Novel food information: Porcine Reproductive and Respiratory Syndrome Virus-Resistant Pigs

Background

Health Canada has notified Genus PLC that it has no objection to the food use of Porcine Reproductive and Respiratory Syndrome Virus-Resistant Pigs. Health Canada conducted a comprehensive assessment of these pigs according to the Codex Alimentarius Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Animals. These guidelines are internationally accepted principles for establishing the safety of foods with novel traits.

The following provides a summary of the original notification from Genus PLC and the evaluation by Health Canada and contains no confidential business information.

Introduction

A novel food submission was received from Genus PLC, on December 20, 2023, regarding the acceptability for food use of pigs (Sus scrofa domesticus) that have been genetically modified to be resistant to infection by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

PRRSV-resistant (PRRSV-R) pigs have been genetically modified using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated gene editing to precisely delete a small portion of a native pig gene that is involved in mediating infection by PRRSV. This deletion removes domain 5 of the targeted protein, preventing PRRSV from entering into the pig host cells, and conferring disease resistance in the homozygous-edited pigs.

Genus PLC has requested a letter of no objection for a total of 1406 PRRSV-R pigs from the second generation (E1) of animals split across 4 pig breeding lines (Landrace, Large White/Yorkshire, White Composite, and Duroc). Genus PLC has indicated that they are using this approach in order to maintain herds of edited PRRSV-R pigs with greater genetic diversity and to prevent inbreeding. Genus PLC has provided a complete list of all the animal identification numbers and has confirmed that each of the 1406 PRRSV-R animals has the identical deletion in the target gene and no detectable off-target edits.

Genus PLC provided the Food and Nutrition Directorate (FND) with detailed genetic, phenotypic, and compositional information for a representative sample of animals for the regulatory review. As the molecular characterization has confirmed that the targeted edit is identical and there are no detectable off-target edits in every notified E1 animal, the conclusions from the representative sample of animals are considered to be transportable to the other listed events.

The safety assessment performed by FND evaluators was conducted according to the Codex Alimentarius Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Animals. The assessment considered: how PRRSV-R pigs were developed; how the composition and nutritional quality of PRRSV-R pigs compared to non-modified pigs, what the potential is for PRRSV-R pigs to be toxic or cause allergic reactions, and the health status of PRRSV-R pigs. Genus PLC has provided data which demonstrate that PRRSV-R pigs are as safe as conventional pig varieties used as food in Canada.

The Food and Nutrition Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from PRRSV-R pigs are considered novel foods under the following part of the definition of novel foods: "c) a food that is derived from a plant, animal, or microorganism that has been genetically modified such that

the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

Development of the modified animal

PRRSV is the causative agent of PRRS disease which is a major source of morbidity and mortality in the pig industry worldwide. Genus PLC has explained that currently there are no effective treatments for PRRSV and vaccination has been only partially effective.

PRRSV has a very specific host cell type which is restricted to pig macrophage cells. The CD163 protein has been identified to be the main receptor protein that mediates virus internalization and is expressed at high levels on the surface of macrophages in the respiratory system. CD163 is a type I membrane protein and the extracellular portion forms a “pearls-on-a-string” structure of nine scavenger receptor cysteine-rich (SRCR) domains. Specifically, the SRCR5 domain of the CD163 protein has been shown to be a critical interaction site necessary for infection by the virus. Several proof-of-concept studies have demonstrated that both the full-CD163 and SRCR5 deletions conferred resistance to PRRSV infection by in vitro and in vivo tests.

Genus PLC has used proprietary gene editing technology to develop pigs which are resistant to infection by PRRSV through the deletion of exon 7 of CD163 (CD163^ΔE7) which encodes the SRCR5 domain. PRRSV-R pigs were produced from micro-injection of CRISPR-Cas9 ribonucleoproteins (RNPs) into the cytoplasm of 1-cell fertilized porcine zygotes. Genus PLC used a paired guide RNA (gRNA) approach for gene editing. The first gRNA was designed to target a sequence within intron 6 of the CD163 gene and the second gRNA targeted a sequence within intron 7. Following micro-injection into the porcine zygotes, the RNP complexes function by locating the 2 target sites in the pig genome based on sequence complementarity to the gRNA sequences, then the Cas9 protein induces a double-stranded DNA break. The native cellular DNA repair pathway, non-homologous end joining (NHEJ), occurs in the pig cells to repair the DNA break, removing the DNA sequence between the 2 gRNAs. By this manner, 414 base pairs (bp) of the CD163 gene, including the entire exon 7 which encodes the SRCR5 domain as well as parts of introns 6 and 7, were deleted.

