Filed on behalf of Junior Party Paper No. ___ THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, UNIVERSITY OF VIENNA, AND EMMANUELLE CHARPENTIER By: Eldora L. Ellison, Ph.D., Esq. By: Li-Hsien Rin-Laures, M.D., Esq. Eric K. Steffe, Esq. RINLAURES LLC David H. Holman, Ph.D., Esq. 321 N. Clark Street, 5th floor Byron L. Pickard, Esq. Chicago, IL 60654 John Christopher Rozendaal, Esq. Tel. (773) 387-3200; Fax (773) 929-2391 Paul A. Ainsworth, Esq. [email protected] Michael E. Joffre, Ph.D., Esq. STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C. Sandip H. Patel, Esq. 1100 New York Avenue, NW MARSHALL GERSTEIN & BORUN LLP Washington, D.C. 20005 6300 Willis Tower, 233 South Wacker Tel: (202) 371-2600 Drive Fax: (202) 371-2540 Chicago, IL 60606 [email protected] Tel: (312) 474-6300; Fax: (312) 474-0448 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, UNIVERSITY OF VIENNA, AND EMMANUELLE CHARPENTIER Junior Party (Applications 15/947,680; 15/947,700; 15/947,718; 15/981,807; 15/981,808; 15/981,809; 16/136,159; 16/136,165; 16/136,168; 16/136,175; 16/276,361; 16/276,365; 16/276,368; and 16/276,374), v. THE BROAD INSTITUTE, INC., MASSACHUSETTS INSTITUTE OF TECHNOLOGY, and PRESIDENT AND FELLOWS OF HARVARD COLLEGE Senior Party (Patents 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,840,713; and Application 14/704,551). ______Patent Interference No. 106,115 (DK) (Technology Center 1600)
CVC Reply 2 (for judgment based on priority)
Interference No. 106,115 CVC Reply 2
TABLE OF CONTENTS I. INTRODUCTION ...... 1 II. ARGUMENT ...... 5 A. CVC had a complete conception before Broad, coupled with CVC’s uncontested reasonably continuous diligence through CVC’s P3 filing in January 2013...... 5 1. Reason 1: CVC had a complete conception of Count 1 evinced by CVC’s specific embodiment of delivering the sgRNA and Cas9 components to human cells using specific expression vectors and transfection ...... 7 2. Reason 2: CVC had a complete conception of Count 1 before June 26, evinced by CVC’s specific embodiment of delivering the sgRNA and Cas9 components to zebrafish cells as a pre-formed sgRNA-Cas9 ribonucleoprotein complex using nuclear microinjection...... 11 3. Multiple lab groups used CVC’s system with only ordinary skill and routine techniques, further confirming CVC’s conception...... 16 4. CVC’s reasonably continuous diligence during the critical period through CVC’s P3 filing went unchallenged by Broad ...... 18 5. Broad’s attempt to rewrite conception law fails: conception does not require a reasonable expectation of success...... 19 6. CVC proved multiple actual reductions to practice of Count 1...... 25 B. Reason 3: CVC prevails under derivation law...... 28 III. CONCLUSION ...... 30 APPENDIX 1 – LIST OF EXHIBITS ...... 1-1 APPENDIX 2 – STATEMENT OF MATERIAL FACTS ...... 2-1 Broad’s Additional Facts 64 – 200 ...... 2-14 CVC Additional Facts 201 - 276 ...... 2-45
i Interference No. 106,115 CVC Reply 2
TABLE OF AUTHORITIES Cases
Agilent Tech. Inc. v. Dionex Softron GMBH, Interference 106,087 (PTAB, Feb. 25. 2021) ...... 18
Alexander v. Williams, 342 F.2d 466 (C.C.P.A. 1965) ...... 30
Applegate v. Scherer, 332 F.2d 571 (C.C.P.A. 1964) ...... 4, 20, 30
Burroughs Wellcome Co. v. Barr Labs., Inc., 40 F.3d 1223 (Fed. Cir. 1994) ...... 1, 4, 5, 9, 20, 22
Coleman v. Dines, 754 F.2d 353 (Fed. Cir. 1985) ...... 1, 4, 20
Dana-Farber Cancer Inst., Inc. v. Ono Pharm. Co., 964 F.3d 1365 (Fed. Cir. 2020) ...... 20
Field v. Knowles, 183 F.2d 593 (C.C.P.A. 1950) ...... 11
Haskell v. Colebourne, 671 F.2d 1362 (C.C.P.A. 1982) ...... 7
Hedgewick v. Akers, 497 F.2d 905 (C.C.P.A. 1974) ...... 30
Hitzeman v. Rutter, 243 F.3d 1345 (Fed. Cir. 2001) ...... 4, 20, 21
In re Tansel, 253 F.2d 241 (C.C.P.A. 1958) ...... 7
MacMillan v. Moffett, 432 F.2d 1237 (C.C.P.A. 1970) ...... 30
Meade v. McKirnan, 585 F.2d 504 (C.C.P.A. 1978) ...... 30
Mergenthaler v. Scudder, 11 App. D.C. 264 (D.C. Cir. 1897) ...... 4, 11
i Interference No. 106,115 CVC Reply 2
Polye v Uhl, 328 F.2d 893 (C.C.P.A. 1964) ...... 30
Regents of Univ. Calif. v. Broad Inst., Inc., 903 F.3d 1286 (Fed. Cir. 2018) ...... 17
Rey-Bellet v. Engelhardt, 493 F.2d 1380 (C.C.P.A. 1974) ...... 3, 11, 17, 18, 19
Sewall v. Walters, 21 F.3d 411 (Fed. Cir. 1994) ...... 1, 11, 18
Spero v. Ringold, 377 F.2d 652 (C.C.P.A. 1967) ...... 7
Summers v. Vogel, 332 F.2d 810 (C.C.P.A. 1964) ...... 11
Trumbull v. Kirschbraun, 67 F.2d 974 (C.C.P.A. 1933) ...... 11,18
ii Interference No. 106,115 CVC Reply 2 1 I. INTRODUCTION
2 The CVC inventors revolutionized the field of genome editing, giving the world a new
3 system capable of cleaving and editing genes in eukaryotic cells. By identifying for the first time
4 the necessary and sufficient components of the Type II CRISPR-Cas9 cleavage complex and
5 further modifying the RNAs into a single-guide (sgRNA) format, the CVC inventors provided a
6 simple, programmable tool for site-specific gene editing in eukaryotic cells. By the time Zhang
7 learned of CVC’s single-guide CRISPR-Cas9 system, the CVC inventors had already conceived
8 multiple specific embodiments of the invention and were diligently working to reduce the
9 invention to practice.
10 Count 1 recites a composition: a system or a eukaryotic cell comprising a sgRNA
11 CRISPR-Cas9 system. Conception of that composition is complete “when the idea is so clearly
12 defined in the inventor's mind that only ordinary skill would be necessary to reduce the invention
13 to practice, without extensive research or experimentation.” Burroughs Wellcome Co. v. Barr
14 Labs., Inc., 40 F.3d 1223 (Fed. Cir. 1994) (citing Sewall v. Walters, 21 F.3d 411 (Fed. Cir. 1994)
15 and Coleman v. Dines, 754 F.2d 353 (Fed. Cir. 1985)). In accordance with that standard, the
16 CVC inventors unquestionably conceived of the invention before Broad’s alleged June 26
17 conception. By then, CVC had already documented at least two specific embodiments that they
18 later reduced to practice: (1) vectors to express the sgRNA and Cas9 protein to cleave the
19 CTLA6 gene in human cells, and (2) a pre-formed sgRNA/Cas9 ribonucleoprotein (RNP)
20 complex that CVC had already demonstrated works in vitro and subsequently simply
21 microinjected into fish cells to cleave DNA. During the critical period, CVC worked diligently
22 to reduce to practice both embodiments of the invention, achieving multiple actual reductions to
23 practice in a matter of months and requiring no further inventive steps.
24 Now that the record is developed, the facts and the law are clear that CVC is entitled to
- 1 - Interference No. 106,115 CVC Reply 2 1 favorable judgment on priority. Corroborated evidence shows that before Broad’s conception,
2 CVC had already documented the details of both embodiments that CVC then diligently reduced
3 to practice. There is new testimony – from Barrangou, Sontheimer, and even Zhang’s
4 collaborator Marraffini – that scientists at the time expected it to be “straightforward” and
5 “routine” to implement CVC’s sgRNA CRISPR-Cas9 system in eukaryotic cells. Moreover, the
6 record now shows that Zhang first learned of the sgRNA CRISPR-Cas9 system from CVC and
7 exercised no inventive skill in reducing it to practice, confirming that CVC’s conception was
8 complete. Zhang merely used publicly known expression vectors and protocols to implement
9 CVC’s system in eukaryotic cells. Thus, CVC prevails for any of three independent reasons:
10 Reason 1: CVC conceived of the invention of Count 1 before Zhang’s earliest alleged
11 June 26 conception date, and exercised reasonable diligence during the critical period through
12 CVC’s reductions to practice. By June 26, CVC had not only conceived of a sgRNA CRISPR-
13 Cas9 system for cleaving DNA in a eukaryotic cell, but had also identified the specific reagents
14 and methods used in the working example described in its P3 application: chimera A sgRNA
15 targeting the CLTA gene at a site adjacent a PAM, specific sgRNA and Cas9 vectors with
16 specific promoters (U6 and CMV), a specific nuclear localization signal (NLS) added to Cas9,
17 and human kidney (HEK293T) cells. The PTAB has already concluded this example is a
18 constructive reduction to practice of Count 1. CVC’s definite and permanent idea never changed,
19 as the inventors used these same sgRNA, Cas9, vectors, promoters, and NLS successfully to
20 cleave the CLTA target gene in HEK293T cells.
21 Reason 2: By June 26, CVC had also conceived of and documented a sgRNA CRISPR-
22 Cas9 system in which the sgRNA and Cas9 components are assembled as a pre-formed RNP
23 outside of the eukaryotic cell, and the RNP is delivered via microinjection into the cell nucleus.
- 2 - Interference No. 106,115 CVC Reply 2 1 Microinjection was a well-known technique for introducing RNA, DNA and/or protein into cells.
2 This is precisely the embodiment the inventors reduced to practice on August 9, 2012, by
3 microinjecting zebrafish embryos with a sgRNA-Cas9 RNP complex that resulted in an rx3
4 mutation. CVC’s embodiment using a pre-formed RNP complex and nuclear microinjection
5 obviated any obstacles that Broad imagines would have prevented a person of ordinary skill in
6 the art from achieving CRISPR-Cas9-mediated target DNA cleavage.
7 The PTAB should enter judgment in CVC’s favor because CVC has demonstrated a
8 complete conception before Zhang, as shown by either of the foregoing embodiments, and
9 reasonably continuous diligence from before Zhang’s alleged conception date through each of
10 CVC’s multiple actual reductions to practice and CVC’s constructive reduction to practice in P3.
11 See Rey-Bellet v. Englehardt, 493 F.2d 1380, 1386 (C.C.P.A. 1974). Broad presented no credible
12 challenge to CVC’s diligence, neither disputing that CVC began its diligence just before June 26,
13 nor pointing to any evidence to challenge CVC’s diligence thereafter. Should the PTAB agree
14 that CVC exercised reasonably continuous diligence from before Zhang’s alleged conception
15 date through the filing of CVC’s P3 in January 2013, a finding that CVC’s actual reductions to
16 practice (ARTPs) succeeded is not required to grant CVC’s motion. Id.
17 Reason 3: CVC is independently entitled to judgment on priority because, as fully
18 explained in CVC’s Opposition 5 (Paper 2567), Zhang derived the invention of Count 1 from the
19 CVC inventors, via Marraffini. Zhang had no concept of a single-guide RNA CRISPR-Cas9
20 system for genome editing in eukaryotes until Marraffini relayed it to him, based on what
21 Marraffini learned from CVC. Before then, Zhang had only toiled with a cumbersome RNA
22 processing system, resulting in experiments that Zhang’s graduate student summarized in June
23 2012 as having “all failed” before concluding that “[m]aybe other factors need to be identified.”
- 3 - Interference No. 106,115 CVC Reply 2 1 See e.g., Ex. 5013, ¶¶145, 147, 156, A74. In contrast, as Marraffini testified, using CVC’s
2 system would be “straightforward.” Recognizing a race to publish, Zhang dropped his RNA
3 processing experiments and rushed to use well-known, published vectors and protocols to
4 implement CVC’s chimera A sgRNA system. That was not invention; that was derivation.
5 Applegate v. Scherer, 332 F.2d 571, 573 (C.C.P.A. 1964) (1964).
6 In the face of such strong evidence in CVC’s favor, Broad invites the PTAB to commit
7 legal error by interpreting the law of conception to require a reasonable expectation of success.
8 The PTAB should reject that interpretation. Conception has been consistently defined for over a
9 century as “the formation, in the mind of the inventor of a definite and permanent idea of the
10 complete and operative invention, as it is thereafter to be applied in practice.” Coleman, 754 F.2d
11 at 359 (citing Mergenthaler v. Scudder, 11 App.D.C. 264, 276 (1897)). It is a bedrock principle
12 of conception law that “an inventor need not know that his invention will work for conception to
13 be complete.” Burroughs, 40 F.3d at 1228. Indeed, “[w]hether or not [the inventors] believed the
14 invention[s] would in fact work … is irrelevant.” Id. at 1229 (emphasis added). “[T]he discovery
15 that an invention actually works is part of its reduction to practice.” Id. at 1228. Hitzeman v.
16 Rutter, 243 F.3d 1345 (Fed. Cir. 2001) does not support Broad’s interpretation of conception
17 law. Hitzeman at most held that conception requires an inventor to expect to produce the claimed
18 invention, not have a reasonable expectation that the invention will work for its intended
19 purpose. Id. at 1358. The evidence unambiguously shows that the CVC inventors not only
20 expected to produce the invention of the Count, but did indeed produce it. Their subsequent
21 demonstration that the system worked for its intended purpose was part of its reduction to
22 practice, not conception. Burroughs, 40 F.3d at 1228.
23 Broad blatantly attempts to have the PTAB’s ’048 no interference-in-fact decision dictate
- 4 - Interference No. 106,115 CVC Reply 2 1 the outcome of this priority dispute. But that, again, would be an error, because that decision
2 involved a different count (method), different legal issue (obviousness, not conception), and
3 vastly different and limited evidentiary record. The facts and relevant law compel the PTAB to
4 award priority to CVC here, and doing so is consistent with the PTAB’s previous decisions.
5 II. ARGUMENT
6 A. CVC had a complete conception before Broad, coupled with CVC’s uncontested 7 reasonably continuous diligence through CVC’s P3 filing in January 2013.
8 Conception requires a “definite and permanent idea” of the invention and its intended
9 purpose, not a reasonable expectation that the inventor will succeed in achieving it, as Broad
10 argues. See Burroughs, 40 F.3d at 1228. Broad concedes that its earliest possible conception date
11 is June 26, 2012. Paper 2118, 42:9-10; MF201. Broad could not assert an earlier date because
12 June 26 is the date Zhang first learned of the invention of Count 1 from CVC via Marraffini.
13 Paper 2567, 33:12-41:24; Ex. 5265, 66:7-67:7; 68:13-21; Ex. 3713, 27-29; MF225-231.
14 CVC’s evidence of conception before June 26 expressly meets all the elements of Count
15 1. See Ex. 4381, 63-65; Ex. 4382, 7; Ex. 4349, ¶¶124-128, 155, 167-172; Ex. 4603; Ex. 5105,
16 16-29; Ex. 4345, ¶¶61, 158-163, 167-174; Paper 1579, 7:17-19:9; MF26-30, 32-39. In addition
17 to the March 1 notebook entry, by April 11, 2012, CVC’s invention disclosure form described
18 attaching an NLS to Cas9, confirmed that the system should target a DNA sequence adjacent a
19 PAM, confirmed that a chimera A sgRNA system worked in vitro, and described approaches for
20 using the system in eukaryotic cells, including (i) microinjecting the system’s components into
21 zebrafish embryos and (ii) producing the system’s components from expression vectors in cells,
22 both of which approaches CVC later used in its reductions to practice. Paper 1579, 14:2-17:10;
23 Ex. 4382, 4, 7; Ex. 4349, ¶¶72-74; Ex. 5105, 16-29; Ex. 4345, ¶¶158-163; MF32-36. By May 28,
24 the CVC inventors had produced a pre-formed complex of chimera A sgRNA and S. pyogenes
- 5 - Interference No. 106,115 CVC Reply 2 1 Cas9, and demonstrated it worked in vitro to cleave multiple eukaryotic DNA targets. Paper
2 1579, 17:11-19:9; Ex. 4382, 4-7; Ex. 4603; Ex. 4349, ¶¶124-128; Ex. 4345, ¶¶167-174. Also by
3 May 28, the CVC inventors had documented a specific way to make their invention with
4 expression vectors, and had documented the specifics of the sgRNA and Cas9 vectors, promoters
5 (U6 and CMV), sgRNA (CLTA6 chimera A), target sequence (CLTA site adjacent to PAM),
6 Cas9 (S. pyogenes), target cell (human kidney cells), and delivery method (transfection)—i.e.,
7 the very details Broad alleges were lacking for CVC’s conception on March 1. See id.; Paper
8 2569, 10:5-8, 36:12-17; MF37-39, 202-208, 211-214.
9 CVC’s inventors documented each of the approaches discussed above, corroborating
10 their complete conception of at least two specific embodiments of Count 1 before June 26. See
11 e.g., Paper 1579, 7:17-19:9; Paper 2567, 31:9-33:11. CVC prevails on priority by showing
12 conception of either embodiment before Broad, coupled with CVC’s reasonably continuous
13 diligence to a reduction to practice. Broad failed to raise any plausible challenge to CVC’s
14 diligence through CVC’s P3 filing date. Thus, the law compels judgment in CVC’s favor.
15 Broad nitpicks CVC’s March 1 notebook entry, arguing “descriptions of delivery
16 technique, promoter selection, NLS selection and placement, codon optimization approach,
17 vector selection … [or] planned experimental conditions….” and “other parameters, such as the
18 U6 promoter, are not found….” Paper 2569, 10:5-8, 36:12-17. The response is that Count 1
19 neither recites nor requires a specific delivery technique, promoter, NLS, codon optimization, or
20 vector. These aspects are routine and involve exercising only ordinary skill; none are required for
21 complete conception of the Count. MF5-21. Indeed, complete conception does not require that
22 “the final size and shape of every part and the location of every nut, screw, and bolt must be
23 exactly foreseen before the conception of an apparatus can be said to be complete.” In re Tansel,
- 6 - Interference No. 106,115 CVC Reply 2 1 253 F.2d 241, 243 (C.C.P.A. 1958). Even if such details were required for conception, CVC had
2 those, too—all before June 26. See e.g., Paper 1579, 17:11-19:9; MF32-39, 202-208, 211-214.
