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Company Statement & FAQs on Plasmid Insertion Found in First Gene-Edited Bull

Recombinetics, Inc. has recently been the focus of a series of media stories highlighting the discovery by the FDA of a plasmid insertion in one of the founding lines of its gene-edited, polled (hornless), cattle generated during a pilot study published in Nature Biotechnology (Carlson et al, 2016). The company did not directly screen for the presence of the plasmid, it should have.

Recombinetics regrets the consequences this error could have on fellow scientists working diligently to deploy gene-editing to help improve agriculture, medicine, and environmental stewardship. The company wants to ensure our partners, collaborators, shareholders, funders, advocates, regulators, and the public that it is committed to transparency, collaboration, and continuous improvement to its processes, products, services, and business practices.

Since the polled pilot project, Recombinetics put an improved development and deployment protocol to the test for several products submitted to the FDA through the INAD process. The FDA has rigorously evaluated and granted discretionary enforcement to Recombinetics to market these products. This protocol is also being used to help progress the polled project towards commercialization.

The company asserts that future polled animals developed with gene-editing technology will: 1) be free of plasmids; 2) harbor the intended polled allele that has been present in cattle for thousands of years; and 3) be veterinarian-certified and registered with the appropriate breed association as to good health and possessing the genetic polledness trait, respectively. Though never intended for commercial use due to sub-standard genetic potential of the donor animal, the discovery of plasmid in one founder line during the testing stage disqualifies half the progeny from further evaluation and potential regulatory approval. Null segregants would still be eligible due to the non-novel state of their genome.

 

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FAQs Regarding Plasmid Found in Buri

 

How did the FDA find the plasmid?
A FDA bioinformatician used publicly-available data from the gene-edited hornless cow’s genome and ran an alignment program against a publicly available plasmid database. That is when the plasmid was found for the first time.

How did RCI miss the plasmid?
RCI did not originally look for plasmid integrations back in 2013 when these cells were first made. We should have. Our goal for this proof-of-concept project was to screen for accurate targeting of our gene edits (we successfully hit the target), screen for off-target edits caused by inappropriate cutting by TALENs (there was none), and for the presence of the Celtic allele duplication which causes a bovine to be polled (it was present). Our screens all showed positive results in the areas we looked at. Some of these analyses used Next-Generation sequence data of the entire genome, where plasmid sequence is typically pre-filtered from the data set because almost all sequence runs contain reads from contaminating plasmid. We now know not to pre-screen reads when the plasmid template is used for DNA repair steps of gene editing.

How are we ensuring this doesn’t happen again?
We have long since changed our quality controls and evolved our methods to deploy polled that have improved significantly. As part of our ongoing deployment process, we can specifically screen for plasmid integrations, and we have developed methods that no longer use plasmid as a donor template for polled.

With any new technology, the QC protocols significantly improve over time. To put in perspective how much technology has changed since 2013, it would be like going from an iPhone 6 to iPhone 11. Recombinetics updated our system to review the sequence data a couple of years ago. We have run this updated system and have since had approval with FDA on a porcine biomedical model. (The polled calves Buri and Spotigy were made and analyzed before this updated system was in place.) 

When and How did Recombinetics first learn of the plasmid integration?
In March, when UC Davis was informed by FDA of their sequence similarity search results from the plasmid database. UC-Davis scientists then quickly confirmed the results in Buri’s progeny. Final validation took much longer, as third-generation long-read sequencing was used to clearly define the results of a single unintended plasmid integration at the gene-editing target site in the genome. The unintended plasmid integration still caused a polled phenotype in Buri’s offspring, and this work was done at UC-Davis.

Has Recombinetics submitted an INAD for its polled cattle with the FDA?
No, we did not submit an INAD. This is a proof-of-concept and not intended for commercial use.

In developing these gene-edited polled clones from dairy animals of relatively low commercial value, there never was a commercial intent for these animals or their offspring. They were purely research animals made to prove our edit would cause a polled phenotype and that this edit was identical to the polled mutation found in non-edited cattle. The INAD process was not something critical to the Recombinetics business developments since our intention is to only make animals with commercial partners with elite genetics.  However, testing regulatory systems with our experimental gene edited animal(s) is of interest.

What happened when you applied for GRAS in 2016? What’s the status of that?
We submitted the GRAS application as all four naturally occurring versions of polled allele have been eaten by consumers for well over 100 years. Due to the timing of our GRAS submission (about a month before draft guidance 187) and FDA publishing the guidance, our GRAS filing was never reviewed. FDA informed us we needed to submit an INAD for approval instead of GRAS. As answered in the previous question we did not submit the INAD.

Has Recombinetics produced any additional polled cattle since Buri and Spotigy were born?
No new founder animals have been generated that transmit the polled trait, only cells and embryos have been made. We have bred two sets of the progeny of Buri through conventional breeding. The first set bred by researchers at UC Davis was done to show the expected experimental outcome of normal stable inheritance of gene edits for the polled trait. The set bred in Australia was done as an observational demonstration of gene-editing technology for polled, and these animals could be used for regulatory approval under the new regulatory frameworks set down by the Office of the Gene Technology Regulator and Food Standards of Australia and New Zealand (FSANZ), respectively.

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