Depending on the methodology used for gene editing, there is the potential for integration of foreign DNA (fDNA) into the host's genome. Genus PLC described that they selected a methodology that would reduce the risk of fDNA integration and provided data to support that there was no fDNA present in the gRNAs or Cas9 protein used in gene editing. Genus PLC provided PCR analysis to confirm that no gRNA sequences were inserted into the pig genome.

The first generation of gene edited animals (referred to as the E0 generation) is often mosaic (i.e., possess multiple different edited alleles in different cells). Although the gene editing methodology produced a variety of alleles in the E0 generation of PRRSV-R pigs, Genus PLC used a multistep screening process to select only animals that carried the identical CD163^ΔE7 edit. Only E0 boars carrying the intended CD163^ΔE7 edit were bred with wildtype sows to produce offspring, the E1 generation. Since the E1 animals were derived from a single sperm cell from the E0 boar, the E1 animals were heterozygous for the gene edit and not mosaic.

The E1 generation was analyzed again by detailed molecular characterization. The submission describes in detail how each individual animal was analyzed by robust molecular characterization and only select E1 generation animals, which all have the exact intended gene edit and no detectable off-target edits, were used to further advance the breeding lines and produce the E2 generation. The E2 generation animals were then characterized for zygosity and inheritance pattern.

Genus PLC indicates that the homozygous E2 animals and the subsequent generations will be both inter-mated and mated with wildtype animals to maintain homozygosity and increase population genetic diversity, respectively. Similar to a conventional breeding program, subsequent generations of PRRSV-R pigs will not be strictly bred within generations but will overlap to manage zygosity and diversity. Genus PLC will maintain separate herds of PRRSV-R pigs for each of the 4 breeding lines. The PRRSV-R pigs will be bred through conventional pig breeding structures to produce the animals that will be used for food production.

The gene editing, animal breeding, molecular characterization, and production processes described by Genus PLC were extremely tightly controlled to ensure consistent results. Genus PLC has demonstrated that every animal selected from the E1 generation to be used in their breeding program carries the identical 414 bp deletion of exon 7 from CD163.

Characterization of the modified animal

Genus PLC used multiple DNA sequencing methods in a step-wise approach to characterize the CD163 locus of the PRRSV-R pigs:

Short-read sequencing to screen animals for the presence of the intended 414 bp CD163^ΔE7 edit (E0 and E1);
Long-read Nanopore sequencing to evaluate the structural integrity adjacent to and encompassing the gene edit (E0 only); and
Hybridization-based sequence capture followed by short-read sequencing to identify any unintended sequence variations across the whole CD163 gene (E0 and E1).

These molecular characterization methods were used to screen thousands of animals in Genus PLC's breeding program. According to the provided reports, ~21% of E0 animals contained the intended CD163^ΔE7 edit. As expected due to the NHEJ repair mechanism, other edited CD163 alleles were identified, consisting of various exon 7 deletions and/or indels at either gRNA cut site. Also as expected, some of the E0 animals were identified to be mosaic and contained multiple different alleles at the CD163 locus. The Nanopore long-read sequencing identified that ~20% of animals from the first round of editing had large (>450 bp) deletions or inversions. As has been described, only selected E0 boars that contained the exact intended CD163^ΔE7edit were used to breed the E1 generation. In the E1 generation, sequencing confirmed the transmission of the CD163^ΔE7edit from the E0 sires with no unexpected changes to the CD163 sequence.

Genus PLC used a similar hybridization-based sequence capture method to assess the E0 and E1 animals for off-target edits (i.e., unintended genetic changes at other locations in the pig genome). 182 potential off-target sites across the pig genome were identified experimentally for the 2 gRNAs that were used in the gene editing process. Sequence capture probes were designed to capture 1000 bp on either side of the identified off-target sites. Genus PLC performed a validation study using whole genome sequencing on a subset of pigs to validate the hybridization-based sequence capture methods and to uncover potential new off-targets not previously captured. Based on the results of that study, the hybridization-based sequence capture method had greater sensitivity to off-target edits with a low cellular frequency and no new off-target edits were identified by WGS. Genus PLC indicates that it is not practical to perform WGS on all of the animals in the breeding program and that the hybridization-based sequence capture methodology they have used is sufficient to identify off-target edits. Additionally, the animal breeding process used by Genus PLC is extremely tightly controlled and any animals exhibiting unexpected genetic or phenotypic traits would not be used in further breeding.