3 1. Reason 1: CVC had a complete conception of Count 1 evinced by CVC’s 4 specific embodiment of delivering the sgRNA and Cas9 components to 5 human cells using specific expression vectors and transfection
6 By June 26, CVC had documented conception of a specific embodiment of Count 1 that
7 includes the same sgRNA sequence, Cas9 sequence, promoters, NLS, cell type, and methods
8 described in Example 2 in CVC’s P3 application, which the PTAB concluded is a constructive
9 reduction to practice of Count 1. Ex. 3004, ¶¶408-450; Paper 877, 106:1-14; MF49, 206-210. If
10 CVC’s disclosure of those elements in its P3 application was sufficient to establish constructive
11 reduction to practice, CVC’s description of that same combination of elements by May 28 is
12 necessarily sufficient to demonstrate conception. Haskell v. Colebourne, 671 F.2d 1362, 1365-66
13 (C.C.P.A. 1982) (disclosure “adequate to establish a constructive reduction to practice” is
14 sufficient to establish conception); Spero v. Ringold, 377 F.2d 652, 660 (C.C.P.A. 1967) (same).
15 By May 28, 2012, the CVC inventors had constructed the pMJ874 vector, which uses the
16 U6 promoter to drive expression of the CLTA6 sgRNA targeting a specific CLTA target gene
17 sequence next to a PAM. See e.g., Paper 1579, 17:11-19:9; Ex. 4382, 4-7; Ex. 4349, ¶¶124-128;
18 Ex. 4603; Ex. 4345, ¶¶167-172; Ex. 3004, ¶¶408-450; MF37-39, 202. This is the same pMJ874
19 vector that Jinek and East would later transfect into human HEK293T cells with S. pyogenes
20 Cas9 to successfully target and cleave the CLTA target DNA, and the same vector used in
21 Example 2 of CVC’s P3 application. Id. And, as the contemporaneous evidence shows, CVC
22 specifically chose the CLTA locus because their colleagues Drubin and Cheng had already
23 shown successful genome editing using zinc finger nucleases (ZFNs) targeting the same locus.
24 See e.g., Paper 1579, 27:20-28:17; Ex. 4475; Ex. 4775; Ex. 4776; Ex. 4350, ¶62; Ex. 4349, ¶¶75,
25 78; Ex. 4352, ¶12; Ex. 5013, ¶¶45-47; MF23-24. Also by May 28, CVC had constructed an S.
- 7 - Interference No. 106,115 CVC Reply 2 1 pyogenes Cas9 expression vector with the same elements in P3’s Example 2, i.e., a strong CMV
2 promoter, NLSs, and a GFP tag, and CVC had successfully expressed the Cas9 protein in human
3 HEK293T cells and visualized nuclear localization of Cas9. See e.g., Paper 1579, 18:17-19:4;
4 Ex. 4349, ¶¶109, 123-127, 155; Ex. 4444; Ex. 4484; Ex. 4536; Ex. 4345, ¶173; Ex. 3004, ¶¶408-
5 450. The CVC inventors were also codon optimizing the Cas9 gene to improve expression levels.
6 Id. CVC’s specific vector embodiment undeniably meets all the elements of Count 1:
CVC’s Specific embodiment Elements of Count 1 • Human HEK293T cells with Eukaryotic cell comprising target DNA CLTA target gene molecule • Type II CRISPR System Type II CRISPR System comprising: • S. pyogenes Cas9 - Cas9 • CLTA6 chimera A sgRNA - sgRNA • System is capable of cleaving or System is capable of cleaving or editing a target editing CLTA locus DNA molecule or modulating transcription of a gene encoded by the target DNA molecule
7 See Paper 1579, 7:17-19:9; Ex. 4345, ¶¶167-174; MF32-39, 202-207.
8 Before June 26, CVC also understood how to obviate Broad’s theoretical “obstacles” to
9 implementing the invention in eukaryotic cells. MF3-24. According to Broad’s expert, Breaker,
10 Zhang allegedly overcame theoretical obstacles in eukaryotic cells, such as RNA degradation,
11 localization, complex formation, and chromatin, by making certain “adaptations” to CRISPR –
12 using strong promoters to express the sgRNA and Cas9 protein, adding an NLS to Cas9, and
13 codon optimization. Ex. 5264, 69:8-70:2, 77:18-79:8, 84:21-85:13, 97:3-9. The contemporaneous
14 evidence summarized below shows that CVC had already made each of Zhang’s so-called
15 “adaptations” before June 26 – i.e., before Zhang ever learned of CVC’s sgRNA CRISPR-Cas9
16 system – and understood that these were routine methods to address the purported obstacles.
- 8 - Interference No. 106,115 CVC Reply 2 Broad’s theoretical CVC’s evidence of conception before June 26 Citations to record obstacle Paper 1579, 17:11-19:9; • CVC made the pMJ874 vector using the Ex. 4382, 1-7; Ex. 4349, RNA strong Pol III promoter, U6, to drive ¶¶124-128; Ex. 4603; Ex. degradation expression of CLTA6 sgRNA. 4345, ¶¶167-172; Ex. 5264, 69:8-70:2. • CVC made an expression vector using the strong Pol II promoter, CMV, to express S. Paper 1579, 18:17-19:4; Localization pyogenes Cas9 with two NLSs and a GFP Ex. 4349, ¶155; Ex. 4444; and tag. Ex. 4484; Ex. 4536; Ex. complex • CVC demonstrated nuclear localization of 4345, ¶173; Ex. 5264, formation NLS-Cas9 in human HEK293T cells. 77:18-79:8, 79:11-80:2, • CVC was codon optimizing the Cas9 gene 82:17-84:5, 84:21-85:13. sequence. • CVC designed a series of sgRNA guides to Paper 1579, 27:20-28:17; target Cas9 to the human CLTA locus. Ex. 4382, 1-5; Ex. 4469; • Chromatin CVC selected the CLTA locus because the Ex. 5040; Ex. 4350, ¶¶33- same locus had been previously shown to be 34; Ex. 4384; Ex. 5264, accessible by ZFNs, indicating to the CVC 97:3-9. inventors that chromatin was not a concern.
1 “An idea is definite and permanent when the inventor has a specific, settled idea….”
2 Burroughs, 40 F.3d at 1228. CVC’s contemplated expression vector embodiment was definite
3 and permanent, and its design did not change over time. Paper 1579, 12:9-13:2, 17:11-19:9,
4 29:8-32:10. CVC’s vector using the U6 promoter to express CLTA chimera A sgRNA (pMJ874)
5 was constructed by May 28 and is the same chimera A vector Jinek and East used when reducing
6 CVC’s invention to practice in October 2012, only a few months later. Id., 29:8-32:10; MF37-39,
7 202-208. Likewise, CVC’s S. pyogenes Cas9 vector was constructed by May 28 and uses the
8 same CMV promoter, encodes the same Cas9 amino acid sequence, and contains the same NLS
9 that Jinek and East used to reduce the invention to practice in October 2012. Id. CVC’s target
10 cell (human HEK293T cells) and vector delivery method (transfection) described by May 28
11 were also the same as what CVC used in its October actual reductions to practice. Id.
12 Broad argues on page 36, lines 18-20, that CVC’s conception by March 1 was incomplete
- 9 - Interference No. 106,115 CVC Reply 2 1 because the chimera A sgRNA in Jinek’s March 1 notebook entry is “not what results when
2 expressed from a U6 promoter.” Paper 2569, 36:18-20. That argument is irrelevant. By May
3 28—before Broad’s earliest alleged conception—CVC had already made the vector that uses the
4 U6 promoter to express the CLTA sgRNA (pMJ874). MF37-39, 202-208. Moreover, Broad’s
5 argument is scientifically wrong. Dr. Doyon explained that adding four U’s to the 3’ end of an
6 RNA transcript made from the U6 promoter was a well-known inherent aspect of using the U6
7 promoter and is not a substantive change in structure or function. Paper 2567, 22:11-24:16; Ex.
8 5013, ¶¶74-76; MF20. Even Broad’s evidence confirms this. Zhang’s internal emails and the
9 Cong 2013 publication repeatedly refer to the sgRNA sequences with no mention of additional
10 U’s on the 3' end because it was so well understood that the U’s would result from the promoter.
11 See e.g., Ex. 3751; Ex. 3537, 3; Ex. 3829; Ex. 3201, Fig. 2B; Ex. 3425, ¶24; Ex. 5013, ¶75.
12 Broad fabricates an illusion of doubt in the inventors’ minds by cataloging snippets from
13 various CVC documents. See Paper 2569, 24:22-29:11. These simply reflect that the inventors
14 understood and considered these routine implementation issues during the process and, at all
15 stages, had a plan to address them. The inventors all testified as much. Ex. 4350, ¶40; Ex. 4349,
16 ¶¶28, 36, 230; Ex. 4351, ¶26; Ex. 4348, ¶¶22-24; MF3-24. At most, the snippets reflect that the
17 inventors did not have absolute certainty that every single experiment would work. But complete
18 conception does not require certainty that the invention will work; discovery that the invention
19 actually works is part of its reduction to practice. Burroughs, 40 F.3d at 1228.
20 Moreover, “the extent of testing or other research done after the mental formulation of an
21 invention is not a reliable indicator” of a complete conception—instead, it is the “nature of [the]
22 research” that matters. Rey-Bellet, 493 F.2d at 1387; Sewall, 21 F.3d at 415 n.3 (“the existence of
23 research or experimentation does not necessarily indicate, by itself, that complete conception did
- 10 - Interference No. 106,115 CVC Reply 2 1 not exist.”). CVC’s research after June 26 required nothing more than ordinary skill and routine
2 methods, and does not undermine conception. See Rey-Bellet, 493 F.2d at 1387; Summers v.
3 Vogel, 332 F.2d 810, 815-16 (C.C.P.A. 1964).
4 Even for the more demanding standard of enablement, supposed failure by POSAs—or
5 even the inventors themselves—does not disprove invention. Trumbull v. Kirschbraun, 67 F.2
6 974, 980 (C.C.P.A. 1933); Field v. Knowles, 183 F.2d 593, 604-605 (C.C.P.A. 1950). It certainly
7 does not do so where, as here, the inventors as well as multiple POSAs actually reduced the
8 invention to practice using information the CVC inventors disclosed by June 26, “mak[ing] such
9 adjustments and corrections as would occur to those naturally skilled in the art.” Trumbull, 67
10 F.2d at 980; Field, 183 F.2d at 601. “Conception is complete when one of ordinary skill in the art
11 could construct the apparatus without unduly extensive research or experimentation.” Sewall, 21
12 F.3d at 415 (emphasis added); see also, Mergenthaler v. Scudder, 11 App. D.C. 264, 277 (D.C.
13 Cir. 1897) (“‘The true date of invention is at the point where the work of the inventor ceases and
14 the work of the mechanic begins.’”); Applegate, 332 F.2d at 573.
15 2. Reason 2: CVC had a complete conception of Count 1 before June 26, 16 evinced by CVC’s specific embodiment of delivering the sgRNA and 17 Cas9 components to zebrafish cells as a pre-formed sgRNA-Cas9 18 ribonucleoprotein complex using nuclear microinjection.
19 CVC’s alternative second specific embodiment also proves CVC’s complete conception
20 of Count 1 before June 26. Before that date, CVC had conceived of all the elements of Count 1
21 and contemplated delivering a pre-formed sgRNA-Cas9 ribonucleoprotein (RNP) complex to
22 zebrafish embryos using nuclear microinjection. Paper 1579, 14:2-18:4; Ex. 4382, 7; Ex. 4349,
23 ¶¶124-128; Ex. 4603; Ex. 5105, 16-29; MF32-39, 211-214. CVC’s pre-formed RNP complex is
24 the very “system capable of cleaving … the target DNA molecule,” required by the Count.
25 MF32-39, 211-214.
- 11 - Interference No. 106,115 CVC Reply 2 1 For example, before June 26, CVC had conceived of, and indeed already constructed, a
2 working sgRNA and Cas9 system as a pre-formed RNP complex and confirmed that it was
3 capable of cleaving at least five different eukaryotic target DNA sequences within the GFP gene
4 in vitro. See e.g., Paper 1579, 17:11-19:9; Ex. 4382, 2, 7; Ex. 4349, ¶¶124-128; Ex. 4345, ¶¶167-
5 172; MF32-39, 211-214. By April 11, CVC had identified “Example references” for introducing
6 their “RNA-guided DNA endonuclease” into eukaryotic cells for “gene targeting in zebrafish,”
7 citing multiple articles published for ZFN and TALENs that describe nuclear microinjection into
8 zebrafish embryos. See e.g., Ex. 5105, 2-3, 8; Ex. 4349, ¶¶72-74; Ex. 4350, ¶¶57-61; Ex. 4345,
9 ¶¶158-164; MF32-36. And the CVC inventors had already agreed to contact Raible to
10 microinject CVC’s sgRNA CRISPR-Cas9 system into zebrafish. See e.g., Paper 1579, 19:19-24,
11 22:2-6; Paper 2567, 32:21-33:3; Ex. 4799; Ex. 4348, ¶115; Ex. 4351, ¶¶56-58; Ex. 4294, ¶¶9-14;
12 Ex. 5013, ¶¶45-47. CVC’s specific RNP embodiment undeniably meets all the elements of
13 Count 1:
CVC’s Specific embodiment Elements of Count 1 • Zebrafish embryo with target DNA Eukaryotic cell comprising target DNA molecule • Type II CRISPR System Type II CRISPR System comprising: • S. pyogenes Cas9 - Cas9 • Chimera A sgRNA - sgRNA • System is capable of cleaving or System is capable of cleaving or editing a target editing the target DNA DNA molecule or modulating transcription of a gene encoded by the target DNA molecule
14 See Paper 1579, 7:17-19:9; Ex. 4345, ¶¶167-174; Ex. 4343, ¶¶60-70; MF32-39, 211-214.
15 CVC’s RNP specific embodiment further shows the inventors’ idea was definite and
16 permanent, as the sgRNA CRISPR-Cas9 system never changed from conception through the
17 actual reductions to practice in zebrafish on August 9. See e.g., Paper 1579, 12:9-13:2, 17:11-
18 19:9; MF45, 217. For example, Raible prepared CVC’s sgRNA and Cas9 components as a pre-
19 formed RNP complex comprising a chimera A sgRNA and S. pyogenes Cas9 – the same chimera
- 12 - Interference No. 106,115 CVC Reply 2 1 A sgRNA-Cas9 system CVC had already demonstrated to be capable of cleaving eukaryotic
2 target DNA sequence in vitro. Ex. 4294, ¶¶8, 54; Ex. 4382, 1-7; Ex. 4349, ¶¶124-128; Ex. 4603;
3 Paper 1579, 14:2-17:10; MF45, 217. Raible’s routine microinjection of the sgRNA-Cas9 RNP
4 complex to the nucleus of the zebrafish embryo was the same technique CVC had already
5 described, e.g., in their invention disclosure form. Ex. 4294, ¶¶8, 54; Ex. 5105, 2-3, 8. Indeed,
6 the PTAB has previously acknowledged that “direct injection, codon optimization, and targeting
7 of proteins and RNA to the cell nucleus” were “routine and known to be useful in achieving
8 activity of prokaryotic proteins in eukaryotic cells” Ex. 3110, 35:3-7 (emphasis added). By 2008,
9 microinjection was “widely used in the studies of transduction-challenged cells, transgenic
10 animal production, and in vitro fertilization to mechanically transfer DNAs, RNA interferences,
11 sperms, proteins, peptides, and drugs. The advantages of microinjection include the precision of
12 delivery dosage and timing, high efficiency of transduction as well as low cytotoxicity.” Ex.
13 4053, Abstract.
14 Chylinski, Charpentier and Raible all “expected” that the experiments would result in
15 success. Ex. 4348, ¶¶11, 25, 115; Ex. 4351, ¶¶23, 26, 56; Ex. 4294, ¶¶10, 13, 74. Charpentier in
16 particular was “convinced” the system would work, and it did, as Raible observed and
17 documented an eyeless zebrafish by August 9. Ex. 4351, ¶60; Ex. 4294, ¶14. Paper 1579, 22:1-
18 27:15; Ex. 4294, ¶¶50-58, 70-71; Ex. 4338, 79-80; Ex. 4343, ¶¶59-72; MF45, 215-221.
19 Tellingly, Broad’s opposition barely addresses CVC’s pre-formed RNP complex
20 microinjection evidence. Paper 2569, 46:13-14. This is undoubtedly because microinjecting a
21 pre-formed RNP complex directly to the nucleus completely obviates Broad’s theoretical
22 barriers. See Paper 871, 17:3-12 (Judge Katz: “But is there something different about
23 microinjection that sort of negates any of the issues that we talked about in the prior opinion”
- 13 - Interference No. 106,115 CVC Reply 2 1 and “which overcomes any of those problems that were known in the art about using this kind of
2 a complex system in a eukaryotic cell?”); see also, Ex. 4036, ¶¶136-137; Ex. 4343, ¶¶93-106;
3 Ex. 4345, ¶87. As Dr. Moens explained, when microinjecting a pre-formed RNP complex:
4 There is no need for codon optimization, RNA or protein expression, 5 concomitant folding, and co-localization of RNA and protein, because the 6 complex is already formed before injection. The use of pre-formed 7 complexes also minimizes RNA and protein degradation, because the 8 complex is protected from cellular factors, as it is in bacterial host cells.
9 Ex. 4343, ¶95 (emphasis added); see also, Ex. 4345, ¶87. Microinjection of a pre-formed RNP
10 complex into zebrafish also negates any potential impact of chromatin because, e.g., the
11 zebrafish cells are rapidly dividing. MF23-24. As Doyon explains, “cells have time periods when
12 the DNA is unwrapped from chromatin, and this happens more often during cell division.
