR-equivalent to the scrapie resistant allele) bovine PrP gene. In Phase II, fibroblasts derived from fetal or adult donors of each sex will be isolated, PrP-modified, and animals of each sex and allele will be generated from each PrP-modified cell by cloning employing somatic cell nuclear transfer (NT) technologies. The product of a successful phase II study will be cattle carrying a null or PrPARR allele, which will be used to establish heterozygous, hemizygous (PrP0/ARR), and homozygous animals for each allelic variation (PrPwt, PrP0, and PrPARR). In preparation for commercialization, breeding stocks for allele transmission will be established and a portion of the cattle will be challenged with infectious BSE or Bovine scrapie in order to assess their resistance profile. Based on recent studies, the PrPARR allele is a dominant trait, since only one PrPARR allele is sufficient to confer scrapie resistance to heterozygous sheep. Therefore, it is anticipated that PrPARR/wt heterozygote cattle would be also resistant to BSE challenge. If this assumption is correct, generating BSE resistance cattle herds via artificial insemination using a homozygous PrPARR/ARR semen donor would be more readily accomplished. Homozygous knockout, PrP0/0, animals would also be resistant to BSE, although the potential for any negative effects of total PrP disruption on the long-term health of the animal cannot be predicted at this time. Validated BSE-resistant cattle will have immediate and long-term medical, social and economic impact by eliminating the need for extensive and costly BSE screening and evaluation of cattle herds, reducing potential human health risks associated with consuming BSE-infected beef and dairy products, strengthening consumer's confidence in the safety of beef and dairy products, and avoiding possible catastrophic economic consequences for the beef and dairy industries of another or more extensive BSE outbreak.
Project Methods
Our GenEdit strategy for modifying the PrP gene addresses many of the limitations of traditional homologous recombination (HR). The five critical components: (1) Use of microinjection to deliver our targeting molecules to cultured cells, its higher efficiency requiring fewer cells for initial treatment and avoids use of other chemical compounds which may have undesired toxicities. (2) GenEdit technology uses a gene-modification molecule that is at least 50 times more efficient than conventional HR vectors - therefore fewer single cell clones need screening during the isolation process. GenEdit introduces only specified changes in the sequence of the target gene without additional selective genes and therefore leaves no extra DNA sequences or footprints in the genome. (3) A sensitive high throughput screening strategy has been developed enabling prompt identification of PrP-modified cell clones. (4) GenEdit technology is able to simultaneously alter 1 to 5 nucleotides in the target gene, sufficient so that non-functional or resistant alleles can be readily generated. (5) GenEdit technology does not introduce or leave any foreign DNA in the genome and the modified cells are indistinguishable from cells carrying naturally occurring genetic mutations. This point is a key issue relevant to the eventual commercialization of the our BSE-resistant cattle, particularly since the use of genetically modified organisms (GMOs) carrying foreign DNA by the food industry has become a controversial issue, especially in international markets. The first task is establishing primary cell lines. Cattle have been cloned from fibroblast, epithelial, muscle, granulosa, and germ cells. Fibroblast and epithelial cells are our best choices because of tissue availability, microinjectibility and maintenance in culture (40-50 doublings). Fetal skin fibroblasts will be isolated from a 30-60 day fetus and adult fibroblasts from auricular explants. Lines will be tested for population doublings and growth at low densities following single cell sorting. The second task is design of GenEdit molecules specific for the target cell's PrP gene sequence. The existence of a dominant polymorphism barrier for prion disease in animals carrying the PrPARR allele has been shown by a variety of studies including scrapie challenge in sheep naturally carrying this allele. The corresponding amino acid changes will be introduced into the bovine PrP gene. Gene-editing molecules differ from the target sequence by only a few nucleotides. One change affects the gene function while the second change aids identification of the editing event. The third task is optimization of GenEdit molecule delivery. We will compare / contrast direct nuclear microinjection with standard lipofection in each cell line. The final task employs a sensitive screening strategy, allele-preferred PCR followed by restriction fragment length polymorphism analysis, to detect gene-edited events in order to measure their frequency in the various established cell lines, data needed to select the final cloning strategy - single cell sorting versus sequential pooling.