Genus PLC screened 227 E0 animals across the 4 breeding lines for the presence of off-target edits at all 182 sites. ~58% E0 animals had 0 detectable off-target edits, 1 animal was identified to carry 4 off-target edits, and the rest had between 1 and 3 off-target edits. Genus PLC provided detailed information about each off-target edit identified in the E0 animals. In the E1 generation, Genus PLC screened 3270 animals across the 4 breeding lines for the presence of off-target edits at all 182 sites. They found that ~1% of screened E1 animals carried off-target edits. As the cellular frequency of off-target edits varied in the E0 animals from <1% to 55% and not all of the screened E0 animals were selected to sire offspring, it is reasonable that only a small proportion of E1 animals were identified to contain off-target edits.

Genus PLC indicates that 1406 E1 animals from the 4 breeding lines, all carrying the exact same CD163^ΔE7 deletion and no off-target edits, were selected for breeding the E2 generation. As each of the notified animals contains the exact same genetic modification and no detectable off-target edits, the extrapolation of phenotypic and composition data from representative samples of animals, rather than testing each animal, is strongly supported.

The stable inheritance of the gene edit in the PRRSV-R pigs is built into the selection process as only animals containing the gene edit were desirable for inclusion in the breeding program. Genus PLC provided data demonstrating the inheritance and stability of the CD163^ΔE7gene edit in PRRSV-R pigs. It was demonstrated through sequencing that the CD163^ΔE7 gene edit on chromosome 5 is stably inherited with the identical sequence from the E0 generation to the E1 generation.

The pattern of inheritance of the CD163^ΔE7 gene edit was assessed in the E2 generation of PRRSV-R pigs using a TaqMan genotyping assay. The E2 generation was derived from crossbreeding heterozygous E1 parents, therefore the E2 progeny were expected to segregate according to Mendelian rules of genetics for a single gene with 2 alleles (i.e., 1 homozygous-edited : 2 heterozygous : 1 homozygous-wildtype). Inheritance data was provided for four representative E2 populations derived from four different E0 animals, as well as summary data for the whole breeding program. Based on the results provided, the CD163^ΔE7 gene edit was found to segregate according to Mendelian rules of genetics.

In order to support the phenotypic stability, the PRRSV resistance phenotype was assessed in E2 and E3 generation animals. Genus PLC performed disease challenge studies on E2 and E3 homozygous-edited, heterozygous, and homozygous-wildtype animals. The results demonstrated that the homozygous-edited pigs were resistant to the PRRSV strains used in the study, while the heterozygous and homozygous-wildtype pigs were susceptible to the virus. These results confirm that the trait is genetically recessive and support the intergenerational phenotypic stability of the PRRSV-R pigs.

Further supporting the trait stability and genotype-phenotype relationship, western blot analysis of the CD163 protein in homozygous-edited, heterozygous, and homozygous-wildtype E2 generation animals confirmed that the expression of the modified CD163^ΔE7and/or full-length CD163 protein isoforms was in alignment with the genetic makeup of each animal tested.

Genus PLC indicates that each animal selected for commercial sale, for production of semen for sale, and each animal selected for further breeding will be genetically tested to confirm the presence of the CD163^ΔE7 edit before sale or further breeding. A subset of these tested animals will also be tested to confirm the expression of the gene by reverse-transcription-PCR. Any animals with unexpected results will be removed from the herd and no longer bred. Genus PLC notes that both of these confirmatory tests are required by the US FDA.

Product information

The deduced amino acid sequence of the CD163^ΔE7 gene results in the deletion of 105 amino acids (Δ474-578) and 1 amino acid substitution (A473G) in the modified CD163^ΔE7 protein compared to the wildtype CD163 protein.

Based on the information provided, the CD163^ΔE7 protein is expressed at an equivalent level to the unmodified CD163 protein in PRRSV-R pigs. The CD163 protein is expressed in macrophage cells that are found at low levels in many tissue types. Genus PLC states that as there were no changes to the gene regulatory sequences, and therefore, the expression pattern of the CD163^ΔE7 gene is not expected to change.

Genus PLC provided western blot analysis of the CD163 and CD163^ΔE7 proteins derived from monocyte-induced macrophages in all 4 breeding lines and from pulmonary alveolar macrophages (PAMs) from 2 breeding lines. The results demonstrated that both the full-length CD163 and shortened CD163^ΔE7 isoforms were observed at the expected sizes and that the expression levels of CD163 and CD163^ΔE7 were not significantly different.