13 Opening of access to chromatin happens more frequently when cells are dividing frequently,
14 such as zebrafish embryos.” Ex. 4345, ¶113 (emphasis added); see also, Ex. 4343, ¶103
15 (“chromatinization changes dynamically in rapidly dividing cells and across developmental
16 time.”). Further, Dr. Zamore testified, “DNA is constantly alternating between states where it's
17 wrapped around the nucleosome and states where it's not completely wrapped around the
18 nucleosome,” and “nucleosome[s] slide” along the DNA “leaving areas open for processes like
19 gene expression.” Ex. 5349, 196:2-198:2. Breaker admitted he did not consider publications
20 disclosing nucleosome sliding and the open and closed dynamics of chromatin. Ex. 5347,
21 213:22-223:11, 226:20-228:18; Ex. 5323, Ex. 4570; Ex. 5325.
22 The inventors knew that microinjection rendered Broad’s “obstacles” irrelevant. For
23 example, in a June email to Charpentier, Chylinski stated that injecting RNPs into zebrafish
24 meant there was “no need for in vivo expression of neither RNA nor protein, codon bias
25 optimization and so on.” Ex. 4796; Ex. 4348, ¶116; Ex. 4351, ¶¶58-61. Charpentier also
26 understood that RNA degradation was not an issue due to stability of the RNP complex and that
- 14 - Interference No. 106,115 CVC Reply 2 1 chromatin was not an issue in rapidly dividing cells. Ex. 4351, ¶¶26, 32; MF3-4, 23-24, 218-219.
2 Of the 22 quotes Broad highlights, only two involve microinjecting a pre-formed RNP
3 into a zebrafish embryo. See Paper 2569, 27 (citing Exs. 4799 and 4916). Neither quote indicates
4 the inventors encountered any perplexing difficulties or unduly extensive experimentation. In
5 Exhibit 4799, Raible merely states that “off target sites” and “toxicity” are relevant to “human
6 applications” (i.e., clinical therapeutics). Id. (emphasis added). Broad crops out the remainder of
7 Raible’s sentence, which says these are “less of an issue in zebrafish or medaka.” Id. (emphasis
8 added). Exhibit 4916 merely acknowledges that CVC “observed” toxicity in some fish, far from
9 a “perplexing intricate difficulty” undermining conception. Raible stated that toxicity from the E.
10 coli-produced protein preparation was not unexpected and that there were “conventional way[s]
11 of proceeding” including using “lower concentrations” or a “fresh preparation with more highly
12 concentrated Cas9” – which was exactly what he did. Ex. 4294, ¶¶39-40.
13 Broad’s witness, Mourrain, attempts to brush aside CVC’s RNP microinjection approach,
14 opining with no support that “no one was injecting gene editing nuclease proteins in fish at the
15 time.” Ex. 3447, ¶115. But Mourrain is contradicted by evidence he did not consider: that
16 artisans were routinely injecting gene-editing proteins such as I-SceI meganucleases into
17 zebrafish and medakafish embryos. See Ex. 4328; Ex. 4622; Ex. 5344; Ex. 4343, ¶87; MF222-
18 224; Ex. 5348, 182:20-183:4; Ex. 4053, 507 (“direct microinjection of proteins is most powerful
19 to deliver cytosolic and nuclear proteins.”); MF222-224.
20 Before June 26, the CVC inventors conceived of the sgRNA CRISPR-Cas9 system of
21 Count 1, had made a pre-formed RNP complex containing a chimera A sgRNA and S. pyogenes
22 Cas9 and demonstrated its ability to cleave DNA in vitro, and had a settled approach for
23 microinjecting the sgRNA-Cas9 system as a pre-formed RNP complex into zebrafish embryos.
- 15 - Interference No. 106,115 CVC Reply 2 1 Paper 1579, 7:17-19:9; MF32-39, 211-214. Given the “stunning efficiency” in vitro, Raible
2 predicted that he would have results within 1-2 months. MF215. Within six weeks, as predicted,
3 Raible successfully reduced the invention to practice. Paper 1579, 22:1-27:15; MF45-47, 215-
4 217. East repeated that success by November 1, 2012, by introducing a pre-formed RNP into
5 human cells with a commercially available transfection reagent. Paper 1579, 34:1-13; Ex. 4353,
6 ¶102; Ex. 4366, 38; Ex. 4349, ¶252; MF50. Thus, CVC’s CRISPR “system” was not only
7 “capable of cleaving or editing,” as Count 1 recites, but it indeed cleaved and edited the target
8 DNA. Id.
9 3. Multiple lab groups used CVC’s system with only ordinary skill and 10 routine techniques, further confirming CVC’s conception.
11 The record now includes testimony from multiple scientists about the state of the art in
12 2012. See Exs. 4343 (Moens), 4345 (Doyon), 4348 (Chylinski), 4349 (Jinek), 4350 (Doudna),
13 4351 (Charpentier), 4354 (Sternberg), 5013 (Doyon), 5014 (Zamore), 5016 (Barrangou), 5018
14 (Sontheimer), and 5265 (Marraffini). See Paper 1579, 36:5-42:4; Paper 2567, 10:1-15:20; see
15 also, Exs. 4036, 4189, and 4193. For example, Sontheimer stated, “scientists would be able to
16 quickly apply [CVC’s system] for genome editing in eukaryotic cells, because the process for
17 doing so was straightforward and required only routine genome-editing techniques.” Barrangou
18 and Marraffini agreed that it would be “straightforward.” Ex. 5016, ¶17; Ex. 5265, 31:8-19. And
19 Barrangou stated, “[t]he question at the time was not whether [CVC’s] CRISPR-Cas9 system
20 would work in eukaryotes—my colleagues and I all expected it would work and someone would
21 get that first paper—the real question was whether it could outcompete the existing genome-
22 editing technologies, such as TALENs and ZFNs.” Ex. 5016, ¶17; see also, id., ¶¶18-20; Ex.
23 5018, ¶21; Ex. 4294, ¶74. But, outcompeting existing technologies or any particular efficiency
24 are not part of the Count.
- 16 - Interference No. 106,115 CVC Reply 2 1 The state of the art in 2012 was confirmed by the fact that numerous lab groups rapidly
2 applied CVC’s sgRNA-Cas9 system in eukaryotic cells using ordinary skill and the same
3 reagents and methods they had previously published for use with ZFNs or TALENs. Paper 1579,
4 35:10-36:4; Ex. 4076, Fig. 1A; Ex. 4079, Fig. 1B; Ex. 3623, Fig. 1A; Ex. 4084, Fig. 1C; Ex.
5 4233, Fig. 1; Ex. 5020; Ex. 4075; Ex. 4080; Exs. 4082-4087; Ex. 4089; Ex. 4090; Ex. 4093; Ex.
6 4106; see also, Ex. 5350, 54:3-58:1; MF232-237. This is also discussed in CVC’s Opposition 5.
7 See Paper 2567, 19:14-20:19. As the Federal Circuit noted in Regents of Univ. Calif. v. Broad
8 Inst., Inc., evidence of near simultaneous ARTPs can be used to show “the level of skill in the
9 art” and “constitutes objective evidence that persons of ordinary skill in the art understood the
10 problem and a solution to that problem.” 903 F.3d 1286, 1295 (Fed. Cir. 2018).
11 CVC’s complete conception is further bolstered by the new evidence in the record
12 regarding Zhang’s purported reductions to practice. Once he learned of CVC’s sgRNA system
13 for gene editing in eukaryotes, he quickly used it, along with published reagents and protocols,
14 exercising just ordinary skill. Paper 2567, 20:20-30:15; Ex. 5013, ¶¶64-104; MF232-233. Zhang
15 made no contributions to Count 1. As Marraffini testified, once he told Zhang about CVC’s
16 sgRNA CRISPR-Cas9 system for genome editing in eukaryotic cells, the next steps of applying
17 CVC’s system were “pretty straightforward.” Ex. 5265, 31:8-19. The PTAB may not ignore the
18 copious objective evidence showing that CVC and the rest of the field understood the alleged
19 “problem” and knew how to solve it. Regents at 1295. That neither CVC nor others encountered
20 “perplexing intricate difficulties arising every step of the way” (Rey-Bellet, 493 F.2d at 1386) or
21 “unduly extensive research or experimentation” (Sewall, 21 F.3d at 415) when applying CVC’s
22 sgRNA CRISPR-Cas9 system in eukaryotic cells confirms the completeness of CVC’s
23 conception.
- 17 - Interference No. 106,115 CVC Reply 2 1 It is irrelevant whether, as Broad argues on page 33, lines 23-24, some experiments
2 performed by CVC’s colleagues in other eukaryotic cell types (e.g., nematodes) had not yet
3 succeeded, whether there was doubt that the positive results in medakafish (“less green”
4 embryos) “might be heterozygotes, might be unspecific,” or whether collaborations in yeast,
5 mice, or plants had not yet started. Paper 2569, 33:23-24; id., 12:16-14:11. The question is not
6 whether some colleagues’ experiments succeeded or failed, but rather whether the inventors’
7 conception of Count 1 was complete. The law does not require a 100% success rate for attempts
8 at reduction to practice. See Trumbull at 980. In fact, the law of conception does not require any
9 success rate for reductions to practice. See Rey-Bellet, 493 F.2d at 1389. There is no evidence
10 that any of CVC’s colleagues engaged in unduly extensive research or experimentation. See
11 Sewall, 21 F.3d at 415. Broad also improperly infers activities not in the record, and which CVC
12 does not rely upon for conception or reduction to practice. See Agilent Tech. Inc. v. Dionex
13 Softron GMBH, Interference 106,087, Paper 267 (P.T.A.B., Feb. 25. 2021) (non-precedential)
14 (finding “evidence that may have been, but was not, relied upon” by junior party irrelevant to
15 “whether [junior party] has met its burden of proof.”) Rather, CVC cites correspondence with its
16 colleagues as evidence of CVC’s reasonable diligence, which, as discussed below, Broad barely
17 challenged.
18 4. CVC’s reasonably continuous diligence during the critical period 19 through CVC’s P3 filing went unchallenged by Broad
20 CVC proved its inventors exercised reasonably continuous diligence from before Broad’s
21 alleged June 26 conception date through CVC’s actual reductions to practice in August, October,
22 and November 2012, and through CVC’s constructive reduction to practice in January 2013. See
23 Paper 1579, 3:11-12, 32:11-33:6, 49:5-21, Appx 3, 3-85. Broad’s opposition only cites pages 44-
24 45 of CVC’s priority motion, which discusses CVC’s diligence activities in March and April
- 18 - Interference No. 106,115 CVC Reply 2 1 2012, outside of the critical period. Id. Broad made no attempt to challenge the fact that CVC
2 was acting diligently from just before Broad’s earliest asserted conception date of June 26.
3 MF37-39, 252-254. Broad provides no expert testimony and cites no evidence to challenge
4 CVC’s diligence during the critical period, instead providing only conclusory attorney argument.
5 Paper 2569, 39:17-24.
6 CVC had a complete conception of Count 1 before June 26, coupled with uncontested
7 diligence from at least just before June 26 through CVC’s P3 filing, which the PTAB has already
8 deemed a constructive reduction to practice of Count 1. Ex. 3004, ¶¶408-450; Paper 877, 106:1-
9 14. Thus, whether CVC has proven its ARTPs in the Fall of 2012 does not matter for priority. At
10 a minimum, ARTP efforts constitute diligence. See Rey-Bellet, 493 F.2d at 1389 (even repeated
11 attempts and failures at reduction to practice count as diligence).
12 5. Broad’s attempt to rewrite conception law fails: conception does not 13 require a reasonable expectation of success.
14 Broad’s attempt to import a “reasonable expectation of success” into conception law
15 lacks any credible basis. Binding precedent in Applegate, 332 F.2d at 573, Burroughs, 40 F.3d at
16 1230-1231 and, more recently, Dana-Farber Cancer Inst., Inc. v. Ono Pharm. Co., 964 F.3d
17 1365, 1372 (Fed. Cir. 2020), have made it clear that conception does not require a reasonable
18 expectation of success. See also, Paper 2567, 37:14-39:14; Paper 1579, 42:5-43:21. Rather, it
19 requires a specific, settled idea of the invention, which the CVC inventors possessed as evinced
20 by their documentation of the specific embodiments discussed above. Coleman, 754 F.2d at 359.
21 Broad mischaracterizes Hitzeman v. Rutter as requiring both the inventors and a POSA to
22 have a reasonable expectation of success for the inventors to have had a complete conception.
23 Paper 2569, 19:1-4. Broad’s theory would turn conception on its head, failing to recognize that
24 inventors are persons of extraordinary creativity, in contrast to POSAs, who merely have
- 19 - Interference No. 106,115 CVC Reply 2 1 ordinary creativity. Instead of assessing whether a complete conception has formed in the mind
2 of the inventor, Broad would require a complete conception in the mind of a POSA. This is not
3 the law, nor does it even make sense. The Federal Circuit explicitly rejected Broad’s theory:
4 “[f]or conception, we look not to whether one skilled in the art could have thought of the
5 invention, but whether the … inventors actually had in their minds the required definite and
6 permanent idea.” Burroughs, 40 F.3d at 1232. Broad’s theory is found nowhere in Hitzeman.
7 In Hitzeman, the count required a DNA expression vector capable of replicating in yeast
8 such that it (i) expresses hepatitis B virus S protein in yeast and (ii) the yeast assembles the S
9 protein into virus particles with a sedimentation rate identical to authentic HBsAg 22nm
10 particles. Hitzeman, 243 F.3d at 1352. Hitzeman held that the purported inventors had not yet
11 conceived the invention because they had no “reasonable expectation that they would produce
12 the claimed invention” in the first place– particularly the recited 22 nm particles. Id. at 1358. See
13 Burroughs, 40 F.3d at 1229 (conception requires only (1) inventors have in their minds “the
14 specific structure of the compound” and (2) “an operative method of making it”). CVC’s
15 conception satisfies Hitzeman because before June 26, CVC had already produced the sgRNA
16 CRISPR-Cas9 system of Count 1 as a pre-formed RNP complex outside of a cell and had a way
17 of delivering the complex into a eukaryotic cell via microinjection. Paper 1579, 14:2-17:10; Ex.
18 4382, 7; Ex. 4349, ¶¶124-128; Ex. 4603; Ex. 5105, 16-29; MF214. A pre-formed RNP complex
19 obviates any issues related to expressing or assembling the sgRNA or Cas9 in the eukaryotic cell,
20 because the complex is already formed outside the eukaryotic cell prior to delivery. Ex. 4343,
21 ¶95; MF214. And there is no dispute that CVC’s pre-formed sgRNA-Cas9 RNP complex,
22 demonstrated to be “capable of” cleaving eukaryotic DNA, could be delivered into a eukaryotic
23 cell using routine techniques and ordinary skill, such as microinjection into a zebrafish embryo
- 20 - Interference No. 106,115 CVC Reply 2 1 as Raible did. MF16, 217-219. In contrast to the inventors in Hitzeman, the CVC inventors
2 expected to easily produce the invention of Count 1. The same holds true for CVC’s expression
3 vector embodiment. Before June 26, the inventors had made specific vectors with specific
4 promoters, specific CLTA sgRNA, and specific Cas9, and expected to produce the invention
5 using those same elements, and that is all that Hitzeman requires.
6 Broad also argues that the court’s disposition of priority on the sixth patent in Burroughs
7 supports its reasonable expectation of success theory. Paper 2569, 17:24-18:22. But Broad
8 overlooks critical facts in Burroughs. The sixth patent in Burroughs claimed a “method of
9 increasing the number of T-lymphocytes in a human infected with the [HIV] virus comprising
10 administering to said human an effective amount of AZT.” Burroughs, 40 F.3d at 1231. In
11 contrast to the inventions in the first through fifth patents, there was a factual dispute as to
12 whether the inventors even contemplated that AZT would cause the increased T-cell numbers
13 claimed in the sixth patent. Id. As the court noted, “the record [was] devoid of any statement that
14 the inventors thought AZT could raise a patient’s T-cell levels.” Id. (emphasis added). The court
15 mentioned reasonable expectation of success only because such an expectation might imply that
16 the inventors themselves likely contemplated that claimed element. This is in marked contrast to
17 the CVC inventors, who clearly documented before June 26 their idea for a sgRNA-Cas9 system
18 that was capable of cleaving a target DNA in a eukaryotic cell. Paper 1579, 7:17-19:9. And, the
19 CVC inventors expected it to work based on their in vitro successes, and later demonstrated the
20 use of this same system in their actual reductions to practice. Id., 22:1-35:9. There is no dispute
21 that eukaryotic cells comprising CVC’s sgRNA-CRISPR Cas9 system "capable of" cleaving
22 DNA could be produced, and no evidence to support Broad’s argument that POSAs and the
23 inventors would expect a system with “stunning efficiency” to lose all of its activity upon
- 21 - Interference No. 106,115 CVC Reply 2 1 microinjection.
2 For example, in emails to the editors of Science and Nature, Doudna and Charpentier
3 each stated that the inventors expected this gene-editing capability, stating “[w]e foresee
4 considerable exploitation of this system for targeted genome editing in cells of the three
5 kingdoms of life for biotechnological biomedical and gene-therapeutic purposes.” Ex. 4598 and
6 Ex. 4583 (emphasis added). Similarly, Charpentier stated in a June 28 draft email to Raible that
7 the CVC inventors were “indeed convinced” that the system would work in a zebrafish model,
8 and were eager to test it. Ex. 4351, ¶60; Ex. 4807; Ex. 4294, ¶14; see also, Ex. 4469 (Doudna
9 email to Jinek stating, “a lot of the pieces are already in place … we could compare efficiencies
10 [between Cas9 and ZFN]”). Thus, the facts here are nothing like the sixth patent in Burroughs;
11 rather, the facts here mirror Burroughs patents 1-5, where the court said:
12 The question is not whether Burroughs Wellcome reasonably believed that the 13 inventions would work for their intended purpose…, but whether the inventors had 14 formed the idea of their use for that purpose in sufficiently final form that only the 15 exercise of ordinary skill remained to reduce it to practice.
16 Burroughs, 40 F.3d at 1231 (emphasis added). Here, the inventors’ idea of Count 1 was “in
17 sufficiently final form,” as evinced by their documented specific embodiments discussed above.