Progress 05/15/04 to 12/31/05

Outputs
During the time of this proposal we successfully accomplished four of our key objectives, the fifth and final objective remained difficult (see below): In tasks 1-4. We characterized and maintained several bovine cell lines derived from male and female donor animals (Holstein). We also determined the conditions necessary to derive a cell clone from a single cell. SDF targeting molecules were generated for introducing the knockout 25Xv PrP0 and 179R PrPARR alleles as proposed and suitable conditions, lipofection and/or microinjection, for SDF delivery to bovine cells were established. We validated the AP-PCR/RFLP genotypic screening approach using a phenotypic gene editing reporter system and also demonstrated that the 25Xv PrP allele can be detected when present at a frequency of 0.01 % (or 1 in 5,000 genomes). The PrPARR allele was able to be detected at 1 in 1,000 genomes. The complication in the fifth and final task became too difficult to over comeTask 5. Assessing the Frequency of Gene Editing events (Unsuccessful-completed) Using one of the SDF targeting molecules (PrP25Xv) we demonstrated successful targeting of bovine PrP in bovine fibroblasts. Based on our pooling strategy approximately 2X105 SDF transfected cells were screened in pools of 500 cells/well (~ 400 samples). We detected approximately 28 positives, estimating that the targeting frequency was approximately 0.014% (~1 in 7,000 cells). The SDF directed sequence modification was confirmed by pyrosequencing. Unexpected Results: During the analysis and cell processing we determined that the cells needed to be cultured for at least 14 days prior to AP-PCR analysis to eliminate the risk of PCR artifacts from SDF carryover. We then devised an alternative screening strategy whereby smaller pools of treated cells were screened from which single cell clones were grown and maintained for secondary analysis. This strategy prove to be difficult, whereby the expansion of faster growing non-converted clones dominated the smaller cell pools and confounded the enrichment and isolation of single converted clones. We reasoned that it will be necessary to plate at single cell per well at the onset of the experiment in order to isolate a pure converted clone. At a frequency of 1 editing event per 7000 cells, we would need to plate and maintain over 210 X 96 well plates in order to isolate one converted clone. We reasoned that labor and reagent/material cost would be prohibitive and our remaining time and effort on this project would be better served by our attempting to increase the over all frequency. Alternative Strategies: To this end we decide to employ a new approach to gene editing technology which employs targeted double strand breaks in order to facilitate and enhance homologous recombination. This approach is beyond the scope of this current application, however our preliminary data suggest that zinc-finger nucleases targeting the PrP gene sequence enhance gene editing 10 to 100 fold over the SDF-mediated genome editing process. We intend to explore further this rapidly developing technology for the eventual isolation of bovine cell clones carrying an altered PrP gene.

