Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 1 of 28 PageID #: 22865
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`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
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` Civil Action No. 22-311-WCB
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`JURY TRIAL DEMANDED
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`IMPOSSIBLE FOODS INC.,
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`Plaintiff,
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`v.
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`MOTIF FOODWORKS, INC., and
`GINKGO BIOWORKS, INC.,
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`Defendants.
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`DECLARATION OF DR. GEOFFREY LIN-CEREGHINO
`IN SUPPORT OF MOTIF’S SUR-REPLY CLAIM CONSTRUCTION BRIEF
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`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 2 of 28 PageID #: 22866
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`Table of Contents
`Introduction ............................................................................................................................. 3
`I.
`II. Terms for Construction ............................................................................................................ 3
`A. Promoter Element ................................................................................................................ 3
`1. Mischaracterization of “Promoter Element” and POSA Understanding ......................... 4
`2. Mischaracterization of the Yeast Patents ......................................................................... 7
`3. Mischaracterization of “Promoter Element” in Technical Literature .............................. 8
`4. Dr. Alper’s Research Illustrates the Lack of Reasonable Certainty Regarding “Promoter
`Element” ............................................................................................................................... 15
`B. Mxr1 Terms (Terms 2-3) ................................................................................................... 18
`1. Term 2.a ......................................................................................................................... 20
`2. “From” Pichia (P.) pastoris (Term 3) ............................................................................. 20
`C. Term 6 ................................................................................................................................ 22
`D. Term 8 ................................................................................................................................ 23
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`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 3 of 28 PageID #: 22867
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`I, Geoffrey Lin-Cereghino, hereby declare:
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`I.
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`INTRODUCTION
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`1.
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`I submitted a declaration in this Litigation on January 26, 2024 (“Declaration” or
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`“Decl.”) in support of Motif’s Answering Claim Construction Brief. I incorporate that Declaration
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`and accompanying Exhibits herein entirely by reference.
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`2.
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`I may refer to my Declaration and accompanying Exhibits as additional context for
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`my testimony in this “sur-reply” declaration (“Sur-Reply Declaration”), which I submit in support
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`of Motif’s Sur-Reply Claim Construction Brief.
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`3.
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`I have considered the declaration of Impossible’s expert Dr. Hal Alper submitted
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`in this Litigation on January 26, 2024 (“Alper Declaration” or “Alper Decl.”). I have also
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`considered the declaration of Ginkgo’s expert Dr. Carl Batt submitted on February 2, 2024 (“Batt
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`Reply”). In my Sur-Reply Declaration, I respond to positions taken in these declarations and
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`address claim construction issues that I understand to be in dispute in this Litigation. I may offer
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`additional views or opinions regarding the testimony of Drs. Alper and Batt and/or related case
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`matters.
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`II.
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`TERMS FOR CONSTRUCTION
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`A.
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`4.
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`Promoter Element
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`My understanding of this term, and the parties’ respective proposed constructions
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`of this term, is reproduced below for reference. I understand Impossible has articulated a different
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`construction than the construction it previously applied. E.g., Decl. ¶18.
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`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 4 of 28 PageID #: 22868
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`Impossible
`Plain and ordinary meaning. No
`construction is necessary.
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`To the extent that this term is construed:
`a polynucleotide that regulates (e.g.,
`drives) transcription of a polynucleotide
`sequence (e.g., gene)
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` a
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` promoter element is upstream of, and
`adjacent to or in close physical proximity
`to the gene
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`All asserted claims
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`Ginkgo/Motif
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`indefinite
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`5.
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`I previously opined that the term “promoter element” is indefinite (Decl. §VIII.A),
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`and I continue to have that opinion. Dr. Alper’s contrary view is incorrect, as I explain below.
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`1.
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`Mischaracterization of “Promoter Element” and POSA Understanding
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`6.
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`Dr. Alper disagrees with Dr. Batt’s analysis on the grounds that the term “element”
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`is not being construed. Alper Reply ¶25. I understand that “promoter element” is the patent claim
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`term being construed, but I believe it is important to understand the role of “element” within the
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`claim term.
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`7.
