Category: Biotechnology

This is part 4 of a 1, 2, 3, 4 part series on Regulation of Genetically Modified Animals

I am in perhaps a somewhat rare position regarding the contemplated USDA regulatory framework, as I actually have several animals from amenable species (sheep, cattle) on the ground that were developed using “techniques that use recombinant, synthesized, or amplified nucleic acids to modify or create a genome” for “agricultural purposes”.  I have not published these data and they are the dissertation research projects of graduate students, and so I will only say that these animals were produced using CRISPR/Cas9 genome editing and a sgRNA to produce a gene knockout. This was done by introducing a Cas9-sgRNA ribonucleoprotein (RNP) into single cell zygotes following in vitro fertilization of in-vitro matured oocytes.  The resulting double strand break (DSB) was repaired by the non-homologous end joining (NHEJ) pathway, and subsequent embryo transfer to surrogate dams. In other words, each animal is a unique individual, and there was no foreign template or intergeneric combination of genetic material introduced into the editing process at any time. None of the genome editing target genes were intended to impact animal health, nor was the application intended to have an animal health claim. So as I read through the contemplated regulatory framework, I cant help but personalize how it would affect my research, and how it compares to the existing regulations.

By way of background, it is perhaps important to emphasize that the animals that make up the research herds and flocks at land-grant universities are typically entering the food supply at the end of experiments.  Our dairy cattle getting fed different experimental rations keep getting milked, the calves from a beef cattle crossbreeding study are finished in our feedlot and enter commerce at the end of the study, and our poultry produce eggs that are sold to the public, along with meat products from all our animal facilities (except the horses!).  There we sell foals at the UC Davis foal auction. The food animals are actually processed at our USDA-inspected slaughter facility, and butchered by meat science students. The income from the “meat lab” sales goes back into the operational account for the animal facilities.

So back to the genome-edited knockout animals I have on campus. Under the existing FDA regulations, they are considered unapproved animal drugs and cannot enter the food supply without a new animal drug approval or prior authorization. According to the FDA, I would need to apply for an investigational new animal drug (INAD) for these animals, and then request a Food Use Authorization (FUA) for these experimental animals to enter the food supply at the end of the experiment. I have actually already started to apply for that and the FDA have requested an answer to the following questions before they can even open an INAD, let alone tell me what information I need to provide to them to potentially obtain a FUA for my experimental gene knockout sheep and cattle. I have started on this paperwork, but it will take some time to prepare it all and submit it through their electronic portal.

• Description of the project, including the goal of the intentional genomic alterations (IGAs), any relevant information from other animals in which the same genes have been knocked out (e.g., references to published articles characterizing knockout mice), and a description of the planned studies
• Description of how the IGAs were produced, including detailed experimental design
  • Include information such as which Cas9 nuclease variant was used (e.g., SpCas9, SpCas9-HF1) and any associated information regarding the variant’s fidelity, criteria used to design and select guide RNAs, and any controls utilized to mitigate the potential for unintended alterations
• Available molecular characterization data, including a description of the methods used (e.g., PCR, Sanger sequencing, long-range PCR, etc.)

So under the USDA contemplated regulatory framework, published in the Federal Register 12/28/2020, these knockout animals would clearly fit under the “amenable species modified or developed using genetic engineering and intended for agricultural purposes and human food”. If they were knockout plants they would have an up-front exemption from regulation under the SECURE revision as they “result from natural cellular repair of a targeted DNA break without any introduced DNA to direct the repair”.

For the USDA animal health safety review I would need to show that “the animal under review is found to pose no greater risk to animal health than the animal from which it was derived”. This is where is gets a little unclear.  The term “the animal from which it was derived” suggests that the animals are being derived from a known unmodified animal, presumably through genetic engineering of a cell line derived from that animal, followed by somatic cell nuclear transfer cloning of the edited cells. But my animals are derived from editing multiple zygotes, so each edited animal has its own unique genomic sequence i.e. there is no animal from which these animals were derived, and conceptually editing outcomes differ between all of these different individuals, especially because the DSB was repaired using the error-prone NHEJ pathway.  So does each individual animal have to have a different evaluation?

The safety review requires “a molecular characterization of the modification and an understanding of the process by which it was introduced, that the intended change was made and that there were no unintended disruptions of endogenous genes, unintended DNA insertions, or off-target changes if the genome was modified without inserting DNA.” It is not hard to prove that the intended change was made, we typically do that with a PCR amplification of the target locus, and sequencing of the amplified PCR product. But to answer the rest of those questions will require, at a minimum, whole genome sequencing of every animal, and proving a negative i.e. proving no unintended disruptions of endogenous genes, unintended DNA insertions, or off-target changes if the genome was modified without inserting DNA.  Both the sequencing and the bioinformatics to try to answer these questions would be very expensive, well beyond the cost of a sheep, or even a cow. This is especially true when every animal has a unique genotype, and there is no way to differentiate off-target changes from spontaneous de novo mutations.