Impacts
During this project we developed and validated a very sensitive assay for the detection of the dominant negative PrPARR allele. This assay can be used as a genotyping tool for the detection of variant cattle strains carrying these mutations. We also made significant progress in the development of modified PrP strains using sequence specific double strand break enhanced homologous repair. This technology, although still in its infancy, utilizes gene editing technology for altering a specific gene in living cultured cells from which animals may be generated using nuclear transfer cloning technologies. As this field matures it will be possible to rapidly develop of any cattle or livestock trait, including disease resistance, enhanced feed utilization, and milk production. The eventual generation of BSE resistant cattle, once validated, will be a significant benefit to the beef and dairy industries. Following the report of only a few BSE cases in Canada and U.S. certain export markets, including Japan and Australia, closed which has dealt a serious economic blow to the affected beef and dairy industries. BSE has also impacted the healthcare industry in other ways, as bovine byproducts are critical components in the production of biopharmaceuticals. The magnitude of the potential risk to the public health posed by these bovine by-products used in pharmaceuticals and cosmetics is difficult to quantify and probably very low, but the FDA and similar National drug control agencies are very concerned and actively seeking solutions. BSE resistant cattle offer one solution.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
Task 1. Establishing Primary Cell lines (Completed) We established and maintained several bovine cell lines. Adult epithelial and fibroblast cell lines, and a fetal bovine fibroblast cell line were derived from male and female donor animals (Holstein). Growth characteristics (Completed): Aliquots of each master seed stock were grown and their doubling and culture maintenance times established. Of note, the adult fibroblast cell lines (male and female) grow for greater than 65 doubling with no signs of senescence. The epithelial cell lines were more difficult to maintain and carry as a pure culture. Single cell plating: (Completed): For all fibroblast cell lines, single cell suspensions were relatively easy to derive and maintain. The epithelial cells were in general difficult to a derive a dispersed suspension. We have not yet derived single cell suspensions using the FACS Vantage SE particle sorter. Task 2. SDF molecule design: Generation of SDFs to modify the bovine PrP gene (PrP) (Completed). The PrP gene from each cell line was PCR amplified and sequenced. SDF targeting molecules were generated for introducing the 25Xv PrP0 and 179R PrPARR alleles as proposed. Task 3. SDF Delivery (Completed): Lipofection: Reporter constructs and SDF molecules were delivered into to the primary bovine cells using a panel of commercially available lipids. From these experiments we have determined the best condition for delivering both SDF and reporter gene expression plasmid whereby approximately 35% of the cells received exogenous DNA. Microinjection was also assessed for delivering plasmid and SDF. As many as 8,000 successful injections can be performed per day. The viability following injection was very good, 90%. Task 4. Genetic Analysis (Completed):We validated the AP-PCR/RFLP genotypic screening approach using a phenotypic gene editing reporter system (see preliminary data) and also demonstrated that the 25Xv PrP allele can be detected when present at a frequency of 0.01 % (or 1 in 5,000 genomes). The PrPARR allele was able to be detected at 1 in 1,000 genomes. Task 5. Assessing the Frequency of Gene Editing events (In Progress) Using one of the SDF targeting molecules (PrP25Xv) we demonstrated successful targeting of bovine PrP in bovine fibroblasts. Based on our pooling strategy 200,000 SDF-transfected cells were screened in pools of 500 cells/well ( 400 samples). We detected approximately 28 positives, estimating that the targeting frequency was approximately 0.014% . The SDF directed sequence modification was confirmed by pyrosequencing. Unexpected Results: During the analysis and cell processing it was determined that the cells needed to be cultured for at least 14 days prior to AP-PCR analysis (to permit complete elimination of residual SDF molecules that might result in false positive assay results). Given the estimated frequency of gene editing and considering the population doublings of our primary cells (>65 doublings), we have devised an alternative screening strategy whereby smaller pools of treated cells (50 cell per pool) will be screened initially. Positive pools are currently being isolated.

Impacts
This proposal describes a strategy to achieve the Phase I milestone, which is establishing primary bovine cell lines from various tissues, and assessing their growth properties and competency for undergoing Genome Editing technology (GenEdit). The successful completion of these objectives will determine the ideal primary cell line to initiate Phase II studies, which uses GenEdit to modify the bovine PrP gene in the most suitable cell line, after which nuclear transfer (NT) technologies will be employed for generating animals carrying the modified PrP alleles. The product of a successful phase II study will be cattle, carrying a null or modified PrP gene, that will be used to establish heterozygous, hemizygous, and homozygous animals. Subsequent to commercialization, breeding stocks for allele transmission will be established and a portion of the cattle will be challenged with BSE infectious material to assess their resistance profile. Validated BSE-resistant cattle will have immediate medical, social and economic impact by reducing the potential human health risks associated with consuming BSE-infected beef, strengthening consumers confidence in the safety of beef products, and circumventing possible catastrophic affects on the beef industry as a consequence of an eventual BSE outbreak.

Publications

  • No publications reported this period