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`In particular, it is important to understand “element’s” role in “promoter element”
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`because there is not consistent, accepted, or universal meaning in the scientific literature for what
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`makes any particular sequence a “promoter element.” As I previously noted (Decl. ¶65), I am not
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`aware of any technical definition of this term such as a dictionary definition, or that any art-
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`accepted definition exists. I have not seen Dr. Alper point to a scientific dictionary or treatise that
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`defines this term.
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`8.
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`I also disagree with Dr. Alper that Dr. Batt’s analysis fails to account for POSAs’
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`knowledge. Alper Reply ¶25. In fact, Dr. Batt’s opinions, and mine, acknowledge the
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`complexities of research in the genetic engineering field. As explained in my Declaration, I have
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`considered whether a POSA would be reasonably certain of whether something is a “promoter
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`element” in light of the how that terminology is used in the Yeast Patents and scientific
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`publications.
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`9.
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`In particular, a review of the literature shows that the term “promoter element” or
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`similar terminology (“element” of a promoter) is used inconsistently or idiosyncratically,
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`depending on the needs or interests of the particular author(s). E.g., Decl. ¶¶65-66. For example,
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`this term is not used consistently to refer to any specific or particular structural features within a
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`polynucleotide sequence. The literature cited by Dr. Alper is consistent with my prior observations
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`on this point. In consequence, the varied usage of the term “promoter element” in the art does not
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`provide clarity or reasonable certainty to a POSA considering the scope of the Yeast Patent claims.
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`10.
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`Dr. Batt and I have also considered the practical challenges facing a POSA who is
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`designing a genetic expression system. E.g., Decl. ¶¶67-69. As discussed, a sequence that has
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`some functionality in some contexts—including a potential transcription factor binding site, such
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`as a “consensus” sequence—may not be functional in other contexts.
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`11.
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`For example, as Dr. Batt and I previously explained, the transcription factor Mxr1p
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`does not bind to its recognized “consensus” sequence (5’-CYCCNY-3’) in all cases. E.g., Decl.
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`¶¶68-69. In fact, Mxr1p does not always bind to consensus sequences even when those sequences
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`are present in known promoters, such as the DHAS and PEX8 gene promoters. This was shown
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`early on, for example, in the research of Kranthi 2010. E.g., Decl. ¶69 & n.8. As I explained, the
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`factors that govern binding and activity for any particular site are poorly understood, even to this
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`day. Decl. ¶122. Even Dr. Alper’s research reflects this situation, as I discuss below.
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`12.
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`The lack of clarity regarding sequences that bind Mxr1p is further exemplified in a
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`review article discussed by Dr. Batt, Vogl 2013.1 Vogl 2013 discusses research findings regarding
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`Mxr1p’s ability to bind the promoter of the AOX1 gene, which contains “consensus” sequences
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`disclosed in Kranthi 2010. Scientists study the AOX1 promoter by performing “deletion studies,”
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`which artificially truncate portions of the promoter, to study the ability of Mxr1p to bind to parts
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`of the promoter and regulate expression.
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`13.
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`Vogl 2013 reports that “[g]enerally, the results of the different studies match fairly
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`well, but there are also a few notable discrepancies.” Ex. B-8, page 389. As one example, Vogl
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`2013 refers to two different research groups, Inan and Xuan, which “deleted approximately the
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`same fragment” in the AOX1 promoter (“XuanD” and “InanC”). Vogl 2013 teaches: “However,
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`the dropout deletion of XuanD decreased the activity to 16% of the wild-type promoter, whereas
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`the dropout deletion of InanC retained wild type like activity (109%).” Id. Vogl 2013 speculates
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`that “[c]onsequences to the DNA structure influencing the interaction of DNA-binding proteins
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`[e.g., Mxr1p] might be one possible explanation” for the observed differences in activity. Id.
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`14.
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`Vogl 2013 also reports on “band shift” studies indicating that Mxr1p does not
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`always bind to portions of a promoter even though “MXRE1 [containing the consensus sequence]
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`identified by Kranthi [2009] lies within” the same portions being studied. Ex. B-8, page 389. Vogl
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`2013 tries to explain these findings by theorizing that “different protein[s]” may be binding and
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`differentially affecting Mxr1p’s ability to bind these regions.
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`15.
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`At various points in his declaration, Dr. Alper states that the Mxr1 consensus
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`sequence was “well-characterized” and “consistently” binds Mxr1. Relatedly, he asserts that
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`1 Ex. B-8, Vogl & Glieder, “Regulation of Pichia pastoris promoters and its
`consequences for protein production,” 30 New Biotechnol. 385 (2013).