Further, at a minimum, the animal health risk assessment would include an evaluation of the following issues:

• Molecular Characterization: What is the genetic modification(s) in the animal, how was the genetic modification(s) introduced, and how does the genetic modification(s) alter protein or ribonucleic acid (RNA expression)?
• Animal Health: Is there scientific evidence that the modified animal could plausibly, either directly or indirectly, increase susceptibility of livestock, including of the animal itself, to pests, non-infectious diseases, or infectious diseases of livestock, including zoonotic diseases? Is there scientific evidence that the modified animal could plausibly increase the spread of pests or infectious diseases of livestock, including zoonotic diseases?  When a plausible pathway to such an increased risk is identified, further analysis would be conducted to evaluate the pathway.  When an animal health claim is made or a modification is known to adversely affect animal health, the review would assess the animal health claim.
• Environmental Factors: Is there scientific evidence that introduction of the modified animal into the environment may result in environmental impacts that would warrant review pursuant to the National Environmental Policy Act (NEPA) or other statutes?

And in addition, the FSIS review would include an evaluation of the following issues (theoretically pre-slaughter, although obtaining meat for compositional analysis from animals pre-slaughter is obviously problematic):

• Evaluation of expressed substances: Is there scientific evidence that the genetic modification could result, directly or indirectly, in toxins, chemical residues, or other potentially deleterious substances in meat or poultry products?
• Allergenicity: Is there scientific evidence that the genetic modification would directly or indirectly alter the allergenic potential of meat or poultry products derived from the animal?
• Food storage and processing: Is there scientific evidence that meat or poultry products derived from the modified animal could mislead consumers regarding wholesomeness or the need for appropriate storage (e.g., meat that maintains a red appearance even when spoiled)?
• Compositional analyses of key components: Is there scientific evidence that meat or poultry products from the modified animal are compositionally (e.g., nutritionally or functionally) no different than meat from conventional animals, such that it meets any regulatory definition, standard of identity or other labeling requirement, and consumer expectations for the applicable product?

Needless to say, providing answers to all of these questions for each individual knockout animal would be an unsurmountable hurdle for an academic laboratory. Throughout the USDA contemplated regulatory framework the term “developer” is used. For example, “Under the contemplated regulatory framework, developers could request that USDA conduct a risk-based and science-based safety review focused on animal health”. Historically, developers of genetically engineered plants have been large multinational companies who have the resources to provide the regulatory data to support commercialization and ultimately deregulation of their products. Research universities are not developers in this sense, but we still produce amenable species modified using genetic engineering and intended for agricultural purposes and human food.

One of the questions the USDA poses in the request for comments is,

“How often does a start-up company or not-for-profit university or research organization modify or develop an animal using genetic engineering?”

My answer would be never.  Researchers at universities often modify animals using genetic engineering but never has one developed and commercialized an “amenable species modified or developed using genetic engineering and intended for agricultural purposes and human food.” The two food approvals for genetically engineered animals were both obtained by companies, albeit small companies. AquaBounty for the “AquAdvantage” salmon, and Revivicor for the “GalSafe” pig.  To date neither of these products has been sold for food in the United States, although AquaBounty is getting mighty close.

In closing, as long as animals produced using genetic engineering, even those that could have been produced using conventional breeding,  are subjected to unique regulatory scrutiny not required of identical products produced using conventional breeding, research in food animals using genetic engineering for agricultural applications will be cost-prohibitive in the United States.

Another question the USDA poses is,

“Should USDA exempt certain types of genetic modifications of amenable species intended for agricultural use from regulation?  If so, what types of modifications and why?”

I would suggest that the SECURE up-front exemptions for certain types of modifications in plants, specifically a modification (1) resulting from natural cellular repair of a targeted DNA break without any introduced DNA to direct the repair;  (2) that is a targeted single base pair substitution; or (3) that introduces a gene known to occur in the plant’s gene pool, or causes a change in a gene that corresponds to a version of that gene present in the organism’s gene pool should be extended to genetically engineered animals.  These exemptions were primarily designed to exclude from regulatory oversight organisms that could have been achieved using conventional breeding. Just like the Coordinated Framework calls for.

If you have comments on the contemplated regulatory framework, there is a 60 day public comment period that closes 2/26/2021. I would encourage all people working in this field to post comments. Comments can be posted Federal eRulemaking Portal: https://beta.regulations.gov/comment/APHIS-2020-0079-0005. Go to  Supporting documents and any comments that are received on this Advance Notice of Proposed Rulemaking may be viewed at https://beta.regulations.gov/comment/APHIS-2020-0079-0005.