18 Moreover, evidence in the record from Raible, Sontheimer, Barrangou, and Marraffini
19 show that POSAs expected success with CVC’s sgRNA CRISPR-Cas9 system specifically once
20 they learned of it. Ex. 5016, ¶¶17-20; Ex. 2018, ¶21; Ex. 4294, ¶74; Ex. 5265, 31:8-19; MF268,
21 271-275. Broad’s reasonable expectation of success theory is premised on supposed issues with
22 non-analogous systems: group II introns, ribozymes, and riboswitches, all of which Broad admits
23 are RNA enzymes. Paper 2569, 22:27-24:21, 26:1-29:11. As Zamore and Doyon explain, such
24 systems differ from CRISPR-Cas9 because the RNA, rather than protein, is the catalytic
25 component that cuts the DNA, and that RNA is vastly larger and more complex than the simple
- 22 - Interference No. 106,115 CVC Reply 2 1 stem-loop structure of sgRNA. Ex. 4345, ¶114; Ex. 5349, 193:3-194:18; MF238-239. For
2 example, Breaker testified that the catalytic RNA of group II introns is 800-1000 nucleotides.
3 Ex. 5347, 177:22-178:9. Additionally, the protein component of group II introns is required to
4 help the RNA fold so that it can cut DNA. Ex. 5349, 193:3-194:18; MF238-239. In contrast,
5 protein folding occurs much more readily; therefore, systems in which the catalytic component is
6 a protein, e.g., ZFN, TALEN, and Cas9, are more straightforward to use. Id. Breaker admitted he
7 failed to consider that group II introns are larger and more complex than sgRNA, or that they use
8 RNA as the catalytic component unlike CRISPR-Cas9, ZFNs, TALENs, and meganucleases. Ex.
9 5347, 169:12-171:16, 172:19-177:11, 177:22-183:18; Ex. 5318; MF238-239. Moreover,
10 Breaker’s testimony is unreliable. He failed to consider relevant art and evidence related to using
11 prokaryotic RNPs in eukaryotic cells and siRNA/shRNA successes that contradict his opinions in
12 his declaration. Ex. 5347, 158:5-165:7, 192:5-198:22, 233:9-238:7, 242:17-245:5; Ex. 5316; Ex.
13 5317; Exs. 5319-5322; Ex. 4097; Ex. 5328; Ex. 5321; Ex. 5327; MF242-246. Thus, Breaker’s
14 views and Broad’s arguments lack scientific merit.
15 On the contrary, the evidence shows that the field at the time considered protein
16 nucleases like ZFNs, TALENs, and meganucleases to be the relevant art for CRISPR-Cas9:
Evidence Description Ex. 3521, 3 Zhang’s February 2011 invention disclosure memorandum stating “related art include anything pertaining to Zinc Finger or TAL Effector-based genome targeting.” Ex. 3424, ¶50 Zhang’s 2020 interference declaration stating “[ZFNs] and TALENs ... were the predominant methods used for eukaryotic genome editing as of 2011” Ex. 4381, 63 Jinek March 1 lab notebook comparing CRISPR-Cas9 with ZFNs and TALENs Ex. 4598 and Doudna’s and Charpentier’s May 27 emails to Science and Nature stating that Ex. 4583 “RNA-programmed Cas9 offers significant advantages over zinc-finger nucleases or TALEN proteins.” Ex. 5255, 2 Jinek 2012 reviewer comments stating that CRISPR-Cas9 is “desirable for a number of genome editing applications that is impossible to obtain with TALENs or ZFNs.”
- 23 - Interference No. 106,115 CVC Reply 2 Evidence Description Ex. 5324, Science December 2012 publication stating that CRISPR-Cas9 “may one day 1527 challenge zinc finger nucleases and TALENs as the core genome engineering technology.”
1 See also, Ex. 3001, ¶2; Ex. 3201, 819; Ex. 3233, 5-6; Ex. 3832, 1; Ex. 3623, 825; Ex. 4233, 229;
2 Ex. 4085, 1; Ex. 4075, 1179; Ex. 4076, 230; Ex. 3655, 2; Ex. 4257, 838; Ex. 4343, ¶¶12, 85, 93;
3 Ex. 4345, ¶¶64, 149; Ex. 5016. ¶¶17-20; Ex. 5018, ¶21; Ex. 5105, 3, 19; Ex. 5314, 1; Ex. 5347,
4 223:12-226:17. And ZFNs, TALENs, and meganucleases all had been shown to work in
5 eukaryotic cells, notwithstanding the prokaryotic origin of the FokI nuclease included within
6 ZFNs and TALENs. MF260-262.
7 Broad cannot rely on unsworn, improperly submitted statements, filed as Exs. 3435-3443,
8 3446, 3449-3452, from scientists who have not offered testimony in this proceeding. These
9 statements should be afforded no weight if they are admitted into evidence. First, the statements
10 are not sworn testimony. See Standing Order, ¶157.2. Second, Broad refused to make any of the
11 12 scientists available for cross-examination. Paper 2706, 2:2-6. Third, the PTAB refused to
12 order Broad to make the scientists available. Fourth, the PTAB denied CVC’s request to counter
13 the statements with new declarations. Paper 2708, 2. Broad’s 14 statements were submitted in
14 USPTO ex parte examination or in EP oppositions of uninvolved applications, where the
15 scientists were never cross-examined. Giving any weight to these statements would therefore be
16 highly prejudicial to CVC. Fifth, Broad in presenting these statements, failed to inform the
17 PTAB or even its own expert that there were counter-statements from other scientists. See Ex.
18 5347, 140:7-148:9. In other words, Breaker’s “selection” of the witness statements was not
19 objective; it was one-sided and deliberately spoon-fed to him by Broad’s counsel. Id. In contrast,
20 CVC provides sworn testimony from numerous experts and fact witnesses explaining the state of
21 the art as of 2012. See Exs. 4343, 4345, 4348, 4349, 4350, 4351, 4354, 5013, 5014, 5016, 5018,
- 24 - Interference No. 106,115 CVC Reply 2 1 and 5265; see also, Paper 1579, 36:5-42:4; Paper 2567, 10:1-15:20.
2 6. CVC proved multiple actual reductions to practice of Count 1.
3 CVC achieved successful ARTP in zebrafish. By August 9, as expected, Raible
4 detected the specific chokh phenotype, which is diagnostic of a fish having a mutation in its rx3
5 gene, and Raible conveyed that finding to Chylinski. Paper 1579, 21:1-34:9; MF45-47, 240.
6 CVC showed that this was an ARTP, meeting all the elements of Count 1, and that the inventors
7 appreciated the invention worked for its intended purpose. See e.g., Paper 1579, 23:8-26:24; Ex.
8 4294, ¶¶55-58; Ex. 4338, 79-80; Exs. 4912-4915; Ex. 4916, 10; Ex. 4343, ¶¶45-58; Ex. 4351,
9 ¶70; Ex. 4348, ¶127; MF45-47, 240. Indeed, Chylinski testified that he “recall[ed] having the
10 conversation with Dr. Raible and him describing [the fish embryo] – him describing the
11 phenotypes he observed” and that he “remember[ed] seeing pictures” of fish embryos. Ex. 6202,
12 101:14-102:3. And Raible testified that “I also communicated with Dr. Chylinski [about the
13 result].” Ex. 6211, 146:1-4. Under a rule of reason, the CVC inventors’ testimony is corroborated
14 by contemporaneous documentary evidence, e.g., Raible’s lab notebook and photographs of the
15 mutant fish, and testimony from non-inventor Raible. See e.g., Ex. 4294, ¶¶55-58; Ex. 4338, 79-
16 80; Exs. 4912-4915; Ex. 4916, 10; MF45-46. Raible wanted to publish data comparing
17 efficiencies to ZFNs or TALENs, but “believed that other labs with more resources” were likely
18 to generate those data first. Ex. 4294, ¶74-75. MF276.
19 Mourrain fails to undermine CVC’s successful ARTP. The loss of the pigmented retina
20 and the reduced eye lens Raible observed are sufficient to diagnose an rx3 mutant fish, because
21 they are the hallmarks of the mutant. Ex. 4294, ¶¶55-56; Ex. 4343, ¶¶3-40, 45-48. Ex. 4343, ¶52;
22 MF46. Indeed, these two unique characteristics were used to identify and propagate the mutant
23 zebrafish line before scientists even determined the gene where the mutation was located, and
24 thus before scientists could even perform any molecular assays. Ex. 4306. In other words,
- 25 - Interference No. 106,115 CVC Reply 2 1 scientists have long used the same criteria Raible used to identify fish as mutants, without
2 requiring molecular confirmation. Mourrain admitted that the remaining characteristics were all
3 identified after publication of the reference identifying the mutated gene, and thus could not have
4 been required for identification of an rx3 mutant. Ex. 4306; Ex. 4343, ¶¶32-39; MF247-250. And
5 as Mourrain acknowledged, a 2019 article reported CRISPR-Cas9 cleavage of the rx3 gene based
6 solely on the visual phenotype, without molecular assays. Ex. 5348, 179:6-21 (they “did a good
7 job documenting the phenotypes.”); Ex 5342. Mourrain admittedly failed to consider prior art
8 publications in which phenotypes alone were commonly used to conclude successful
9 mutagenesis in zebrafish, including the rx3 mutant phenotype. Ex. 5348, 173:3-174:22; 178:6-
10 179:21 (admitting he failed to consider Ex. 5340 or Ex. 5342).
11 While Mourrain noted that Raible did not obtain molecular confirmation of the rx3
12 mutant, Raible’s molecular analysis was simply inconclusive because it was not sensitive enough
13 to detect all possible mutations. As Mourrain admitted, Raible’s 2% agarose gel would not have
14 “distinguish[ed] [mutations] of eight base pairs or fewer,” even though a single base mutation
15 had been shown to disrupt the rx3 gene. Ex. 5348, 120:19-123:7. And while Mourrain criticized
16 Raible’s fish as showing some signs of developmental delay, he admitted that a mutant could
17 show some toxicity-related developmental delay and successful targeted mutagenesis at the same
18 time. Ex. 5348, 40:2-11, 185:4-186:16; Ex. 5345; MF251. Moreover, Moens, who has personally
19 injected tens of thousands of zebrafish embryos with gene targeting reagents, testified that in
20 over 25 years of zebrafish injections, she has “never seen a fish that looked like a rx3/chokh
21 mutant result from nonspecific mutation or toxicity.” Ex. 4343, ¶54. And Mourrain admitted that
22 he was unaware of any literature disclosing a chokh phenotype occurring spontaneously or from
23 nonspecific toxicity or microinjection injury. Ex. 5348, 49:3-52:18, 61:20-64:21.
- 26 - Interference No. 106,115 CVC Reply 2 1 CVC achieved successful ARTPs in mammalian cells in Fall 2012. CVC also achieved
2 multiple successful ARTPs of Count 1 in mammalian cells in October and November 2012,
3 when non-inventor corroborator East obtained CRISPR-Cas9-mediated DNA cleavage products
4 in human HEK293T cells, and Doudna and Jinek appreciated those results. Paper 1579, 27:17-
5 35:9; Ex. 4345, ¶¶87-101, 201-218; Ex. 4349, ¶¶245-251; Ex. 4353, ¶¶87-142; Ex. 4366, 31-53;
6 48-52, 241. East’s October 31 and November 20 ARTPs are described in Example 2 of CVC’s
7 P3 application, which is a constructive reduction to practice of Count 1. Ex. 3004, ¶¶416-423,
8 Fig. 36E; Paper 1579, 32:11-33:6; Paper 877, 106:11-14; MF49, 209-210.
9 Breaker attempted to cast doubt on CVC’s experiments through his conjecture about
10 CVC’s cell lysis protocol, but his criticisms lack merit. CVC’s same lysis protocol was reviewed
11 and accepted by multiple peer-reviewers of the Jinek 2013 publication, which the reviewers
12 described as an “excellent paper,” and has since been cited numerous times by others (including
13 Zhang) as demonstrating CRISPR-Cas9 cleavage in human cells. See e.g., Ex. 5329; Ex. 5335,
14 348; Ex. 5336, 404; Ex. 5337, 139; Ex. 5347, 251:21-257:19; MF255. And, Breaker’s “gentle
15 lysis buffer” theory that CVC’s CRISPR-Cas9-mediated DNA cleavage occurred in the cell
16 lysate rather than in the cells is completely debunked by the record evidence clearly showing the
17 lysates were maintained at 4°C or kept frozen. See e.g., Ex. 4382, 26-27 (cells were lysed “at
18 4°C” and separated using a “refrigerated centrifuge”); Ex. 4366, 26 (cells lysed by “shak[ing] at
19 4°”); EX. 4366, 66 (lysate “place[d] @ -20 [°C]”). S. pyogenes Cas9 is only active between 20
20 and 44°C and cannot cleave DNA at the cold temperatures Jinek and East kept the lysates,
21 meaning that CVC’s DNA cleavage occurred in the cells before the cells were lysed. See Ex.
22 5334, 4, 6; Ex. 5333, 23103; MF256-257. Breaker admitted that he did not know Cas9’s
23 permissible temperature range and did not consider references that explicitly disclose the
- 27 - Interference No. 106,115 CVC Reply 2 1 temperature range for Cas9 activity (Exs. 5333 and 5334) when forming his opinions Ex. 5347,
2 264:7-269:4; Ex. 5333; Ex. 5334. Moreover, after DNA cleavage, the NHEJ process (required to
3 detect indels in a Surveyor assay) requires the addition of ATP. Ex. 6335, 14067; Ex. 6336, 4;
4 Ex. 6343, 850; Ex. 6347, 1; MF258-259. Breaker admitted that neither Jinek nor East added any
5 ATP to the lysates, again proving that NHEJ could not have occurred in the lysates. Ex. 5347,
6 276:7-16; Ex. 4382, 26-27; Ex. 4366, 26. Breaker also admitted he did not consider the fact that
7 Jinek sonicated the genomic DNA in the lysates, shearing the DNA into countless fragments
8 with non-homologous ends that would “swamp” any NHEJ system theoretically active in the
9 lysate. Ex. 5347, 276:21-279:7; Ex. 5338, Ex. 5339. Breaker’s conjecture should not be credited,
10 and his testimony is entitled to little or no weight under 37 C.F.R. § 41.158, because he
11 repeatedly fails to provide any underlying evidence to support his speculations. See e.g., Ex.
12 3448, ¶¶269-271, 278-280, 301-302. In sum, more than a preponderance of the evidence
13 establishes that CVC conceived the invention before Broad and exercised reasonably continuous
14 diligence during the critical period. Broad’s priority motion throws out a fallback argument
15 (Paper 2118, 46:18-22), hoping it would stick. But it, too, lacks any credible foundation in law
16 and should be rejected. See Paper 2567, 53:21-59:19.
17 B. Reason 3: CVC prevails under derivation law.
18 As shown in CVC’s Opposition 5, Zhang derived the invention of Count 1 from CVC,
19 compelling judgment in CVC’s favor. See Paper 2567, 30:19-37:13. CVC’s priority motion
20 could not have raised derivation, because Broad had hidden the fact that Marraffini provided
21 Zhang CVC’s sgRNA CRISPR-Cas9 system, including instructions to use it in eukaryotic cells.
22 After CVC’s cross-examination of Marraffini, CVC promptly notified the PTAB of CVC’s intent
23 to raise derivation in its Opposition 5. See Paper 2474, 4; Paper 2567. And the PTAB instructed
24 CVC not to file a separate motion. Id. Regardless, the facts amply show derivation.
- 28 - Interference No. 106,115 CVC Reply 2 1 The CVC inventors had a complete, corroborated conception of Count 1 before June 26,
2 2012. See Sections A.1-A.3, supra and Paper 1579, 7:17-19:9; see also, Paper 2567, 30:17-
3 33:11. And, CVC communicated the entirety of Count 1, including the eukaryotic element, to
4 Zhang via Zhang’s collaborator, Marraffini. Paper 2567, 33:12-34:19; MF225-231, 263-270.
5 Marraffini—an independent third party witness whom Broad elected not to question at
6 deposition—testified that he first learned about CVC’s sgRNA-Cas9 genome-editing system and
7 its projected use for genome editing in eukaryotic cells by serving as a reviewer of the
8 confidential Jinek 2012 draft manuscript. Ex. 5265, 23:19-24:9, 24:18-25:3, 27:4-16, 29:20-30:3,
9 64:9-18, 66:7-67:7, 68:13-21; see also, Ex. 5013, ¶¶48-55; Ex. 5254, 2, 12; Ex. 4768, 27; Ex.
10 5016, ¶¶11-15; Ex. 5018, ¶¶10-18; Ex. 5266, 187:21-188:13; Ex. 5255, 2; Ex. 3713, 27-29;
11 Paper 2567, 13:5-18:17, 33:12-37:13; MF270. Promptly following the June 21 CRISPR
12 Research Conference where CVC disclosed its invention, Marraffini relayed CVC’s invention to
13 Zhang on June 26. MF225-231, 263-269. Before then, Zhang admittedly had no concept of the
14 Count’s sgRNA CRISPR-Cas9 system, which is unsurprising since Zhang did not yet know that
15 tracrRNA was a necessary component of the DNA-cleavage complex—a concept that had been
16 unknown even to the CRISPR community before CVC disclosed it at the June 21 conference.
17 MF263-267. (Zhang’s assertions to the contrary are unsupported by the contemporaneous
18 evidence and are scientifically unfounded. Ex. 5014, ¶¶56-76; see also, Ex. 5265, 23:6-18,
19 24:17-25:3, 29:20-30:3; Ex. 5018, ¶16; Ex. 5016, ¶14.) On June 26, Marraffini instructed Zhang
20 to change course from his prior RNA processing experiments and use “pre-processed RNAs”
21 instead as CVC did. Marraffini testified unequivocally that he communicated to Zhang the
22 “single-guide RNA system described in Jinek 2012” and that “it would be an important tool for
23 genome editing in eukaryotes, specifically.” Ex. 5265, 68:13-21; MF267. CVC’s communication
- 29 - Interference No. 106,115 CVC Reply 2 1 to Zhang was sufficiently enabling for “one of ordinary skill in the art to construct and
2 successfully operate the invention.” Meade v. McKirnan, 585 F.2d 504, 507 (C.C.P.A. 1978). As
3 Marraffini testified, the next steps of putting CVC’s system into eukaryotic cells would be
4 “pretty straightforward,” and Zhang purportedly obtained results with CVC’s system in a matter
5 of weeks. Ex. 5265, 31:8-19; Paper 2118, 42:9-10. Ex. 5013, ¶¶56-58; Paper 2567, 18:18-20:19;
6 MF232-233, 267-268. Binding precedent in Applegate and Alexander v. Williams, 342 F.2d 466
7 (C.C.P.A. 1965), compels judgment against Broad; see also, Hedgewick v. Akers, 497 F.2d 905,
8 n.4 (C.C.P.A. 1974); Polye v Uhl, 328 F.2d 893 (C.C.P.A. 1964). Finally, Broad implies, but
9 never expressly argues, that a simultaneous conception and reduction to practice standard
10 applies, but that rare exception cannot apply in cases such as this “where the issue is originality
11 or derivation.” MacMillan v. Moffett, 432 F.2d 1237, 1240 (C.C.P.A. 1970).