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`promoters of the AOX1 gene (which contain Mxr1p consensus sequences) are well characterized.
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`Alper Decl. ¶18. For reasons explained above, consistent with Dr. Batt’s observations (Batt Reply
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`¶¶45-49), such statements by Dr. Alper are factually inaccurate.
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`16.
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`In sum, the consequence of these empirical findings is that, absent structural or
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`other defining features of a “promoter element,” a POSA would not be reasonably certain about
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`whether any particular expression system infringes the “promoter element” term under Dr. Alper’s
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`interpretation (assuming all other limitations are satisfied) if that system has some functionality.
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`17.
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`Thus, contrary to Dr. Alper’s suggestion that promoter elements were “known” in
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`the art (Alper Reply ¶30), a POSA would not be reasonably certain whether any particular
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`sequence or structure is a “promoter element.”
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`2.
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`Mischaracterization of the Yeast Patents
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`18.
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`Dr. Alper suggests that the Yeast Patents’ specification contains examples of
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`promoter elements and describes “what makes a given nucleotide sequence a ‘promoter element’.”
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`Alper Reply ¶¶26, 30. Not true. The portions of the specification that Dr. Alper cites to refers
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`expressly to full promoters. Ex. L-2, ’656 patent 4:60-5:11, 6:36-37. The patents’ teaching of
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`full-length promoters, which have been identified for endogenous genes, is not the same as
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`teaching a POSA the use, boundaries, or any specific structural features of any particular subparts
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`or portions within those promoters.
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`19.
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`Dr. Alper also incorrectly suggests that Examples in the Yeast Patents’
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`specification contain examples of promoter elements. Alper Reply ¶33 (discussing Examples 22
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`and 25). These Examples do not contain the term “promoter element.” Example 22 discloses
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`cutting a promoter into halves. Example 25 discloses the use of the full AOX1 promoter. They
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`do not teach how to differentiate “promoter elements” from a full native gene promoter.
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`20.
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`Dr. Alper mischaracterizes the Yeast Patents by suggesting that they use the term
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`“promoter element” “consistent with [its] ordinary usage.” Alper Reply ¶30. As I explain below,
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`there is no ordinary, consistent usage of “promoter element” in the technical literature. Even Dr.
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`Alper’s usage of this term is idiosyncratic, as shown below regarding the “Redden” reference.
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`21.
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`Relatedly, I note that the Yeast Patents’ specification does not contain the words
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`“adjacent” or “positioned relative to” at all, much less use these terms to refer to a “promoter
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`element” in particular. Even assuming a “promoter element” were positioned upstream of a coding
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`sequence (e.g., as part of a full promoter), that does not explain the boundaries, scope, or other
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`structural features that distinguish a “promoter element” from a full promoter. I see nothing in the
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`Yeast Patents that addresses this issue.
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`3.
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`Mischaracterization of “Promoter Element” in Technical Literature
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`22.
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`Dr. Alper incorrectly asserts that Dr. Batt “completely fails to consider” a POSA’s
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`familiarity with the term “promoter element.” Alper Reply ¶26. This is untrue, as reflected in Dr.
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`Batt’s declarations. Additionally, Dr. Batt's opinion is consistent with my opinion, which is based
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`on my own review and consideration of the state of the art and a POSA’s knowledge.".
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`23.
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`In fact, the literature cited by Dr. Alper confirms that “promoter element” is not
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`used consistently in the technical literature. He discusses the use of the “promoter element” term
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`in certain patents and scientific articles. As explained below, and as Dr. Batt points out (Batt Decl.
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`¶¶19-30), these references each teach a different meaning for that term. Those references provide
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`context for the authors’ audience to understand what is meant by “promoter element” in each
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`reference. Dr. Alper suggests that these references indicate that a POSA would understand what
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`“promoter element” means, but in fact they show that a POSA would need to read each reference
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`individually to understand what is being described. Put another way: if “promoter element” had
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`a consistent meaning, there would be no need for independent explanations of its meaning in the
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`references, and that term would be used consistently across the different references.
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`24.