In the words of one commentator

“It, of course, remains to be seen what will be the Biden Administration’s approach to ag biotech; however, given the consistency of approach to the evolving ag biotech regulatory structure by both the Obama and Trump Administrations, it would not be surprising if the Biden Administration also continued the evolution of a more science-based, evidence-based, and risk-based approach to regulation of ag biotech – including genetically engineered animals. It would behoove stakeholders that would support such consistency of regulatory approach to submit detailed comments that would assist USDA in developing such a notice of proposed rulemaking (NPRM).

As with all opportunities to provide comments on Federal agency proposals, it is important to submit concise, substantive, and well-supported and well-documented comments to the administrative docket. In this case, given the trans-Administrations nature of the action, it is important that interested stakeholders use this opportunity to best advantage to urge the Biden Administration to continue a 21st Century approach to regulating ag biotech.”         Keith Matthews, Wiley Rein LLP

(*****This series of 4 BLOG posts (1,2,3,4) is extracted from my paper from the Proceedings of the Range Beef Cow Symposium XXVI, November 18-20, 2019. Mitchell, NE. pages 37-49.*****)

The alternative animal product arena is complex and quite varied. Some products are entirely plant product derived and employ only plant origin proteins or metabolites. Other endeavors are using cells of animal origin to derive a more structurally similar meat yet still with animal genomes. Most of the analysis and discussion, though, has focused on bovine alternatives because of the iconic position of cattle in many climate and sustainability discussions.

There are two ‘alternative meat’ sources that are often confused. One is so called “plant-based” or “vegan meat replacements” (e.g. Beyond Burger). These types of “veggie” burgers have been around a long time (e.g. Morningstar Farms, Boca Burgers), and now have some bells and whistles like genetically-engineered heme to make them bleed (e.g. Impossible Burger), but at the end of the day they consist of plant-sourced material being molded into a meat substitute type product. They are currently being sold at some fast food restaurants, and in supermarkets. Impossible Burger recently received safety approval for its genetically engineered heme as a color additive in ground beef analogue products, opening the way for its sale in supermarkets.

These are DIFFERENT to cultured meat, which is the term I am going to use to refer to animal cells grown in cell culture. This technology has other terminology – some appealing (e.g., in vitro meat, cellular meat, fermented meat, or slaughter-free meat, clean meat), and some derogatory (e.g. artificial meat, synthetic meat, zombie meat, lab-grown meat, non-meat, or artificial muscle proteins). This is discussed in my 2018 article “Why cows are getting a bad rap in lab-grown meat debate”.

Cultured meat requires the initial collection of stem cells from living animals and then greatly expanding their numbers in a bioreactor, a device for carrying out chemical processes. These living cells must be provided with nutrients in a suitable growth medium containing food-grade components that must be effective and efficient in supporting and promoting muscle cell growth. A typical growth medium contains an energy source such as glucose, synthetic amino acids, antibiotics, fetal bovine serum, horse serum and chicken embryo extract. Some of these components are problematic for consumers wishing to avoid animal products. The status quo for culturing tissue involves the use of fetal bovine serum, a byproduct of the livestock industry collected from fetuses in pregnant cows that are being slaughtered.  Large uncertainties remain in what a viable, animal-free, growth media may look like.

The world’s first lab-grown beef burger is unveiled in London on Aug. 5, 2013. (David Parry/AFP/Press Association)

If cultured meat is to match or exceed the nutritional value of conventional meat products, nutrients found in meat not synthesized by muscle cells must be supplied as supplements in the culture medium. Conventional meat is a high-quality protein, meaning it has a full complement of essential amino acids. It also provides a source of several other desirable nutrients such as vitamins and minerals, and bioactive compounds. Therefore to be nutritionally equivalent, cultured meat medium would need to provide all of the essential amino acids, along with vitamin B₁₂, an essential vitamin found solely in food products of animal origin. Vitamin B₁₂ can be produced by microbes in fermentation tanks, and could be used to supplement a cultured meat product. It would also be necessary to supplement iron, an especially important nutrient for woman of reproductive age that is also high in beef.

The process for making cultured meat has technically challenging aspects. It includes manufacturing and purifying culture media and supplements in large quantities, expanding animal cells in a bioreactor, processing the resultant tissue into an edible product, removing and disposing of the spent media, and keeping the bioreactor clean. Each are themselves associated with their own set of costs, inputs and energy demands.

Cultured meat production will likely require more industrial energy than do livestock to produce equivalent quantities of meat. The reason is that all of the biological structures avoided in cellular agriculture play important roles in meat production. An animal’s skin regulates temperature; internal organs digest food, circulate nutrients, and distribute oxygen; and the immune system destroys pathogens. When meat is grown in a bioreactor, all the same functions must still be accomplished, but at the expense of industrial energy. A bioreactor regulates temperature, food is predigested and fed to cells as simple sugars and amino acids, oxygen is pumped into the bioreactor, and all equipment is sterilized to prevent the growth of pathogens. Hence, a shift from livestock production to cellular agriculture could be a transition toward greater reliance on industrial energy.