CD163 mRNA transcripts were reverse transcribed and sequenced to verify that the gene was being expressed as expected. The results confirmed that the expression of CD163 and/or CD163^ΔE7 mRNA transcripts matched the zygosity of the tested animals and Genus PLC reported that no unexpected sequence variation was observed, including changes in mRNA splicing. Although protein sequence data was not provided, the CD163^ΔE7 mRNA sequencing results combined with the western blot analysis are considered to support the conclusion that there are no unexpected changes to the CD163^ΔE7 protein sequence.

Based on the available data provided, Health Canada has no safety concerns regarding PRRSV-R pigs from a molecular perspective.

Dietary exposure

PRRSV-R pigs represent an alternative to other pork products in the marketplace. Dietary exposure will be relying on existing meat preferences. The consumption of PRRSV-R pigs is expected to represent a small proportion of the pork already being consumed by the Canadian public. The introduction of PRRSV-R pigs to the market is not expected to affect the total consumption of pork by Canadian consumers.

Nutrition

The petitioner conducted a comparative study of the compositional and nutritional components of the Longissimus dorsi (loin) muscle tissue from four zygosity groups (10 per group, 5 males and 5 females): homozygous edited animals for the CD163 gene, heterozygous animals for the edited CD163 gene, a control group of animals that were homozygous null for the edited CD163 gene, and a reference group of wildtype animals. The animals were from the E2 generation of the Large White/Yorkshire breeding line, and were reported to be healthy and raised under normal husbandry and feeding practices until the appropriate market weight of approximately 205 days.

Longissimus dorsi (loin) muscle samples were collected from each animal 24 hours post-mortem, trimmed of visible fat, frozen and stored at -80°C, and shipped frozen for analysis. Samples were analysed for proximates (ash, calories, carbohydrate, moisture, protein and total fat), 36 fatty acids, 18 amino acids, 10 minerals, six vitamins and cholesterol using acceptable methods. There were no statistically significant differences (P>0.05) between the four groups for any of the analytes measured. The results indicate that the nutritional composition of muscle tissue from the homozygous edited CD163 gene animals is comparable to that of the nonmodified (wildtype) animals.

In response to a deficiency letter from the Canadian Food Inspection Agency (CFIA), the petitioner provided additional data comparing compositional analysis for proximates (ash, calories, carbohydrates, moisture, protein and total fat) from the Longissimus dorsi (loin) muscle for each of the four breeding lines. For each line, two groups of animals (10 animals per group, 5 males and 5 females) were assessed, a test group consisting of E2 pigs homozygous for the CD163 gene edit and a wildtype group (control). There were no statistically significant differences between the test and wildtype groups for any of the proximates tested.

The petitioner states that there is no evidence to suggest that the edited CD163 gene, or the gene editing process affects the nutritional composition of tissues from any of the four breeding lines. The only effect of the CD163 gene edit is the resistance to infection by PRRSV in the homozygous animals.

Based on the review of the information provided by the petitioner, Health Canada has not identified any nutritional safety concerns associated with the proposed food use of the Genus PRRSV-R pigs.

Chemistry

Chemical contaminant residue data were not provided, nor were any unique contaminant considerations been identified with respect to Genus PLC PRRSV-R pigs. As well, there are no maximum levels for contaminants specific to this food set out in Health Canada's List of Contaminants and Other Adulterating Substances in Foods or its List of Maximum Levels for Chemical Contaminants in Foods.

The petitioner provided analytical data for 10 different nutritive trace elements measured in pig meat from 10 animals (5 males, 5 females) in each of the following 4 zygosity groups of the E2 generation: animals homozygous for the edited CD163 gene, animals heterozygous for the edited CD163 gene, a control group of animals that were homozygous null for the edited CD163 gene, and a reference group of wildtype animals from an elite breeding line.

There were no statistically significant differences in any of the nutritive trace elements measured in the meat of the gene-edited PRRSV-R pigs, the null homozygotes and animals from the original line. This information suggests that it would also not be expected that the edited CD163 gene would impact the concentrations of trace elements that are of greater concern to human health (e.g., lead, arsenic) in gene-edited pigs.

Considering the above information, Health Canada is of the opinion that PRRSV-R pigs are not expected to pose a health concern from a chemical contaminants perspective relative to pigs without an edited CD163 gene.

As with any food or food ingredient sold in Canada, it is the responsibility of the food manufacturer to ensure that its use does not result in a violation of Section 4(1)(a) and (d) of the Food and Drugs Act, which states that no person shall sell an article of food that has in or on it any poisonous or harmful substance or is adulterated. If an elevated concentration of any chemical contaminant is found in any type of food, Health Canada may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

Toxicology

Health Canada evaluated the safety of the gene edited PRRSV-R pigs by assessing the potential toxicity of the modified CD163 protein.