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`Redden (Alper Ex. 2). Redden is a review article published in July 17, 2015 by
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`Dr. Alper and a co-author, titled “The development and characterization of synthetic minimal yeast
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`promoters.” I note that Redden was published after May 11, 2015, which I have been asked to
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`assume is the effective filing date of the Yeast Patents,2 and would therefore have not been known
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`to a POSA of the Yeast Patents.
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`25.
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`Redden acknowledges the complexity of eukaryotic promoter sequences relative to
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`prokaryotic promoter sequences. It states (Alper Ex. 2 at 2):
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`The innate complexity of eukaryotic transcription makes organisms like S.
`cerevisiae quite distinct from their bacterial counterparts. This complexity is
`instantiated by considerably longer eukaryotic promoters compared to bacterial
`ones. E. coli promoters typically span well under 100 nucleotides7, whereas native
`yeast promoters (especially those used in synthetic biology efforts) can stretch
`hundreds of base pairs8,9. Mechanistically, a longer stretch of DNA is needed to
`load and stabilize a bulkier (~100 kDa larger) and more highly regulated eukaryotic
`RNA polymerase II (RNAP)10,11.
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`26.
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`Redden states (Alper Ex. 2 at 2, emphasis added) that “two of the most commonly
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`used yeast promoter elements, the GAL1 inducible promoter and the strong GPD (TDH3)
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`promoter span over 400 and 600 nucleotides respectively.” Thus, as used in Redden, “promoter
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`element” means full native gene promoters. Dr. Alper relies on Redden for this particular teaching
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`of the meaning of “promoter element.” Alper Reply ¶27.
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`27.
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`Notably, Redden’s “promoter elements” are relatively long nucleotide sequences,
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`spanning hundreds of base pairs (i.e., nucleotides). Redden also discusses smaller “elements,”
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`2 Decl. ¶33, n.1.
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`e.g., “core” elements, which are artificially engineered into various systems. Redden states (Alper
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`Ex. 2 at 2):
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`This core element scaffold was built on distinct, essential sequences for promoter
`function—a TATA box with consensus sequence of TATAWAWR24 followed by
`a
`transcription
`start
`site
`(TSS) with
`consensus
`sequence
`of
`A(Arich)5NYAWNN(Arich)6 (ref. 25). The TSS is found in native promoters at a
`distance of 40–120 bp downstream of the TATA box25,26. However, to establish
`minimal promoters, we created and evaluated ensembles of variably spaced TATA
`box-TSS core elements. Although the core elements contain all sequence
`components necessary for transcription initiation, very low expression is expected
`without UAS elements, which provide the overall strength and regulation of a
`promoter5,27.
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`28.
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`Redden thus teaches that certain TATA boxes and UAS sequences are necessary
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`for the function of the promoter at issue in that reference. However, it does not describe these
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`relatively short sequences as “promoter elements.” Rather, it describes the full promoters as
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`“promoter elements.” Dr. Alper does not explain whether, contrary to the articulation in his own
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`paper, these shorter sequences (or other parts of his synthetic promoters) would be considered
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`“promoter elements” as claimed in the Yeast Patents.
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`29.
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`Dr. Alper cites Redden as disclosing “minimal” promoter elements for the
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`proposition that “a promoter element may lack sequences determined to be dispensable for driving
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`transcription.” Alper Reply ¶28. Here again, Dr. Alper articulates another example of how
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`researchers in the field may use “promoter element” terminology differently and arbitrarily.
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`30.
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`In particular, Dr. Alper’s statement is confusing because it suggests that a
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`“promoter element” may be defined by what it does not have. He does not explain how the
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`absence of structure in a polynucleotide would make a POSA reasonably certain of the scope of a
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`“promoter element.” For example, he does not explain how a smaller sequence could be a
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`“promoter element” but a larger sequence containing that smaller sequence would not be. (It is
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`unclear from Dr. Alper’s vague assertions whether that is actually his opinion.) Nor does Dr. Alper
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`explain how a POSA would recognize the smaller but not the larger sequence as a “promoter
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`element” (if that is his opinion).
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`31.
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`Li (Alper Ex. 3). Li is a review article published in 2007, titled “Expression of
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`Recombinant Proteins in Pichia Pastoris.”
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`32.
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`Li mentions “promoter element” once. Li states: “Most P. pastoris expression
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`systems use the methanol-induced alcohol oxidase (AOX1) promoter [1]. Upon induction by
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`methanol, the fraction of total soluble protein that is composed of alcohol oxidase can typically
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`rise to 30% [2], indicating the power of this promoter element.” Alper Ex. 3, page 106 (emphasis
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`added).