It has been more than two years since the FDA released its draft “Guidance for Industry #187” “Regulation of Intentionally Altered Genomic DNA in Animals”, that proposes to regulate ANY “intentionally altered genomic DNA” in food animals as a veterinary drug, irrespective of product novelty or risk. Human intention does not equate to risk. This regulatory trigger seems to be aimed squarely at some taint associated with human intervention . Despite the fact that the public comment period for the FDA’s draft guidance for industry #187 closed on June 19, 2017, there has not yet been a formal response to the many comments by public sector scientists working in this field who see that this regulatory approach will make food animal research in this field cost prohibitive, and effectively preclude the use of gene editing in food animal breeding programs.

Dietary DNA is generally regarded as safe to consume, and is a routine ingredient of food obtained from any species. Millions of naturally-occurring DNA variations are observed when comparing the genomic sequence of any two healthy individuals of a given species. Breeders routinely select desired traits resulting from this DNA variation to develop new cultivars and varieties of food plants and animals. Regulatory agencies do not evaluate these new varieties prior to commercial release.

To put this in perspective, one study of whole genome sequence data from 2,703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA  (A, C, G, T) had been changed to a different one.

A small fraction of these mutations have been selected by breeders owing to their beneficial effects on characteristics of agronomic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety. All non-processed foods harbor DNA as a natural component and that DNA is different in every individual of every food species (both plants and animals).

Gene editing tools now allow plant and animal breeders to precisely introduce useful genetic variation into agricultural breeding programs. The U.S. Department of Agriculture (USDA) announced that it has no plans to place additional regulations on gene-edited plants that could otherwise have been developed through traditional breeding prior to commercialization. However, the U.S. Food and Drug Administration (FDA) has proposed mandatory premarket new animal drug regulatory evaluation for all food animals whose genomes have been intentionally altered using modern molecular technologies including gene editing technologies.

Mandating premarket regulatory approval for deletions, mutations, and the conversion of one wild-type allele to another wild-type allele in the same species (cisgenic) that could have been obtained using conventional breeding runs counter to the approach that was outlined by OSTP in the 1992 policy announcement (following the 1986 Coordinated Framework document).  There it is stated that, “Exercise of oversight in the scope of discretion afforded by statute should be based on the risk posed by the introduction and should not turn on the fact that an organism has been modified by a particular process or technique”. Additionally, it was clarified that “(O)versight will be exercised only where the risk posed by the introduction is unreasonable, that is, when the value of the reduction in risk obtained by additional oversight is greater than the cost thereby imposed. The extent and type of oversight measure(s) will thus be commensurate with the gravity and type of risk being addressed, the costs of alternative oversight options, and the effect of additional oversight on existing safety incentives.”

At the end of the day, food animals with intentional genomic alterations produce food, and if the food they produce is not biologically active via an oral route of administration it does not make sense to regulate these intentional genomic alterations as drugs. Referring to a DNA sequence variant as a “drug” is likely to confuse or frighten consumers who might infer that there are biologically active substances in their food.

A DNA alteration is not a drug, but rather part of the genetic code uniquely associated with any organism. Through its natural function within a cell, DNA controls how an organism grows and its unique form and function. The phenotype will ultimately be determined by the interaction of an organism’s genomic DNA sequence and the environment in which it lives. We do not regulate the millions of spontaneous genetic alterations that are in our food because DNA is generally regarded as safe to consume, and it is a routine ingredient of food obtained from any species, irrespective of its sequence.

The FDA’s draft “Guidance for Industry #187” entitled “Regulation of Intentionally Altered Genomic DNA in Animals” is not fit for purpose as it relates to food animals that could otherwise have been developed through traditional breeding techniques.   Myself and fellow academic researchers reject the idea that intentional genomic DNA alterations should be regulated as a veterinary drug in food animals, and consider that the proposed approach will thwart the development of genetic approaches by public sector researchers and small companies to use gene editing to solve zoonotic disease and animal welfare problems in the United States.

https://www.gopetition.com/petitions/harmonize-us-gene-edited-food-regulations.html

Please support the petition above calling for harmonization of the U.S. biotechnology regulatory system so that both plants and food animals that could otherwise have been developed through traditional breeding techniques are not subject to additional premarket regulatory requirements based solely on the fact that intentional genomic alterations were introduced using modern biotechnologies or rDNA techniques in the breeding process.

Harmonizing the regulations associated with gene editing in food species is imperative to allow both plant and animal breeders access to gene editing tools to introduce useful sustainability traits like disease resistance, climate adaptability, and food quality attributes into U.S. agricultural breeding programs.