12 III. CONCLUSION
13 The law rewards inventors, such has CVC, not those who merely derive and then reduce
14 to practice using ordinary skill. The CVC inventors, not Zhang, invented the subject matter of
15 Count 1. Whether applying priority of invention law or derivation law, the PTAB should
16 recognize what the scientific community recognized in awarding the Nobel Prize to Charpentier
17 and Doudna: it was their teamwork that made the tremendous scientific leap forward to allow the
18 world to reap the benefits of a single-guide RNA CRISPR-Cas9 system for genome editing in
19 eukaryotes. The extensive corroborated evidence of record shows this to be the case, and the
20 PTAB should award priority to CVC.
21 Respectfully submitted, 22 By /Eldora L. Ellison/ By /Li-Hsien Rin-Laures/ Eldora L. Ellison, Ph.D., Esq. Li-Hsien Rin-Laures, M.D., Esq. Lead Attorney for UC and UV Lead Attorney for EC
- 30 - Interference No. 106,115 CVC Reply 2 Registration No. 39,967 Registration No. 33,547 STERNE, KESSLER, GOLDSTEIN & FOX RINLAURES LLC P.L.L.C. 321 N. Clark Street, 5th floor 1100 New York Avenue, NW Chicago, IL 60654 Washington, D.C. 20005
Date: May 6, 2021 Date: May 6, 2021 1
- 31 - Interference No. 106,115 CVC Reply 2
APPENDIX 1 – LIST OF EXHIBITS
Exhibit No. Description
Exhibit 3001 U.S. Application 61/736,527, Zhang et al., December 12, 2012.
Exhibit 3004 U.S. Application 61/757,640, Jinek et al., January 28, 2013. Paper 893, Decision on Motions 37 C.F.R. § 41.125(a), Interference Exhibit 3110 106,048, February 15, 2017. Cong et al., Multiplex Genome Engineering Using CRISPR/Cas Systems, Exhibit 3201 339(6121) SCIENCE 819-823 (2013) with Supplemental Material. Jinek et al., RNA-Programmed Genome Editing in Human Cells, 2 ELIFE Exhibit 3233 e00471 (2013) (“Jinek 2013”) (Ex. 1057). Dana Carrol, A CRISPR Approach to Gene Targeting, 20 Molec. Therapy Exhibit 3286 1658 (2012) (Ex. 1152) Exhibit 3424 Declaration of Feng Zhang, dated December 17, 2020
Exhibit 3425 Declaration of Le Cong, dated December 17, 2020
Exhibit 3435 Declaration of Benjamin Davies, dated July 17, 2020
Exhibit 3436 Declaration of Mark Kay, dated July 22, 2020
Exhibit 3437 Declaration of Alan Lambowitz, dated July 14, 2020 First Declaration of Paul Simons, dated May 23, 2016 ('048 Interference Ex. Exhibit 3438 2001) Exhibit 3439 Declaration of Lieberman Aiden, dated July 22, 2020
Exhibit 3440 Declaration of Paul Simons, dated July 21, 2020
Exhibit 3441 Declaration of Greg Hannon, dated July 20, 2020
Exhibit 3442 Declaration of Gregory Hannon, dated December 11, 2018
Exhibit 3443 Declaration of Mark Isalan, dated July 19, 2020
Exhibit 3446 Declaration of Caixia Gao, dated February 24, 2021
Exhibit 3447 Declaration of Philippe Mourrain, dated March 26, 2021
Exhibit 3448 Third Declaration of Ronald Breaker, Ph.D., dated March 26, 2021
- 1 -1 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description
Exhibit 3449 Declaration of Adam Bogdanove, dated July 21, 2020
Exhibit 3450 Declaration of Thierry VandenDriessche, dated July 21, 2020
Exhibit 3451 Declaration of Dr. Bryan Cullen with Exhibits 1-5, dated February 8, 2018
Exhibit 3452 Declaration of Dr. Paula Cannon, Dated March 27, 2019
Exhibit 3521 Invention Disclosure Memorandum submitted on February 13, 2011
Exhibit 3537 Evernote File "CRS notes (FZ)", created June 28, 2012 Mali et al., RNA-Guided Human Genome Engineering via Cas9, 339 (6121) Exhibit 3623 SCIENCE 823-826 Sanders, Cheap and easy technique to snip DNA could revolutionize gene Exhibit 3655 therapy, Berkeley Press Release (Jan. 7, 2013) Exhibit 3693 David Cox Lab Notebook Excerpted CRISPR 3.0/Surveyor Assay Gels/thermophilus surveyor 20120930: 2012- Exhibit 3698 09-30 18hr 45min quant.scn Exhibit 3713 String of emails between Feng Zhang and Luciano Marraffini in 2012
Exhibit 3751 Email from Feng Zhang to Le Cong, dated June 27, 2012, 1 page Email from Christina Kelly to Feng Zhang and Kristen Pearse, dated March Exhibit 3752 14, 2012, 1 page Exhibit 3777 Email from Feng Zhang to Le Cong, dated July 23, 2012, 1 page
Exhibit 3829 Email from Le Cong to David Cox, dated July 25, 2012, 2 pages
Exhibit 3832 Email from Feng Zhang to Le Cong, dated February 7, 2011, 2 pages
Exhibit 4036 Declaration of Randall T. Peterson, Ph.D. Zhang, Y. and Yu, L-C., “Microinjection as a tool of mechanical delivery,” Exhibit 4053 Curr. Opin. Biotechnol., 19:506-510 (2008) Cho, S.W., et al., “Heritable Gene Knockout in Caenorhabditis elegans by Exhibit 4075 Direct Injection of Cas9-sgRNA Ribonucleoproteins,” Genetics, 195:1177- 1180, Supplementary Information (2013) Cho, S.W., et al., “Targeted genome engineering in human cells with the Exhibit 4076 Cas9 RNA-guided endonuclease,” Nature Biotechnol., 31(3):230-232, Supplementary Information (2013)
-1-2 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description Shen, B., et al., “Generation of gene-modified mice via Cas9/RNA-mediated Exhibit 4079 gene targeting,” Cell Research, 23:720-723, Supplementary Information (2013) Zuris, J.A., et al., “Efficient Delivery of Genome-Editing Proteins In Vitro Exhibit 4080 and In Vivo,” Nat. Biotechnol., 33(1):73-80, pp. 1-26 (2015) Sung, Y.H., et al., “Highly efficient gene knockout in mice and zebrafish Exhibit 4082 with RNA-guided endonucleases,” Genome Res., 24:125-131, Supplementary Information (2014) Gagnon, J.A., et al., “Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide Exhibit 4083 RNAs,” PLOS ONE, 9(5):e98186, pp. 1-8, Supplementary Information (2014) Hwang, W.Y., et al., “Efficient In Vivo Genome Editing Using RNA-Guided Exhibit 4084 Nucleases,” Nat. Biotechnol., 31(3):227-229, pp 1-12 (2013) Hwang, W.Y., et al., “Heritable and Precise Zebrafish Genome Editing Exhibit 4085 Using a CRISPR-Cas System,” PLOS ONE, 8(7):e68708, pp. 1-9, Supplementary Information (2013) Chang, N., et al., “Genome editing with RNA-guided Cas9 nuclease in Exhibit 4086 Zebrafish embryos,” Cell Research, 23:465-472 (2013) Hruscha, A., et al., “Efficient CRISPR/Cas9 genome editing with low off- Exhibit 4087 target effects in zebrafish,” Development, 140:4982-4987, Supplementary Information (2013) Bassett, A., et al., “Highly Efficient Targeted Mutagenesis of Drosophila Exhibit 4089 with the CRISPR/Cas9 System,” Cell Reports, 4:220-228, Supplementary Information (2013) Yu, Z., et al., “Highly Efficient Genome Modifications Mediated by Exhibit 4090 CRISPR/Cas9 in Drosophila,” Genetics, 195:289-291, Supplementary Information (2013) Nakayama, T., et al., “Simple and efficient CRISPR/Cas9-mediated targeted Exhibit 4093 mutagenesis in Xenopus tropicalis,” Genesis, 51(12):1-15 (2013) Elbashir, S.M., et al., “Duplexes of 21-nucleotide RNAs mediate RNA Exhibit 4097 interference in cultured mammalian cells,” Nature, 411:494-498 (2001) Kim, S., et al., “Highly efficient RNA-guided genome editing in human cells Exhibit 4106 via delivery of purified Cas9 ribonucleoproteins,” Genome Res., 24:1012- 1019, Supplementary Information (2014) Hwang, W.Y., et al., “Efficient genome editing in zebrafish using a CRISPR- Exhibit 4233 Cas system,” Nature Biotechnology, 31(3):227-229, Supplementary Information (2013) Barrangou, R., “RNA-mediated programmable DNA cleavage,” Nature Exhibit 4257 Biotechology, 30(9):836-838 (2012) Stigloher, C., et al., “Segregation of telencephalic and eye-field identities Exhibit 4291 inside the zebrafish forebrain territory is controlled Tx3,” Development, 133:2925-2935 (2006)
-1-3 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description
Exhibit 4294 Declaration of Florian Raible Loosli, F., et al., “Loss of eyes in zebrafish caused by mutation of Exhibit 4306 chokh/rx3,” EMBO Rep, 4(9):894–899 (2003) Grabher, C., et al., “Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI,” Chapter 21 in Detrich, III, H.W., et al., (eds) Methods Exhibit 4328 Cell Biol., The Zebrafish: 2nd Edition Genetics, Genomics and Inormatics, vol. 77, pp. 381-401 (2004) Exhibit 4338 Florian Raible Laboratory Notebook No. 835, November 21, 2008, 12 pages
Exhibit 4343 Declaration of Cecilia Moens, Ph.D.
Exhibit 4345 Declaration of Yannick Doyon, Ph.D.
Exhibit 4348 Declaration of Krzyzstof Chylinski, Ph.D.
Exhibit 4349 Declaration of Martin Jinek, Ph.D.
Exhibit 4350 Declaration of Jennifer A. Doudna, Ph.D.
Exhibit 4351 Declaration of Emmanuelle Charpentier
Exhibit 4352 Declaration of Aaron Cheng, Ph.D.
Exhibit 4353 Declaration of Alexandra East-Seletsky, Ph.D.
Exhibit 4354 Declaration of Samuel Sternberg
Exhibit 4366 Alexandra East-Seletsky Laboratory Notebook
Exhibit 4381 Martin Jinek, Ph.D., laboratory notebook
Exhibit 4382 Martin Jinek, Ph.D., second laboratory notebook Doyon, J.B., et al., “Rapid and efficient clathrin-mediated endocytosis Exhibit 4384 revealed in genome-edited mammalian cells,” Nat Cell Biol., 13(3):331-337 (2011) Exhibit 4444 Email from Martin Jinek to Jennifer Doudna, dated June 26, 2012, 1 page Email from Jennifer Doudna to Martin Jinek, dated April 14, 2012, with Exhibit 4469 attachments, 33 pages
-1-4 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description Email from Aaron Cheng to Jennifer Doudna, Jamie Cate and D. Rubin, Exhibit 4475 dated April 16, 2012, 1 page Exhibit 4484 Email from Martin Jinek to Jennifer Doudna, dated June 26, 2012, 2 pages Email from Martin Jinek to himself, dated May 18, 2012, with attachments – Exhibit 4536 gfp pics, 7 pages Liu, P-Q., et al., “Regulaion of an Endogenous Locus Using a Panel of Exhibit 4570 Designed Zinc Finger Proteins Targeted to Accessible Chromatin Regions,” The Journal of Biological Chemistry, 276(14):11323-11334 (2001) Email from Emmanuelle Charpentier to Claudia Lupp, Angela Eggleston and Exhibit 4583 Jennifer Doudna, dated May 28, 2012, 2 pages Email from Guy Riddihough to Jennifer Doudna and Emmanuelle Exhibit 4598 Charpentier, dated May 29, 2012, 2 pages Email from Martin Jinek to himself, dated May 28, 2012, with attachment – Exhibit 4603 Exp. 40, 2 pages Thermes, V., et al., “I-SceI meganuclease mediates highly efficient Exhibit 4622 transgenesis in fish,” Mechanisms of Development, 118:91-98(2002) Powerpoint presentation by Krzysztof Chylinski and Martine Jinek entitled, Exhibit 4768 “A programmable dual RNA-guided DNA endonuclease in a Type II CRISPR system,” 32 pages Email from David Drubin to Jennifer Dounda, Aaron Cheng and Martin Exhibit 4775 Jinek, dated June 21, 2012, 2 pages Email from Aaron Cheng to Jennifer Dounda, David Drubin and Martin Exhibit 4776 Jinek, dated June 22, 2012, 2 pages Email from Krzysztof Chylinski to Emmanuelle Charpentier, dated June 28, Exhibit 4796 2012, 1 page Email from Florian Raible to Krzysztof Chylinski, dated June 28, 2012, 3 Exhibit 4799 pages Email from Emmanuelle Charpentier to Florian Raible, Kristin Tessmar and Exhibit 4807 Jennifer Doudna, dated June 28, 2012, 4 pages REDACTED Email from Emmanuelle Charpentier to Krzysztof Chylinski Exhibit 4912 and Emmanuelle Charpentier, dated August 9, 2012, 1 page Exhibit 4913 Image - SNAP-185336-0001, 1 page
Exhibit 4914 Image - SNAP-185435-0003, 1 page
Exhibit 4915 Image - SNAP-185406-0002, 1 page Email from Krzysztof Chylinski to Emmanuelle Charpentier and Ines Exhibit 4916 Fonfara, dated August 31, 2012, with attachment, 10 pages Exhibit 5013 Second Declaration of Yannick Doyon, Ph.D.
-1-5 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description
Exhibit 5014 Declaration of Philip D. Zamore, Ph.D.
Exhibit 5016 Declaration of Rodolphe Barrangou, Ph.D.
Exhibit 5018 Declaration of Erik J. Sontheimer, Ph.D. Chen, F., et al., “Methods and Reagents for Modifying Genomes Using Exhibit 5020 RNA-Guided Enonucleases,” U.S. Provisional Application No. 61/734,256 (filed December 6, 2012) Email from Martin Jinek to Jennifer Doudna, dated October 11, 2012, 2 Exhibit 5040 pages Email from Martin Jinek to Jennifer Doudna, dated April 11, 2012, with Exhibit 5105 attachment, 33 pages Email from Gregory Diskant to Paul Ainsworth, dated March 19, 2021, 2 Exhibit 5240 pages Jinek, M., et al., “Programmable dual-RNA guided DNA endonucleases in Exhibit 5254 adaptive bacterial immunity," pp. 1-24 with Supplementary Information (2012 Manuscript) MSID 1225829, Jinek, M., “Programmable dual-RNA guided DNA Exhibit 5255 endonucleases in adaptive bacterial immunity,” Reviewer Comments, 3 pages Program and Conference Logistics provided to the attendees of the Exhibit 5256 CRISPR 2012: 5th Annual CRISPR Research Meeting held at the University of California, Berkeley, CA (June 2012), 5 pages. Deposition transcript of Feng Zhang, Ph.D., with errata, Patent Interference Exhibit 5262 No. 106,115 (February 9, 2021) Deposition transcript of Ronald Breaker, Ph.D., Patent Interference No. Exhibit 5264 106,115 (March 5, 2021) Deposition transcript of Luciano Marraffini, Ph.D., Patent Interference No. Exhibit 5265 106,115 (March 11, 2021) Interview transcript of Krzysztof Chylinski, with errata, Patent Interference Exhibit 5266 No. 106,115 (December 29-30, 2020) Carroll, D. and Charo, R.A., “The societal opportunities and challenges of Exhibit 5314 genome editing,” Genome Biology, 16(242):1-9 (2015) Carroll, D., “A Perspective on the State of Genome Editing,” Molecular Exhibit 5315 Therapy, 24(3):412-413 (2016) Keryer-Bibens, C., et al., “Tethering of proteins to RNAs by bacteriophage Exhibit 5316 proteins,” Biol. Cell 100(2): 125–138 (2008) Coller, J. and Wickens, M., “Chapter Fourteen: Tethered Function Assays: Exhibit 5317 An Adaptable Approach to Study RNA Regulatory Proteins,” Methods in Enzymology, 429: 299-321 (2007)
-1-6 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description Lambowitz, A.M. and Zimmerly, S., “Group II Introns: Mobile Ribozymes Exhibit 5318 that Invade DNA,” Cold Spring Harb. Perspect. Biol., 3:a003616, pp.1-19 (2011) O’Carroll, D., et al., “A Slicer-independent role for Argonaute 2 in Exhibit 5319 hematopoiesis and the microRNA pathway,” Genes & Development, 21:1999–2004 with Supplementary Information (2007) Breaker, R., “Natural and engineered nucleic acids as tools to explore Exhibit 5320 biology,” Nature, 432:838-845 (2004) Paddison, P.J., et al., “Short hairpin RNAs (shRNAs) induce sequence- Exhibit 5321 specific silencing in mammalian cells,” Genes & Development, 16:948–958 (2002) Karikό, K., et al., “Small Interfering RNAs Mediate Sequence-Independent Exhibit 5322 Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 31,” J. Immunol., 172:6545–6549 (2004) Becker, P.B., “Nucleosome sliding: facts and fiction,” The EMBO Journal, Exhibit 5323 21(18):4749-4753 (2002) Exhibit 5324 Genomic Cruise Missiles, Science, 338:1526-1527 (2012) Blossey, R. and Schiessel, H., “The dynamics of the nucleosome: thermal Exhibit 5325 effects, external forces and ATP,” FEBS Journal, 278:3619–3632 (2011) Sorrentino, S., “The eight human ‘‘canonical” ribonucleases: Molecular Exhibit 5327 diversity, catalytic properties, and special biological actions of the enzyme proteins,” FEBS Letters, 584:2194–2200 (2010) Silva, J.M., et al., “Second-generation shRNA libraries covering the mouse Exhibit 5328 and human genomes,” Nature Genetics, 37(11):1281-1288 (2005) Email from Jennifer Doudna to Martin Jinek, Alexandra East, Aaron Cheng Exhibit 5329 and Enbo Ma, dated December 31, 2012, 4 pages Schmidt, S.T., et al., “Nucleic acid cleavage with a hyperthermophilic Cas9 Exhibit 5333 from an uncultured Ignavibacterium,” PNAS, 116(46):23100–23105 (2019) Mougiakos, I., et al., “Characterizing a thermostable Cas9 for bacterial Exhibit 5334 genome editing and silencing,” Nature Communs., 8(1647):1-11 (2017) Sander, J.D. and Joung, J.K., “CRISPR-Cas systems for editing, regulating Exhibit 5335 and targeting genomes,” Nat. Biotechnol., 32(4):347-355 (2014) Nelson, C.E., et al., “In vivo genome editing improves muscle function in a Exhibit 5336 mouse model of Duchenne muscular dystrophy,” Science, 351(6271):403- 407 (2016) Nishimasu, H., et al., “Structural Basis for the Altered PAM Recognition by Exhibit 5337 Engineered CRISPR-Cpf1,” Molecular Cell, 67:139–147 (2017) Pchelintsev, N.A., et al., “Critical Parameters for Efficient Sonication Exhibit 5338 and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins,” PLoS ONE, 11(1):e0148023, pp. 1-11 (2016) EpiGenie Guide: Chromatin Shearing, EpiGenie: Informally Informative, Exhibit 5339 www.epigenie.com, March 6, 2020, 6 pages
-1-7 - Interference No. 106,115 CVC Reply 2
Exhibit No. Description Brockerhoff, S., et al., “A behavioral screen for isolating zebrafish mutants Exhibit 5340 with visual system defects,” Proc. Natl. Acad. Sci. USA, Neurobiology, 92:10545-10549 (1995) Hoshijima, K., et al., “Highly efficient methods for generating deletion Exhibit 5342 mutations and F0 embryos that lack gene function in zebrafish,” Author manuscript, Dev Cell., pp. 1-35 (2019) Mueller, K.P. and Neuhauss, S.C.F., “Sunscreen for Fish: Co-Option of UV Exhibit 5343 Light Protection for Camouflage,” PLoS One, 9(1):e87372, pp. 1-5 (2014) Soroldoni, D., et al., “Chapter 8: Simple and Efficient Transgenesis with Meganuclease Constructs in Zebrafish,” Zebrafish, Methods in Molecular Exhibit 5344 Biology, vol. 546, pp. 117-130, Eds., Graham J. Lieschke et al., Humana Press (2009) Rosen, J.N., et al., “Microinjection of Zebrafish Embryos to Analyze Gene Exhibit 5345 Function,” J. Vis. Exp., 25:e1115, pp. 1-5 (2009) Madelaine, R., et al., “Genetic deciphering of the antagonistic activities of Exhibit 5346 the melanin-concentrating hormone and melanocortin pathways in skin pigmentation,” PLoS Genet., 16(12):e1009244, pp.1-21 (2020) Deposition transcript of Ronald Breaker, Ph.D., Vol. 2, with errata, Patent Exhibit 5347 Interference No. 106,115 (April 23, 2021) Deposition transcript of Philippe Mourrain, Ph.D., with errata, Patent Exhibit 5348 Interference No. 106,115 (April 26, 2021) Deposition transcript of Phillip Zamore, Ph.D., with errata, Patent Exhibit 5349 Interference No. 106,115 (April 26, 2021) REDACTED Deposition transcript of Rodolphe Barrangou, Ph.D., with Exhibit 5350 errata, Patent Interference No. 106,115 (April 19, 2021) Transcript of Interview of Krzysztof Chylinski, December 29-30, 2020, with Exhibit 6202 Supplemental Declaration Transcript of Interview of Florian Raible, February 4-5, 2021, with Exhibit 6211 Supplemental Declaration Baumann & West, DNA end-joining catalyzed by human cell-free extracts, Exhibit 6335 95 Proc. Natl. Acad. Sci. USA 14066-14070 (1998). Diggle et al., Development of a rapid, small-scale DNA repair assay for use Exhibit 6336 on clinical samples, Nucleic Acids Res. 31(15):e83 (2003). Budman & Chu, Processing of DNA for nonhomologous end-joining by cell- Exhibit 6343 free extract, 24 EMBO J. 849-860 (2005). Deshpande et al, DNA-dependent protein kinase promotes DNA end Exhibit 6347 processing by MRN and CtIP, Sci. Adv. 6:eaay0922 (Jan. 2020).