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`33.
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`Thus, similar to Redden, Li uses “promoter element” to refer to a full native gene
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`promoter. Dr. Alper relies on Li for this particular teaching of the meaning of “promoter element.”
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`Alper Reply ¶27.
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`34.
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`As cited by Dr. Alper, the Li and Redden references use “promoter element”
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`broadly to refer to specific promoters. Alper Reply ¶27. Their usage of “promoter element” is
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`inconsistent with the disclosures of the Yeast Patents, which, as I previously explained, teach that
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`“promoter elements” are different from “promoters.” Decl. ¶¶63-64.
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`35.
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`Suppmann (Alper Ex. 4). I see that Suppmann is a patent (U.S. Patent No.
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`7,118,901) to Suppmann and others, titled “Recombinant Bovine Pancreatic Deoxyribonuclease I
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`with High Specific Activity,” which issued on Oct. 10, 2006.
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`36.
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`In contrast to the Li and Redden references discussed above, the Suppmann
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`reference internally defines “promoter element” as a “sub-fragment of a larger promoter
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`sequence.” Alper Reply ¶28; Alper Ex. 4, col. 2, lines 65-67.
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`37.
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`Dr. Alper contends that, based on Suppmann, a “POSA would have also understood
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`that a ‘promoter element’ may comprise less than a complete native promoter.” Alper Reply ¶28.
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`Taking Dr. Alper’s statement at face value, Suppmann’s disclosure merely shows how POSAs
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`would understand “promoter elements” could be different things and that, accordingly, the
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`meaning of this term can only be ascertained within the context of an individual reference.
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`Consequently, neither Suppmann nor any other individual reference clarifies the boundaries of the
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`claimed “promoter element” term.
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`38.
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`Zhang (Alper Ex. 5). I see that the Zhang reference is a patent (U.S. Patent No.
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`9,133,463) to Zhang and Armenta, titled “Engineering Microorganisms,” which issued on Sept.
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`15, 2015.
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`39.
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`Zhang defines “promoter” and “promoter element” the same way: “Promoter or
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`Promoter element: As used herein, the terms ‘promoter’ and ‘promoter element’ refer to a
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`polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to
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`the promoter, and that effects expression of the selected polynucleotide sequence in cells.” Alper
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`Ex. 5, col. 10 lines 44-48.
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`40.
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`In his discussion of Zhang, Dr. Alper states that “even if the promoter element did
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`not constitute a full-length native promoter, it would still be capable of driving transcription.”
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`Alper Reply ¶29. I disagree. First, Zhang equates a “promoter” and a “promoter element” in its
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`definition above. As used idiosyncratically in Zhang (similar to the usage in Li and Redden), those
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`terms mean the same thing. Thus, Zhang does not support Dr. Alper’s proposition that a “promoter
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`element” does not constitute a full-length native promoter.
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`41.
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`Second, I disagree because a POSA would not be reasonably certain of what the
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`“promoter element” claimed in the Yeast Patents is, for reasons I have previously explained (Decl.
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`§VIII.A), and Dr. Alper’s general reference to a different reference, with its respective definition,
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`does not clarify that matter.
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`42.
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`Dr. Alper also mischaracterizes certain patent references that I understand are
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`assigned to Ginkgo Bioworks, a co-defendant in this Litigation. I discuss these references below.
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`43.
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`U.S. 11,702,665 (Alper Ex. 6). I see that U.S. 11,702,665 (U.S. ’665) titled
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`“Paenibacillus-Based Endospore Display Platform, Products and Methods,” issued on July 18,
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`2023. I also see that it has a “provisional application” filing date of Nov. 16, 2017, which is after
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`the (assumed) Yeast Patents’ effective filing date of May 11, 2015. Alper Ex. 6, page 1.
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`44.
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`U.S. ’665 does not define “promoter element” in any way. Rather, it mentions
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`“promoter element” twice in passing (as quoted by Dr. Batt). Batt Reply ¶24. Thus, it is not clear
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`how U.S.’665’s usage of this term informs a POSA’s understanding of “promoter element” as that
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`term is recited in the Yeast Patent claims, and Dr. Alper does not explain. Alper Decl. ¶32.