The petitioner used a weight of evidence approach to assess the potential toxicity of the modified CD163 protein. This included an assessment of the amino acid sequence similarity of the modified protein to known toxins, as well as structural properties of the modified protein, including whether it is resistant to heat and/or digestion. This was considered acceptable given that the wildtype protein has a non-toxic biological function in animals and has a history of being consumed safely in food.

Bioinformatic analysis conducted by the petitioner confirmed the absence of significant similarity of the modified CD163 protein to known toxins in the Uniprot Venom and Toxicity Database, containing a total of 7,797 sequences. No biologically relevant match was identified.

Experimental data was provided to demonstrate that the wildtype and modified CD163 proteins are not degraded after exposure to 95°C for 30 minutes, but are expected to be digested in the stomach. This suggests that the CD163 protein has poor bioavailability when consumed orally because it is expected to be hydrolyzed in the stomach and therefore systemic exposure is expected to be negligible (wildtype and modified protein).

Based on the available information, Health Canada did not identify any toxicological concerns with the modified CD163 protein.

Allergenicity

The wildtype CD163 protein has a history of human consumption through the consumption of pork. The wildtype protein is not known to be allergenic.

The petitioner's bioinformatics analyses, done following international guidance, confirmed a lack of homology of the modified CD163 protein with known food allergens. This suggests that the modified CD163 protein is not expected to cross-react with known food allergens.

Based on the available information, Health Canada did not identify any allergenic concerns.

Animal health

The FND requested the Veterinary Drugs Directorate of the Health Products and Food Branch to review the PRRSV-R pig submission as it relates to animal health. The request was made because animal health is a factor which must be considered when assessing the safety of food derived from genetically modified animals, as recommended in the Codex Alimentarius Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Animals.

The data provided demonstrated PRRSV-R pigs were as healthy as non-modified pigs. Genus PLC submitted data on homozygous-edited, heterozygous, and homozygous-wildtype animals from the E2 generation from two breeding lines (Large White/Yorkshire and White Composite). The animals were examined for approximately 20 phenotypic characters over a full life cycle, from birth to female farrowing. Reproductive data including farrowing data was also assessed, along with mortality and morbidity. No significant differences were identified between homozygous-edited and homozygous-wildtype animals in either line, with the exception of a statistically significant difference between ultrasound-predicted 10th rib back fat depth and birth weight in Large White/Yorkshire animals, and weight at day 140 and lifetime daily weight gain in White Composite animals. However, these values for the homozygous-edited pigs were well within the normal range of acceptability when compared to internal historical reference data. Over the study, the pigs were healthy under typical husbandry conditions of a commercial production unit.

Genus PLC additionally submitted data from disease challenge studies using E2 and E3 homozygous-edited, heterozygous, and homozygous-wildtype animals from the Large White/Yorkshire line. The animals were inoculated with Type 1 or Type 2 PRRSV isolates and monitored by diagnostic tests and/or disease presentation. The results demonstrated that the homozygous-edited pigs were resistant to the PRRSV strains used in the study, while the heterozygous and homozygous-wildtype pigs were susceptible to the virus.

Overall, the data provided showed no substantive evidence of a foreseeable impact on phenotypic characteristics that would impede animal health or the safety of foods derived from PRRSV-R pigs.

Conclusion

Health Canada's review of the information presented in support of the use of PRRSV-R pigs does not raise concerns related to food safety. Health Canada is of the opinion that foods derived from PRRSV-R pigs are as safe and nutritious as foods from currently available pigs.

Health Canada's opinion refers only to the food use of PRRSV-R pigs. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

The Canadian Environmental Protection Act, 1999 (CEPA 1999), administered by Environment and Climate Change Canada (ECCC) and Health Canada, is the key authority for the Government of Canada to ensure that all new substances, including organisms, are assessed for their potential harm to the environment and human health. The New Substances Notification Regulations (Organisms) under CEPA 1999 prescribe the information that must be provided to ECCC prior to the import or manufacture in Canada of new organisms that are animate products of biotechnology, including livestock products of biotechnology.

Genus PLC submitted a New Substance Notification for the PRRSV-R pigs to ECCC and the Healthy Environments and Consumer Safety Branch (HECSB) of Health Canada in November 2024.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food and Nutrition Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Codex Alimentarius Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Animals.

For further information, please contact:

Novel Foods Section
Food and Nutrition Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca

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2026-01-23

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