-1-8 - Interference No. 106,115 CVC Reply 2
1 APPENDIX 2 – STATEMENT OF MATERIAL FACTS
2 1. Dr. Doudna and Dr. Charpentier received the 2020 Nobel Prize in Chemistry “for the
3 development of a method for genome editing.” Ex. 4301, 1, 10; Ex. 4350, ¶¶11, 15; Ex.
4 4351, ¶¶5, 16.
5 Broad’s Response: Admitted.
6 2. The inventors identified the necessary and sufficient components of the CRISPR-Cas9
7 DNA- cleavage complex (Cas9, crRNA, and tracrRNA) before March 1, 2012. Ex. 4350,
8 ¶¶19-40; Ex. 4351, ¶¶18-20; Ex. 4349, ¶¶15-23; Ex. 4348, ¶¶13-18; Ex. 4381, 23-25, 53-
9 56; Ex. 4666; Ex. 4490.
10 Broad’s Response: Denied.
11 3. By March 1, 2012, RNA degradation mechanisms in eukaryotic cells, such as RNases
12 and RNA interference (RNAi), were well known in the art. Ex. 4350, ¶¶14-15, 39; Ex.
13 4349, ¶¶6, 27-28; Ex. 4351, ¶26; Ex. 4348, ¶¶22-24; Ex. 4345, ¶¶119-124; Ex. 4343,
14 ¶¶99-100; Ex. 4568; Ex. 4318; Ex. 4319; Ex. 4398; Ex. 4399; Ex. 4400.
15 Broad’s Response: Admitted.
16 4. The inventors published papers describing RNA degradation, ribozymes, and Group II
17 introns in eukaryotic cells before March 1, 2012. Ex. 4398, Ex. 4399; Ex. 4400; Ex.
18 5211; Ex. 4435; Ex. 5210; Ex. 4350, ¶¶14-15, 39; Ex. 4349, ¶¶6, 27-28; Ex. 4351, ¶26;
19 Ex. 4348, ¶¶22-24.
20 Broad’s Response: Denied.
21 5. By March 1, 2012, temperature, pH, and ion concentration were routine optimization
22 parameters in biological research. Ex. 4350, ¶¶27-28; Ex. 4349, ¶¶29, 37; Ex. 4351, ¶33;
23 Ex. 4348, ¶25; Ex. 5209; Ex. 5210; Ex. 4343, ¶¶97-98; Ex. 4345, ¶¶125-131.
-2-1 - Interference No. 106,115 CVC Reply 2
1 Broad’s Response: Denied.
2 6. The inventors understood potential effects of temperature, pH, and ion concentrations on
3 protein structure and function in eukaryotic cells before March 1, 2012. Ex. 4350, ¶¶27-
4 28; Ex. 4349, ¶¶29, 37; Ex. 4351, ¶33; Ex. 4348, ¶25; Ex. 5209; Ex. 5210; Ex. 4820; Ex.
5 4829.
6 Broad’s Response: Denied.
7 7. By March 1, 2012, the art taught that a short, conserved nucleotide motif called the
8 Protospacer Adjacent Motif (“PAM”) must be present downstream (3’) of the target
9 sequence on the non-target strand of the target DNA molecule for the Type II CRISPR-
10 Cas9 system to cleave the target DNA molecule. Ex. 4041, 1395, 1399; Ex. 4043, 736-
11 738, Fig. 1; Ex. 4044, 169, Fig. 2; Ex. 4045, 68-69; Ex. 4046, 468, Fig. 1; Ex. 4047, 284,
12 Fig. 4; Ex. 4048, 9279, Fig. 3; Ex. 4049, 322, 324; Ex. 4050, 333, Fig. 2; Ex. 4345, ¶¶88-
13 98.
14 Broad’s Response: Denied.
15 8. The inventors published papers describing the role of the PAM sequence for CRISPR-
16 mediated DNA cleavage before March 1, 2012. Ex. 4350, ¶¶22, 36; Ex. 4349, ¶¶24-25;
17 Ex. 4351, ¶¶28-29; Ex. 4348, ¶¶19-21; Ex. 4354, ¶17-22; Ex. 4046, 468; Ex. 3221, 535
18 and Fig. 4H; Ex. 4395, methods; Ex. 4050, 333-334.
19 Broad’s Response: Denied.
20 9. Dr. Charpentier is a co-author on Makarova et al. 2011, which discloses that Cas9 targets
21 the DNA “in a process that requires the PAM.” Ex. 4046, 468; Ex. 4351, ¶14.
22 Broad’s Response: Admitted that Dr. Charpentier is listed as an author on the
23 opinion article Makarova, K.S., et al., “Evolution and classification of the
-2-2 - Interference No. 106,115 CVC Reply 2
1 CRISPR-Cas systems,” Nature Reviews Microbiology, 9:467-477 (2011) (Ex.
2 4046) and that the words “in a process that requires the PAM” appear on page 468
3 but otherwise denied.
4 10. Broad’s own publication Cong 2013 cites a publication co-authored by Dr. Charpentier,
5 and other earlier references, when referring to PAM sequences. Ex. 3201, 822 (citing Ex.
6 4046 as reference 17, Ex. 4041 as reference 22, and Ex. 4043 as reference 23).
7 Broad’s Response: Denied.
8 11. Broad does not dispute that PAM sequences were known to play a role in DNA targeting
9 in natural CRISPR systems before the filing of CVC’s P1 application. Paper 877, 93-94.
10 Broad’s Response: Denied.
11 12. The inventors were aware of RNA/protein interactions and co-localization in eukaryotic
12 cells before March 1, 2012. Ex. 4350, ¶¶31-32; Ex. 4349, ¶¶31, 229-230, 246; Ex. 4351,
13 ¶31; Ex. 4348, ¶¶13-14, 22-25; Ex. 4381, 23-25, 53-56; Ex. 4666; Ex. 4490.
14 Broad’s Response: Denied.
15 13. By March 1, 2012, codon optimization was a known, conventional technique for
16 optimizing protein expression in eukaryotic cells. Ex. 4345, ¶¶109-112; Ex. 4343, ¶96;
17 Ex. 4396, 20476; Ex. 4397, 9270; Ex. 4110, Abstract, Table 1; Ex. 4111, 115, 122; Ex.
18 4112, 796-798.
19 Broad’s Response: Denied.
20 14. By March 1, 2012, adding one or more nuclear localization sequences (NLS) to a protein
21 was a known, conventional optimization technique for optimizing protein localization to
22 the nucleus of eukaryotic cells. Ex. 4078; Ex. 4077; Ex. 4113; Ex. 4665, 763; Ex. 4312,
23 methods; Ex. 4384; Ex. 4424; Ex. 4345, ¶¶99-108; Ex. 4343, ¶101.
-2-3 - Interference No. 106,115 CVC Reply 2
1 Broad’s Response: Denied.
2 15. By March 1, 2012, in vitro transcription techniques using, e.g. T7 polymerase, to
3 transcribe RNA from a DNA template were conventional laboratory techniques. Ex.
4 4122; Ex. 4039, p. 19825; Ex. 4054, p. 7; Ex. 4055, p. 6; Ex. 4057, p. 10; Ex. 4056, pp.
5 16-18; Ex. 4058, S2; Ex. 4059, 5; Ex. 4060, S-Methods; Ex. 4081, p. 3-26, Table 3.4.1;
6 Ex. 4345, ¶241.
7 Broad’s Response: Admitted.
8 16. By March 1, 2012, microinjection techniques for injecting DNA, RNA, proteins, and
9 combinations thereof into, e.g. zebrafish embryos, were conventional laboratory
10 techniques. Ex. 4343, ¶84; Ex. 4074, 487; Ex. 4073, 125, 126-131; Ex. 4056, 18-19; Ex.
11 4057, 10; Ex. 4055, 2; Ex. 4054, 7; Ex. 4306.
12 Broad’s Response: Denied.
13 17. By March 1, 2012, techniques for transfecting proteins and/or nucleic acids into human
14 cell lines using, e.g. lipofection or nucleofection, were conventional laboratory
15 techniques. Ex. 4100, 10189-10190; Ex. 4101, 2026; Ex. 4098, 65; Ex. 4099, 8367; Ex.
16 4095, 5562; Ex. 4060, Fig. 1B; Ex. 4102, 2862; Ex. 4103, 3974; Ex. 4104; Ex. 4105; Ex.
17 4053, 507; Ex. 4345, ¶¶136-140.
18 Broad’s Response: Denied.
19 18. By March 1, 2012, culturing the human embryonic kidney cell line HEK293T was
20 conventional and required only ordinary skill. Ex. 4349, ¶189; Ex. 4350, ¶16; Ex. 4351,
21 ¶¶23-24; Ex. 4352, ¶28; Ex. 4353, ¶¶14, 22; Ex. 4345, ¶¶86-87, 136-140; Ex. 4312,
22 Methods, SFig. 2.
23 Broad’s Response: Admitted.
-2-4 - Interference No. 106,115 CVC Reply 2
1 19. By March 1, 2012, expressing NLS-tagged proteins in eukaryotic cells from plasmid
2 DNA vectors with a CMV promoter was conventional and required only ordinary skill.
3 Ex. 4345, ¶¶99-108; Ex. 4312, 144, Methods; Ex. 4665, 763; Ex. 4604, 3927.
4 Broad’s Response: Denied.
5 20. By March 1, 2012, the plasmid vector pSilencer 2.1 was commercially available and
6 routinely used in the art for expressing small non-coding RNAs under control of the U6
7 promoter. Ex. 4349, ¶87; Ex. 4345, ¶171; Ex. 4566, 170-171.
8 Broad’s Response: Denied.
9 21. By March 1, 2012, the plasmid vector pcDNA3.1 was commercially available and
10 routinely used in the art for expressing proteins under the control of the CMV promoter.
11 Ex. 4349, ¶189; Ex. 4345, ¶183; Ex. 4604, 3927.
12 Broad’s Response: Denied.
13 22. The inventors understood and contemplated optimizing their system using nuclear
14 localization sequences and codon optimization before March 1, 2012. Ex. 4350, ¶29; Ex.
15 4349, ¶28; Ex. 4351, ¶30; Ex. 4348, ¶24; Ex. 4381, 64; Ex. 4312, Methods; Ex. 4396,
16 20476; Ex. 4397, 9270.
17 Broad’s Response: Denied.
18 23. By March 1, 2012, the art taught that the chromatin structure of the eukaryotic cell
19 genome is dynamic and variable. Ex. 4165, 554-555; Ex. 4168, 2; Ex. 4345, ¶¶113-118.
20 Broad’s Response: Denied.
21 24. The inventors understood the possibility of chromatin affecting CRISPR-Cas9’s
22 efficiency in a eukaryotic cell. Ex. 4349, ¶¶28, 229; Ex. 4348, ¶24; Ex. 4350, ¶¶33-34;
23 Ex. 4351, ¶32; Ex. 4165, 554-555; Ex. 4168, 2.
-2-5 - Interference No. 106,115 CVC Reply 2
1 Broad’s Response: Denied.
2 25. Before March 1, 2012, the inventors performed crRNA and tracrRNA truncation
3 experiments and determined that crRNA and tracrRNA could be truncated yet retain the
4 ability to form a functional CRISPR-Cas9 DNA cleavage complex. Ex. 4349, ¶¶18-23;
5 Ex. 4350, ¶¶21-24; Ex. 4351, ¶¶25, 34; Ex. 4348, ¶¶16-18; Ex. 4381, 53-56; Ex. 4666.
6 Broad’s Response: Denied.
7 26. The March 1, 2012 Jinek notebook entry depicts a Type II CRISPR-Cas9 system for gene
8 editing in eukaryotic cells. Ex. 4381, 63-65; Ex. 4345, ¶¶70-82; Ex. 4349, ¶¶34-43; Ex.
9 4350, ¶¶41-51.
10 Broad’s Response: Denied.
11 27. The March 1, 2012 Jinek notebook entry shows a CRISPR-Cas9 system comprising a) a
12 Cas9 protein and b) a single molecule DNA-targeting RNA. Ex. 4381, 63-65; Ex. 4345,
13 ¶¶70-82; Ex. 4349, ¶¶34-43; Ex. 4350, ¶¶41-51.
14 Broad’s Response: Denied.
15 28. The March 1, 2012 Jinek notebook entry shows a single-molecule DNA-targeting RNA
16 comprising i) a targeter RNA capable of hybridizing with a target sequence in a target
17 DNA and ii) an activator-RNA capable of hybridizing with the targeter RNA to form a
18 double- stranded duplex, wherein the activator-RNA and the targeter-RNA are covalently
19 linked to one another with intervening nucleotides. Ex. 4381, 63-65; Ex. 4345, ¶¶70-82;
20 Ex. 4349, ¶¶34-43; Ex. 4350, ¶¶41-51.
21 Broad’s Response: Denied.
22 29. The March 1, 2012 Jinek notebook entry shows a single-molecule DNA-targeting RNA
23 capable of forming a complex with the Cas9 protein and thereby targeting the Cas9
-2-6 - Interference No. 106,115 CVC Reply 2
1 protein to the target DNA molecule. Ex. 4381, 63-65; Ex. 4345, ¶¶70-82; Ex. 4349, ¶¶34-
2 43; Ex. 4350, ¶¶41-51.
3 Broad’s Response: Denied.
4 30. The March 1, 2012 Jinek notebook entry shows a Type II CRISPR-Cas system capable of
5 cleaving or editing a target DNA molecule or modulating transcription of at least one
6 gene encoded by the target DNA molecule. Ex. 4381, 63-65; Ex. 4345, ¶¶70-82; Ex.
7 4349, ¶¶34- 43; Ex. 4350, ¶¶41-51.
8 Broad’s Response: Denied.
9 31. The single-molecule DNA-targeting RNA that CVC published in Jinek 2012 (Fig. 5B,
10 “chimera A”) has the same design as the chimeric RNA drawn in Jinek’s March 1, 2012
11 notebook entry. Ex. 4381, 64; Ex. 3202, Fig. 5B; Ex. 4345, ¶¶70-82.
12 Broad’s Response: Denied.
13 32. The April 11, 2012 IDF shows using a Type II CRISPR-Cas9 system in eukaryotic cells
14 for gene editing. Ex. 5105, 18-21; Ex. 4349, ¶¶72-74; Ex. 4350, ¶58; Ex. 4345, ¶¶159-
15 163.