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`45. WO 2015/148680 (Alper Ex. 7): As Dr. Batt points out (Batt Reply ¶25), Ginkgo’s
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`patent application WO 2015/148680 (WO ’680) contains additional explanation of specific
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`promoter elements in prokaryotic (bacterial) systems. See Alper Ex. 7, [0061]. Similar
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`explanation or disclosure is absent from the Yeast Patents.
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`46.
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`I also note that WO ’680’s description of promoter elements focuses on bacterial
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`promoter architecture—a different kingdom of organisms from yeast (a fungus, which is a
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`eukaryote). WO ’680 (Alper Ex. 7 [0061], infra, emphasis added) explains how eukaryotic
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`promoter architecture is more complex than that of prokaryotes. WO ’680 also refers to functional
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`structures that are not described as promoter elements in that reference (e.g., the TATA box and
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`UAS sequences), further complicating what might be considered a “promoter element” in
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`eukaryote promoters.
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`
`In prokaryotes, a core promoter usually includes two consensus sequences, a -10
`sequence or a -35 sequence, which are recognized by sigma factors. The -10
`sequence (10 bp upstream from the first transcribed nucleotide) is typically about
`6 nucleotides in length and is typically made up of the nucleotides adenosine and
`thymidine (also known as the Pribnow box). The presence of this box is essential
`to the start of the transcription. The -35 sequence of a core promoter is typically
`about 6 nucleotides in length. The nucleotide sequence of the -35 sequence is
`typically made up of the each of the four nucleosides. The presence of this sequence
`allows a very high transcription rate. In some embodiments, the - 10 and the -35
`sequences are spaced by about 17 nucleotides. Eukaryotic promoters are more
`diverse than prokaryotic promoters and may be located several kilobases
`upstream of the transcription starting site. Some eukaryotic promoters contain a
`TATA box, which is located typically within 40 to 120 bases of the transcriptional
`start site. One or more upstream activation sequences (UAS), which are
`recognized by specific binding proteins can act as activators of the transcription.
`Theses [sic] UAS sequences are typically found upstream of the transcription
`initiation site. The distance between the UAS sequences and the TATA box is
`highly variable and may be up to 1 kb.
`
`47.
`
`It is unclear, for example, whether a UAS would be considered a “promoter
`
`element” within the meaning of the Yeast Patent claims. For example, as Dr. Alper contends,
`
`“promoters are adjacent to the gene or in close physical proximity.” Alper Decl. ¶13. In contrast,
`
`a UAS, as taught in WO ’680 (above), may be located a thousand base pairs away from the
`
`transcription start site (TATA box).
`
`48.
`
`US 2023/0257793 (Alper Ex. 8). As Dr. Batt points out (Batt Reply ¶¶26-27),
`
`Ginkgo’s patent application U.S. Application No. 2023/0257793 (US ’793) contains additional
`
`explanation of specific kinds of promoter elements and their structural features. E.g., Alper Ex. 8,
`
`[0026]-[0027]. Similar explanation or disclosure is absent from the Yeast Patents.
`
`49.
`
`For example, US ’793 states that “[i]n some embodiments, the core promoter
`
`element of the synthetic output promoter has a nucleic acid sequence that is from about 6 base
`
`pairs to about 300 base pairs, from about 25 base pairs to about 250 base pairs, from about 75 to
`
`about 225 base pairs, or from about 100 base pairs to about 175 base pairs in length.” Alper Ex.
`
`8, [0026]. The “core promoter element” of US ’793 can also comprise specific sequences:
`
`
`
`14
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`

`

`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 15 of 28 PageID #: 22879
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`
`
`• “a core promoter sequence that is at least 90%, at least 95%, or 100% identical to a core
`promoter sequence from P(AOX1) (SEQ ID NO: 162), P(DAS2) (SEQ ID NO: 163),
`P(HHF2) (SEQ ID NO: 164), or P(PMP20) (SEQ ID NO: 165).” Alper Ex. 8, [0027].
`
`• “a core promoter sequence that is at least 90%, at least 95%, or 100% identical to a core
`promoter sequence from P(AOX1).” Id., [0027].
`
`• “a core promoter sequence that is at least 90%, at least 95%, or 100% identical to a core
`promoter sequence from P(DAS2).” Id., [0027].
`
` “a core promoter sequence that is at least 90%, at least 95%, or 100% identical to a core
`promoter sequence from P(HHF2).” Id., [0027].
`
`• “a core promoter sequence that is at least 90%, at least 95%, or 100% identical to a core
`promoter sequence from P(PMP20).” Id., [0027].
`
`50.
`
`The Yeast Patents lack similar detail that might guide a POSA as to the scope of
`
`•
`
`“promoter element” as that term is used in those patents.
`
`51.
`
`Thus, as shown above, a POSA would understand from each individual reference
`
`cited by Dr. Alper how those authors were using the term and what was meant thereby.
`
`52.
`
`These references illustrate, however, that the “promoter element” term itself does
`
`not have a consistent or universal meaning known to POSAs working in the relevant field in 2015.3
`
`In fact, considered collectively rather than individually, the literature reveals there is no such
`
`consistent meaning.
`
`4.
`
`Dr. Alper’s Research Illustrates the Lack of Reasonable Certainty
`Regarding “Promoter Element”
`
`53.
`
`I understand Dr. Alper researches “synthetic” promoters that are engineered to have
`
`certain characteristics. As Dr. Batt notes (Batt Reply ¶¶31-33), Dr. Alper’s research illustrates the
`
`challenges facing a POSA attempting to discern whether any particular sequence is a “promoter
`
`element.” I agree.
`
`
`3 Dr. Batt refers to other literature, such as the Hartner reference, that describes the
`deletion of a few nucleotides as an “element.”
`
`
`
`15
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`

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`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 16 of 28 PageID #: 22880
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`
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`54.
`
`One publication by Dr. Alper, Cheng 2016,4 shows that potentially-functional sites
`
`in one polynucleotide sequence (“putative transcription factor binding sites,” or TFBS) can be non-
`
`functional in others, even when positioned to drive transcription. The Cheng 2016 study identified
`
`“genetic motifs”—e.g., TFBSs—and discusses “building synthetic promoters” using these TFBS
`
`“using conventional DNA synthesis and molecular biology techniques” and “evaluating the
`
`performance of these synthetic promoters.” Id., Abstract.
`
`55.
`
`Cheng 2016 describes the generation of three “synthetic promoters” that have
`
`equivalent numbers of putative transcription factor binding sites. Despite similarities in their
`
`architecture, Cheng 2016 reports that two of the three systems were functional, but the third (v3)
`
`was “barely functional.” Specifically, Cheng states (Ex. L-12, p. 1460, emphasis added):
`
`In comparison to our negative controls (promoterless construct and HT1080 WT
`representing background expression levels), synthetic promoters v1 and v2
`exhibited considerable functionality approaching reference (hCMV IE or full
`EF1α promoter) levels, whereas variant v3 was barely functional (Figure 4b,c).
`
`Annotation of the enhancer/ proximal promoter region of these synthetic promoters
`using the same JASPAR database indicated that these 3 synthetic variants
`contained similar quantities of putative binding sites (53 for v1 and v2, 52 for v3;
`Supporting Information Table S4a−d), thus the variable expression of both
`reporters reflecting the strength of these promoters were not due to a disparity in
`potential TFBSs.
`
`56.
`
`Cheng 2016 recognizes that “[t]hese expression patterns suggest some context
`
`dependency of the core promoter region and the importance of TFBS ordering in promoter
`
`function.” Ex. L-12, p. 1460.
`
`
`4 Ex. L-12, Cheng & Alper, Transcriptomics-Guided Design of Synthetic Promoters for
`a Mammalian System, 5 ACS Synth. Biol. 1455-1465 (2016).
`
`
`
`16
`
`

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`Case 1:22-cv-00311-WCB Document 353 Filed 02/09/24 Page 17 of 28 PageID #: 22881
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`
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`57.
`
`As an example of context-dependency, Cheng 2016 explains that spacing between
`
`the putative transcription factor binding sites can significantly affect the activity of the synthetic
`
`promoter. For example, it states (Ex. L-12, p. 1461-62):
`
`Interestingly, the data suggest that the spacing between annotated TFBSs
`exhibited greater influence on promoter strength (compare native in light bar with
`reference in dark bar) than the particular arrangement of the TFBSs themselves
`(compare native, sequential, random in light colored bars) (Figure