16 Broad’s Response: Denied.
17
18 33. The April 11, 2012 IDF shows a CRISPR-Cas9 system comprising a) a Cas9 protein and
19 b) a single molecule DNA-targeting RNA. Ex. 5105, 18, 28; Ex. 4349, ¶¶72-74; Ex.
20 4350, ¶58; Ex. 4345, ¶¶159-163.
21 Broad’s Response: Denied.
22 34. The April 11, 2012 IDF shows a single-molecule DNA-targeting RNA comprising i) a
23 targeter RNA capable of hybridizing with a target sequence in a target DNA and ii) an
-2-7 - Interference No. 106,115 CVC Reply 2
1 activator-RNA capable of hybridizing with the targeter RNA to form a double-stranded
2 duplex, wherein the activator-RNA and the targeter-RNA are covalently linked to one
3 another with intervening nucleotides. Ex. 5105, 18, 28; Ex. 4349, ¶¶72-74; Ex. 4350, ¶58;
4 Ex. 4345, ¶¶159-163.
5 Broad’s Response: Denied.
6 35. The April 11, 2012 IDF shows a single-molecule DNA-targeting RNA capable of
7 forming a complex with the Cas9 protein and thereby targeting the Cas9 protein to the
8 target DNA molecule. Ex. 5105, 18, 28; Ex. 4349, ¶¶72-74; Ex. 4350, ¶58; Ex. 4345,
9 ¶¶159-163.
10 Broad’s Response: Denied.
11 36. The April 11, 2012 IDF shows a Type II CRISPR-Cas system capable of cleaving or
12 editing a target DNA molecule or modulating transcription of at least one gene encoded
13 by the target DNA molecule. Ex. 5105, 20-21; Ex. 4349, ¶¶72-74; Ex. 4350, ¶58; Ex.
14 4345, ¶¶159-163.
15 Broad’s Response: Denied.
16 37. The May 28, 2012 Jinek notebook entry shows a single-molecule DNA-targeting RNA
17 labeled “CLTA6 (pMJ874)” comprising i) a targeter RNA capable of hybridizing with a
18 target sequence in a target DNA and ii) an activator-RNA capable of hybridizing with the
19 targeter RNA to form a double-stranded duplex, wherein the activator-RNA and the
20 targeter- RNA are covalently linked to one another with intervening nucleotides. Ex.
21 4382, 4; Ex. 4349, ¶¶124-128; Ex. 4345, ¶¶167-174.
22 Broad’s Response: Denied.
-2-8 - Interference No. 106,115 CVC Reply 2
1 38. The May 28, 2012 Jinek notebook entry shows a single-molecule DNA-targeting RNA
2 labeled “CLTA6 (pMJ874)” capable of forming a complex with the Cas9 protein and
3 thereby targeting the Cas9 protein to the target DNA molecule. Ex. 4382, 4; Ex. 4349,
4 ¶¶124-128; Ex. 4345, ¶¶167-174.
5 Broad’s Response: Denied.
6 39. The May 28, 2012 Jinek notebook entry shows a single-molecule DNA-targeting RNA
7 labeled “CLTA6 (pMJ874)” that, when combined with a Cas9 protein, would form a
8 Type II CRISPR-Cas system capable of cleaving or editing a target DNA molecule or
9 modulating transcription of at least one gene encoded by the target DNA molecule. Ex.
10 4382, 4; Ex. 4349, ¶¶124-128; Ex. 4345, ¶¶167-174.
11 Broad’s Response: Denied.
12 40. The June 28, 2012 email from Dr. Raible to Dr. Charpentier refers to using a Type II
13 CRISPR-Cas9 system in eukaryotic cells for cleaving or editing a target DNA molecule.
14 Ex. 4799; Ex. 4348, ¶¶115-116; Ex. 4351, ¶¶57-59; Ex. 4294, ¶¶14-16; Ex. 4345, ¶¶175-
15 177.
16 Broad’s Response: Denied.
17 41. The June 28, 2012 email from Dr. Raible to Dr. Charpentier refers to a CRISPR-Cas9
18 system comprising a) a Cas9 protein and b) a single molecule DNA-targeting RNA. Ex.
19 4799; Ex. 4348, ¶¶115-116; Ex. 4351, ¶¶57-59; Ex. 4294, ¶¶14-16; Ex. 4345, ¶¶175-177.
20 Broad’s Response: Denied.
21 42. The June 28, 2012 email from Dr. Raible to Dr. Charpentier refers to a single-molecule
22 DNA-targeting RNA comprising i) a targeter RNA capable of hybridizing with a target
23 sequence in a target DNA and ii) an activator-RNA capable of hybridizing with the
-2-9 - Interference No. 106,115 CVC Reply 2
1 targeter-RNA to form a double-stranded duplex, wherein the activator-RNA and the
2 targeter-RNA are covalently linked to one another with intervening nucleotides. Ex.
3 4799; Ex. 4348, ¶¶115- 116; Ex. 4351, ¶¶57-59; Ex. 4294, ¶¶14-16; Ex. 4345, ¶¶175-
4 177.
5 Broad’s Response: Denied.
6 43. The June 28, 2012 email from Dr. Raible to Dr. Charpentier refers to a single-molecule
7 DNA-targeting RNA capable of forming a complex with the Cas9 protein and thereby
8 targeting the Cas9 protein to the target DNA molecule. Ex. 4799; Ex. 4348, ¶¶115-116;
9 Ex. 4351, ¶¶57-59; Ex. 4294, ¶¶14-16; Ex. 4345, ¶¶175-177.
10 Broad’s Response: Denied.
11 44. The June 28, 2012 email from Dr. Raible to Dr. Charpentier refers to a Type II CRISPR-
12 Cas system capable of cleaving or editing a target DNA molecule or modulating
13 transcription of at least one gene encoded by the target DNA molecule. Ex. 4799; Ex.
14 4348, ¶¶115-116; Ex. 4351, ¶¶57-59; Ex. 4294, ¶¶14-16; Ex. 4345, ¶¶175-177.
15 Broad’s Response: Denied.
16 45. CVC’s zebrafish experiment of August 9, 2012 meets all the limitations of Count 1. Ex.
17 4294, ¶¶50-58; Ex. 4343, ¶¶59-82; Ex. 4338, 79-80; Exs. 4913-4915.
18 Broad’s Response: Denied.
19 46. Zebrafish with homozygous rx3 gene mutations have a phenotypic loss of pigmented
20 retina while retaining a reduced eye lens. Ex. 4306, Fig. 1; Ex. 4321, Fig. 1; Ex. 4343,
21 ¶¶33-44.
22 Broad’s Response: Denied.
-2-10 - Interference No. 106,115 CVC Reply 2
1 47. The time between the Raible email of June 28, 2012, describing the zebrafish sgRNA-
2 CRISPR-Cas9 protocol, to the observation of rx3-mutant phenotype fish on August 9,
3 2012, was 42 days (6 weeks). Ex. 4799; Exs. 4913-4915; Ex. 4912; Ex. 4351, ¶70; Ex.
4 4294, ¶¶57- 58; Ex. 4348, ¶127.
5 Broad’s Response: Denied.
6 48. CVC’s October 31 ARTP meets all the limitations of Count 1. Ex. 5075; Ex. 4366, 31-
7 33; Ex. 4345, ¶¶201-218; Ex. 4349, ¶¶245-251; Ex. 4353, ¶¶87-101.
8 Broad’s Response: Denied.
9 49. CVC’s October 31 ARTP is described in CVC’s P3 application in Example 2 and
10 published in the Jinek 2013 eLife paper. Ex. 4353, ¶¶141-142; Ex. 4349, ¶271; Ex. 4345,
11 ¶199; Ex. 3004, ¶[00408]-[00423]; Fig. 36E; Ex. 4137, Fig. 1E.
12 Broad’s Response: Denied.
13 50. CVC’s November 1 ARTP meets all the limitations of Count 1. Ex. 4366, 38; Ex. 4353,
14 ¶102; Ex. 4349, ¶¶217, 245; Ex. 4345, ¶¶220-225.
15 Broad’s Response: Denied.
16 51. CVC’s November 5 ARTP meets all the limitations of Count 1. Ex. 4366, 40; Ex. 4353,
17 ¶110-111; Ex. 4345, ¶¶226-229; Ex. 4349, ¶¶217, 245; Ex. 5171; Ex. 4350, ¶135.
18 Broad’s Response: Denied.
19 52. CVC’s November 18 ARTP meets all the limitations of Count 1. Ex. 4366, 48-49, 53; Ex.
20 5107; Ex. 4353, ¶139; Ex. 4366, 53; Ex. 4349, ¶¶217, 245; Ex. 4350, ¶140.
21 Broad’s Response: Denied.
-2-11 - Interference No. 106,115 CVC Reply 2
1 53. Shen discloses a chimeric RNA in Figure 1B with the identical chimera A design
2 disclosed in Jinek 2012, Fig. 5B. Ex. 4079, Fig. 1B; Ex. 3202, Fig. 5B; Ex. 4345, ¶¶145-
3 150.
4 Broad’s Response: Denied.
5 54. Shen discloses making an sgRNA and S. pyogenes Cas9 mRNA using T7 in vitro
6 transcription, and microinjecting the RNAs into a fish cell. Ex. 4079, pp. 720, SI2-SI3;
7 Ex. 4345, ¶¶145-150.
8 Broad’s Response: Denied.
9 55. Cong discloses a chimeric RNA in Figure 1B with the identical chimera A design
10 disclosed in Jinek 2012, Fig. 5B. Ex. 3201, Fig. 2B; Ex. 3202, Fig. 5B; Ex. 4345, ¶¶145-
11 150.
12 Broad’s Response: Denied.
13 56. Cong discloses expressing an sgRNA and S. pyogenes Cas9 protein using standard
14 plasmid expression vectors and transfecting the plasmids into a human cell line. Ex.
15 3201, 820, SI1; Ex. 4345, ¶¶145-150.
16 Broad’s Response: Denied.
17 57. Cho discloses a chimeric RNA in Figure 1A with the identical chimera A design
18 disclosed in Jinek 2012, Fig. 5B. Ex. 4076, Fig. 1A.; Ex. 3202, Fig. 5B; Ex. 4345, ¶¶145-
19 150.
20 Broad’s Response: Admitted.
21 58. Cho discloses expressing an sgRNA and S. pyogenes Cas9 protein using standard
22 plasmid expression vectors and transfecting the plasmids into a human cell line. Ex.
23 4076, SI1-SI2.; Ex. 4345, ¶¶145-150.
-2-12 - Interference No. 106,115 CVC Reply 2
1 Broad’s Response: Denied.
2 59. The CRISPR Journal quotes Dr. Luciano Marraffini as stating, “The Doudna–Charpentier
3 paper is when we all learned about the single-guide RNA.” Ex. 4320, 8.
4 Broad’s Response: Admitted that the words “The Doudna–Charpentier paper is
5 when we all learned about the single-guide RNA” appear in Ex. 4320 at pg. 8 but
6 otherwise denied
7 60. In a 2014 publication, Fei Ran stated that, “[w]hen we started working on this [CRISPR-
8 Cas9], the tracrRNA hadn’t been discovered yet.” Ex. 4269, 71.
9 Broad’s Response: Denied.
10 61. In a 2014 publication, Fei Ran stated that after learning from Jinek 2012 that “(1) you can
11 fuse the spacer and the repeat and the tracrRNA into a single chimeric RNA; and (2) you
12 can use a single chimeric RNA and Cas9 to program the cleavage of DNA targets in an in
13 vitro cell-free lysis reaction,” the Zhang group “built upon these exciting discoveries.”
14 Ex. 4269, 71-73.
15 Broad’s Response: Admitted that the text “(1) you can fuse the spacer and 1 the
16 repeat and the tracrRNA into a single chimeric RNA; and (2) you can use a single
17 chimeric RNA and Cas9 to program the cleavage of DNA targets in an in vitro
18 cell-free lysis reaction” appear in Ex. 4269 at 72-73 and the words “built upon
19 these exciting discoveries” appear in Ex. 4269 at 73 but otherwise denied.
20 62. Broad first learned of the sgRNA design from the Jinek 2012 publication. Ex. 4269, 71-
21 73; Ex. 4320, 8; Ex. 3202, Fig. 5B; Ex. 3201, Fig. 2B.
22 Broad’s Response: Denied.
-2-13 - Interference No. 106,115 CVC Reply 2
1 63. Jinek’s March 1 notebook entry describes supplying the Cas9 protein (“Csn1”) and DNA-
2 targeting RNA to mammalian cells by “plasmid vectors.” Ex. 4381, 65; Ex. 4349, ¶¶34-
3 43; Ex. 4350, ¶51.
4 Broad’s Response: Denied.
5 Broad’s Additional Facts 64 – 200
6 64. Doudna’s research prior to 2012 focused on elucidating the biochemical and structural
7 biology of systems such as CRISPR, ribozymes, RNAi, and group II introns. Ex. 6204,
8 Doudna Tr. 214:12-215:15; 216:20-217:2; 235:4-8.
9 CVC Response: Denied
10 65. At her deposition, Doudna testified that she could not recall ever conducting a single
11 experiment before 2012 where a eukaryotic cell was the experimental vehicle. Id. at
12 63:11-19; 65:9-66:12.
13 CVC Response: Denied
14 66. Before 2012, Doudna never attempted to use any of the CRISPR, ribozymes, RNAi, or
15 group II introns systems in a eukaryotic cell. Id. at 63:11-19; 65:9-66:12; 65:9-21.
16 CVC Response: Denied
17 67. Before 2012, Doudna never conducted a genome editing experiment where, as in
18 eukaryotic cells, it was necessary to overcome chromatin challenges. Id. at 239:12-
19 240:16.
20 CVC Response: Denied
21 68. Doudna had never conducted any experiments using ZFNs and TALENs by 2012. Id. at
22 57:17-58:9, 58:18-59:6.
23 CVC Response: Denied
-2-14 - Interference No. 106,115 CVC Reply 2
1 69. Dr. Doudna and Samuel Sternberg coauthored A Crack in Creation: Gene Editing and the
2 Unthinkable Power to Control Evolution which was first published in 2017. Ex. 6104,
3 excerpts.
4 CVC Response: Admitted
5 70. Doudna’s book A Crack In Creation, states that Doudna “speculated to [a] TV
6 interviewer that these molecules might one day become tools for editing DNA” and
7 might “act like a molecular repair kit and repair defective genes.” Ex. 6104 at 38, ¶2.
8 CVC Response: Denied but acknowledged that the exhibit contains the partially
9 quoted text.
10 71. Dr. Doudna’s book A Crack In Creation states: “As it turned out, this particular
11 development never came to pass, or at least it hasn’t yet.” Id. ¶3.
12 CVC Response: Denied but acknowledged that the exhibit contains the partially
13 quoted text.
14 72. As part of her biochemical research on the CRISPR system, by March 1, 2012, Doudna
15 was working with fellow CVC inventors Jinek, Charpentier, and Chylinski “to resolve the
16 structure and function of Cas9 . . . and characterize the DNA-cleavage mechanism in the
17 Type II CRISPR-Cas9 system” in in vitro experiments. Ex. 4350, Doudna Dec. ¶19.
18 CVC Response: Denied but acknowledged that the exhibit contains the partially
19 quoted text.
20 73. On March 1, 2012, Doudna emailed Jinek a blank Invention Disclosure Form (“IDF”) for
21 completion, suggesting that use of CRISPR in eukaryotic cells would be “fabulous if it
22 works.” Ex. 4406.
-2-15 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Denied but acknowledged that the exhibit contains the partially
2 quoted text.
3 74. Jinek’s notebook entry of March 1, 2012 reads: “New idea: adapt the Csn1/Cas9 system
4 as a gene-targeting tool in mammalian cells, e.g. in embryonic or induced pluripotent
5 stem cells, especially in those where homologous recombination is not efficient..” Ex.
6 4381 at 63.
7 CVC Response: Admitted.
8 75. Jinek’s notebook entry of March 1, 2012 states the aim to “test whether the strategy can
9 be used to induce DSBs in mammalian cells.” Ex. 4381 at 65.
10 CVC Response: Denied but acknowledged that the exhibit contains the partially
11 quoted text.
12 76. Jinek testified that, as of March 1, 2012, the inventors had “considered multiple —
13 multiple approaches and multiple organisms.” Ex. 6207, Jinek Tr. 272:16-17.
14 CVC Response: Admitted.
15 77. Jinek testified regarding approaches to delivery methods as of March 1, 2012: “Well,
16 these would be delivery using — using DNA constructs. These could be delivery using
17 mRNAs, delivery using purified Cas9 protein, plus synthetic or in vitro transcribed
18 sgRNAs. There may have been others, but I don't — I don't recall.” Id. at 273:4-9.
19 CVC Response: Denied but acknowledged that the exhibit contains the partially
20 quoted text.
21 78. Jinek’s April 11, 2012 IDF, lists “several methods [that] can be employed in order to
22 introduce the DNA into cells and whole organisms,” including “direct microinjection,”
-2-16 - Interference No. 106,115 CVC Reply 2
1 “transfection,” “electroporation,” “transduction using viral vectors,” and
2 “Agrobacterium-mediated transformation of plants.” Ex. 5105 at 24 (Section D).
3 CVC Response: Denied but acknowledged that the exhibit contains the partially
4 quoted text.
5 79. Jinek’s April 11, 2012 IDF further states the CRISPR system might be used in “cells or
6 whole organisms,” and will be under the “control of promoters appropriate for the desired
7 cell type or organism and may be inducible to allow for better control or timing of the
8 expression.” Id.
9 CVC Response: Denied but acknowledged that the exhibit contains the partially
10 quoted text.
11 80. Jinek 2012 was said to have “suggested the ‘exciting possibility’ that CRISPR-Cas9
12 complexes might constitute a simple and versatile RNA-directed system” that could be
13 operative in eukaryotic cells. Ex. 3110 at 14:16-20, citing Ex. 3233 at 1-2.
14 CVC Response: Denied but acknowledged that the exhibit contains the partially
15 quoted text.
16 81. In his September 2012 article reviewing the Jinek 2012 work, Dr. Dana Carroll noted that
17 CRISPR-Cas9 may fall prey to RNA degradation or be hindered by chromatin in
18 eukaryotic cell, and that experiments would be necessary to assess whether CRISPR-
19 Cas9 could operate in eukaryotic cells. Ex. 3286 at 1660.
20 CVC Response: Denied
21 82. In 2014, Dr. Doudna stated that: “‘Our [Jinek] 2012 paper was a big success, but there
22 was a problem. We weren’t sure if CRISPR/Cas9 would work in eukaryotes—plant and
23 animal cells.’ Unlike bacteria, plant and animal cells have a cell nucleus, and inside,
-2-17 - Interference No. 106,115 CVC Reply 2
1 DNA is stored in a tightly wound form, bound in a structure called chromatin.” Ex. 3287
2 at 3.
3 CVC Response: Denied.
4 83. In April 2011, the CVC inventors reached out to multiple researchers with experience
5 using ZFNs and/or TALENs to edit genomic DNA in a variety of eukaryotic cells for
6 assistance with CVC’s attempts to use CRISPR in eukaryotic cells.
7 CVC Response: Denied
8 84. The CVC inventors conducted experiments in C. elegans from March to September 2012.
9 CVC Response: Denied
10 85. Regarding the C. elegans work, Jinek expressed that there were “just too many
11 parameters to optimize.” Ex. 5119; see also Ex. 6207, Jinek Tr. 120:10-12.
12 CVC Response: Denied but acknowledged that the exhibit contains the partially
13 quoted text.
14 86. Jinek testified that the parameters to optimize included the concentration of guide RNA,
15 concentration of Cas9 protein, concentration of mRNA, the buffer composition, and the
16 volumes of reagents. Id. at 121:14-21.
17 CVC Response: Admitted.
18 87. Doudna states in an August 20 email that she “wonder[ed] if the Cas9:RNA complex is
19 either falling apart during or after injection, or if the concentration is too low.” Ex. 4941.
20 CVC Response: Denied but acknowledged that the exhibit contains the partially
21 quoted text.
22 88. Doudna and Meyer acknowledged in an October 2013 publication that their initial
23 attempts with sgRNA were not successful. Ex. 3656 at 343 (“Our initial attempts at
-2-18 - Interference No. 106,115 CVC Reply 2
1 genome editing using Cas9-sgRNA complexes to target the ben-1 locus with three
2 different sgRNAs in multiple experiments were unsuccessful.”).
3 CVC Response: Denied
4 89. Doudna and Meyer state in Ex. 3656 that “We reasoned that dual crRNA:tracrRNA RNA
5 guides might be more effective in vivo for C. elegans than single chimeric sgRNA
6 guides, as observed in some cases for mammalian cells (Cong et al. 2013). . . . We found
7 the dual-crRNA:tracrRNA:Cas9 cleavage reactions to be more efficient than the
8 sgRNA:Cas9 cleavage reactions.”
9 CVC Response: Denied but acknowledged that the exhibit contains the partially
10 quoted text.
11 90. Raible testified that he took the photographs documenting the subject fish in the
12 afternoon on August 9, 2012. Raible Tr., Ex. 6211 at 153:10-14.
13 CVC Response: Admitted.
14 91. Raible testified that he told Chylinski of the results of the subject fish “[w]ithin a few
15 days of documenting this fish.” Ex. 4294 Raible ¶57.
16 CVC Response: Admitted that the full quote in Exhibit 4294, ¶57 states, “Within
17 a few days of documenting this fish, I told Dr. Chylinski of the successful
18 results.” (emphasis added).
19 92. The CVC inventors never reported any success in 2012 using CRISPR-Cas9 in yeast,
20 mice, plants, or medaka fish.
21 CVC Response: Denied
22 93. Doudna could not recall anyone telling her in 2012 that the team had achieved success
23 using CRISPR-Cas9 in fish cells. Ex. 6204, Doudna Tr. 169:10-15.
-2-19 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Admitted.
2 94. On June 28, 2012, Raible wrote to Charpentier that if the fish experiments were a
3 success, “At least, one would expect to generate mutations that could be screened by a
4 PCR approach”; “As competition on this will likely be fierce, we feel that the most
5 efficient procedure would be the following . . .”; and “Given the massive interest in
6 simple methods for genome editing, we would expect that the establishment of a
7 CRISPR/CAS-based genome editing system in any fish system would be of broad
8 interest, and therefore a short article in a high-impact journal would not be unlikely as a
9 result (provided the results match the expectations based on the in vivo data).” Exs. 4799;
10 4802.
11 CVC Response: Denied but acknowledged that the exhibit contains the partially
12 quoted text.
13 95. The CVC inventors’ zebrafish experiments were never published.
14 CVC Response: Denied
15 96. Charpentier testified that she “d[id] not have a specific recollection whether I was in
16 correspondence with Jennifer and Martin between the sending by Chris of these slides
17 and my visit in Berkeley. Whether I communicated during that time this data or during
18 my visit, I don’t - - I don’t recollect, but I surely -- I surely provided an update at some
19 point around this time.” Ex. 6206 at 236:15-21.
20 CVC Response: Admitted.
21 97. In setting up the zebrafish experiment, Florian Raible said that, if the experiment were a
22 success, “[a]t least, one would expect to generate mutations that could be screened by a
23 PCR approach.” Ex. 4799.
-2-20 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Denied but acknowledged that the exhibit contains the partially
2 quoted text.
3 98. Both Chylinski and Raible concluded after two sets of PCR analysis in the zebrafish
4 experiments that there was “no effect visible.” Exs. 4916 at 10; 4294, Raible Dec. ¶¶65-
5 68.
6 CVC Response: Denied
7 99. Chylinski’s August 31 presentation states that phenotypic changes “might be unspecific.”
8 Ex. 4916 at 10.
9 CVC Response: Denied
10 100. On August 9, Chylinski reported to Charpentier of the fish experiments that there was “a
11 hint it might work but we shouldn’t be overexcited now.” Ex. 4911.
12 CVC Response: Admitted.
13 101. Dr. Chylinski’s reference to “[p]otentially good news about fish” and the “hint it might
14 work” were made in reference to experiments in medaka not zebrafish. Id. (“It looks like
15 GFP expression in medaka is much lower in the embryo although there are still problems
16 with toxicity and so on, so it will require some more optimization from their site.
17 Anyway, there is a hint it might work but we shouldn't be overexcited now.”).
18 CVC Response: Denied but acknowledged that the exhibit contains the partially
19 quoted text.
20 102. For human cell experiments, the CVC inventors enlisted the help of Drubin and his
21 graduate student Aaron Cheng, who had published results successfully using ZFNs to edit
22 the eukaryotic genome. Ex. 4384.
23 CVC Response: Denied
-2-21 - Interference No. 106,115 CVC Reply 2
1 103. The CVC inventors suffered multiple failures from March to October 2012 attempting to
2 use CRISPR in eukaryotic cells, as documented by Surveyor assay results showing
3 successful cleavage with TALENs but failure with CRISPR-Cas9. Ex. 4352, Cheng Dec.
4 CVC Response: Denied
5 104. From March to October 2012, the CVC inventors tried adjusting and varying features of
6 the CRISPR system and experimental design to get the system to work. Ex. 6201, Cheng
7 Tr. 79:9-17 (expression strategy); 92:11-19 (plasmid concentration); 92:20-93:6 (codon
8 optimized Cas9); 94:8-14 (varying temperatures); 158:17-159:3 (harvesting cells after
9 different time periods); 156:3-10 (staggering transfections); 81:6-20; 82:8-15; Ex. 5070
10 (changing from Pol III promoter to Pol II promoter).
11 CVC Response: Denied.
12 105. On October 11, 2012 at 5:15 AM, Doudna wrote to East and Cheng, copying Jinek, and
13 Ma stating
14 Hi Alex and Aaron – thanks for sending your results, although it’s
15 disappointing not to see Cas9-mediated cleavage in these
16 experiments. Aaron, I’m wondering if you think there is anything
17 different about the way you did the experiment back in August,
18 when it appeared that there was some degree of cleavage with the
19 CLTA6 guide? Or could that result have been due to a
20 contamination, say with the ZFN sample -? And it will be
21 interesting to see the result from the RNA transfection experiment.
22 Is it worth trying the transfection again with the codon-optimized
23 Cas9? As we have discussed, I still think the problem may be with
-2-22 - Interference No. 106,115 CVC Reply 2
1 the assembly and localization of the Cas9 RNP – either due to
2 degradation of the guide RNA, failure to assemble with Cas9 or
3 failure of the RNP nuclear localization. I will think on this on my
4 way back to SF tonight, and we can meet soon to discuss.
5 Ex. 5043.
6 CVC Response: Admitted.
7 106. On October 11, 2012, Jinek stated in part as follows:
8 Based on the latest set of results, I suspect we have a problem with
9 our RNA design. Either we are not targeting the right piece of
10 DNA (due to chromatin structure etc), or the problem lies with the
11 RNA design per se. Given that ZFN has no problems cleaving the
12 same region (+/- 30 bp), the former is probably a lesser concern at
13 this point. On the other hand, there could be a number of reasons
14 for the later including:
15 -RNA is not made at sufficient levels.
16 -RNA is expressed strongly, but turns over too fast to associate
17 with Cas9 - posibly [sic] due to degradation by exonucleases.
18 -RNA is stable but does not associate with Cas9 at the right place
19 and at the right time.
20 Ex. 5040.
21 CVC Response: Admitted.
22 107. In response, Doudna stated in part:
-2-23 - Interference No. 106,115 CVC Reply 2
1 As for Cas9 in mammalian cells, I completely agree with your
2 analysis and suspect that one or more aspects of the RNA
3 expression/stability/Cas9 assembly/localization are problematic.
4 Ex. 5041.
5 CVC Response: Admitted.
6 108. The CVC team reported that they “did not detect any evidence of genomic cleavage” on
7 July 31, 2012 (Ex. 4352 ¶53.).
8 CVC Response: Denied but acknowledged that the exhibit contains the partially
9 quoted text.
10 109. Doudna reported that “it's disappointing not to see Cas9-mediated cleavage in these
11 experiments” on October 11 (Ex. 5043).
12 CVC Response: Admitted.
13 110. Dr. Zhang had submitted his manuscript for the Cong 2013 paper to Science by October
14 5, 2012. See Ex. 3564.
15 CVC Response: Admitted that the date October 5, 2012 appears in Ex. 3564,
16 otherwise denied.
17 111. The PTAB noted in it is Decision on Motions that “Dr. Carroll stated [in 2012] that actual
18 experiments were necessary to address concerns about chromatin structure and RNA
19 stability and to determine if CRISPR-Cas9 systems would work in eukaryotic cells.”
20 Decision at 100:19-22.
21 CVC Response: Admitted.
22 112. The PTAB also concluded TALENs and ZFNs would not have provided a reasonable
23 expectation of success:
-2-24 - Interference No. 106,115 CVC Reply 2
1 Thus, at the relevant time, Dr. Carroll did not consider the ZFN or
2 TALEN systems to be analogous or relevant to the question of
3 whether CRISPR-Cas9 would work in eukaryotic cells. As
4 explained above, we give this contemporaneous evidence
5 significant weight. Accordingly, we are not persuaded that the
6 successful use of ZFN and TALEN systems would have given
7 those of skill in the art a reasonable expectation that CRISPR-Cas9
8 could also have been used successfully in eukaryotic cells.
9 Ex. 3110 at 41:13-19.
10 CVC Response: Denied but acknowledged that the exhibit contains the partially quoted
11 text.
12 113. When asked in his 2020 interview how confident he was in 2012 that CRISPR- Cas9
13 would work in human cells, Marraffini said, “I don’t think we would have stopped doing
14 the experiments just because it seemed unlikely that it was going to work. At the same
15 time, human chromatin is very complex. What if Cas9 is kicked off the human DNA by
16 all these structures that are not present in phages or bacterial chromosomes? It might have
17 not worked.” Ex. 4320 at 8.
18 CVC Response: Denied but acknowledged that the exhibit contains the partially
19 quoted text.
20 114. Dr. Caixia Gao did not expect CRISPR-Cas9 to work in eukaryotic cells after reading
21 Jinek 2012 given the very different way TALENs work vs. CRISPR. Ex. 3446 ¶6.
22 CVC Response: Denied
-2-25 - Interference No. 106,115 CVC Reply 2
1 115. Dr. Gao attempted for months to get the Chimera A of Jinek 2012 to work in eukaryotic
2 cells, to no avail. Id. ¶¶12-13.
3 CVC Response: Denied
4 116. It was only after reading Cong 2013 that Dr. Gao was able to get CRISPR-Cas9 to work
5 in plant cells. Id. at ¶14.
6 CVC Response: Denied
7 117. CVC has not identified a single third-party POSA or expert who in 2012 expected
8 CRISPR-Cas9 to work in eukaryotic cells.
9 CVC Response: Denied
10 118. The 2012 POSA would have known of the many challenges and uncertainties that can
11 and do preclude prokaryotic, RNA-dependent systems from operating in eukaryotic cells.
12 Breaker 3rd Dec. ¶ 104-174.
13 CVC Response: Denied
14 119. A POSA would have appreciated that the protein-only ZFNs and TALENs, whose DNA
15 binding domains are eukaryotic or have evolved to target chromatinized DNA over
16 millions of years, did not face these challenges. Breaker 3rd Dec. ¶¶92-101.
17 CVC Response: Denied
18 120. In an interview titled Major Insights into Microbiology: An Interview with Luciano
19 Marraffini, published in 2020 in The CRISPR Journal, Luciano Marraffini said, “As you
20 can imagine, transporting the [CRISPR-Cas9] system from bacteria into human cells,
21 you’re a little bit in the dark. You need to add nuclear localization signals to target
22 different targets. Everything seems very simple now. But at the time, we did not know.
23 Ex. 4320 at 8.
-2-26 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Admitted.
2 121. Diagnosing in 2012 why a CRISPR system was failing in a eukaryotic cell and what
3 would be the next potential step to attempt would be time consuming, arduous and
4 require a high degree of skill. Breaker 3rd Dec. ¶¶38-43, 104-106.
5 CVC Response: Denied
6 122. The closest prior art system comprising, like CRISPR-Cas9, a protein component and
7 RNA component is the group II intron system. Breaker 3rd Dec. ¶44-58.
8 CVC Response: Denied
9 123. Group II introns are very inefficient in eukaryotes and technical issues encountered with
10 group II introns would have indicated to a 2012 POSA that CRISPR-Cas9 systems would
11 not work in eukaryotic cells without a description of specific conditions needed for
12 adaptation. Decision at 88-91.
13 CVC Response: Denied
14 124. Jinek 2013 eLife article, states that after Jinek 2012 “it was not known whether such a
15 bacterial system [CRISPR] would function in eukaryotic cells.” Decision at 101:17-18
16 (quoting Ex. 4137, 1–2).
17 CVC Response: Denied but acknowledged that the exhibit contains the partially
18 quoted text.
19 125. The 2014 Berkeley Catalyst publication attributes to Doudna the statement that “Our
20 2012 paper [showing that the Cas9 endonuclease family can be programmed with single
21 RNA molecules to cleave specific DNA sites,] was a big success, but there was a
22 problem. We weren’t sure if CRISPR/Cas9 would work in eukaryotes—plant and animal
23 cells. Unlike bacteria, plant and animal cells have a cell nucleus, and inside, DNA is
-2-27 - Interference No. 106,115 CVC Reply 2
1 stored in a tightly wound form, bound in a structure called chromatin.” Id. at 101:19-
2 102:5 (quoting Ex. 3287 at 3).
3 CVC Response: Denied but acknowledged that the exhibit contains the partially
4 quoted text
5 126. The CVC inventors shared with the 2012 POSA numerous concerns about problems that
6 could interfere with a successful, operative eukaryotic system. Ex. 5040; Ex. 5041; Ex.
7 5043; Ex. 4941; Ex. 4988; Ex. 5040; Ex. 5078; Ex. 5060; Ex. 4799; Ex. 4916.
8 CVC Response: Denied
9 127. In October 2012, the CVC inventors considered “various alternate RNA designs for
10 targeting in cells” (Ex. 5041) and needed to test “critical control[s]” such as whether their
11 Cas9 was even active in case they needed to “prepar[e] some of the other Cas9's for
12 mammalian expression . . . ” Ex. 5053.
13 CVC Response: Denied
14 128. Jinek testified that as of March 1 for human cells “Well, these would be delivery using —
15 using DNA constructs. These could be delivery using mRNAs, delivery using purified
16 Cas9 protein, plus synthetic or in vitro transcribed sgRNAs. There may have been others,
17 but I don’t — I don’t recall.” Ex. 6207, Jinek Tr. 272:16-17; 273:4-9.
18 CVC Response: Admitted that the partially quoted text appears in Ex. 6207,
19 otherwise denied.
20 129. The CVC inventors enlisted Dr. Meyer to attempt to use their CRISPR-Cas9 system in
21 worm (C. elegans) cells, but after experimenting from March to September, the project
22 ended in failure.
23 CVC Response: Denied
-2-28 - Interference No. 106,115 CVC Reply 2
1 130. On or about September 14, 2012, in an email entitled “no good news,” Cheng wrote to
2 Doudna and Jinek reporting he saw “no cleavage for any RNA chimeras, despite using
3 codon-optimized Cas9 constructs this time.” Ex. 4988.
4 CVC Response: Admitted
5 131. Doudna responded that “Since there are so many variables in these experiments, I think
6 we have to try to move forward in a stepwise fashion as much as possible.” Id.
7 CVC Response: Admitted.
8 132. Doudna further stated that “[a]s for RNA localization, I think we’re hoping that the Cas9
9 protein binds the RNA such that the RNP is transported into the nucleus. I wonder if
10 having a too-efficient NLS on Cas9 is actually counterproductive, if it means that Cas9 is
11 transported before it has a chance to find and bind the guide RNA… Thoughts?” Id.
12 CVC Response: Admitted.
13 133. The chimera A design showcased by CVC and disclosed in Jinek 2012—a design with a
14 26 nucleotide tracr sequence—is not what results after expression from a U6 promoter
15 driven plasmid in eukaryotic cells.
16 CVC Response: Denied
17 134. The CVC inventors who recruited Dr. Raible as a collaborator to perform experiments in
18 zebrafish (Dr. Chylinski and Dr. Charpentier) never told their co-inventors in 2012 that
19 any zebrafish experiment succeeded.
20 CVC Response: Denied
21 135. In the prior 048 Interference, CVC never alleged that the August 9 zebrafish experiment
22 was a successful ARTP, even though the 048 count was directed to use of CRISPR in a
23 eukaryotic cell. Ex. 3114.
-2-29 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Denied
2 136. In 2012, the zebrafish model was a “well-established” system for gene editing
3 experiments. Ex. 4799; Ex. 3447, Dec. ¶¶78-91.
4 CVC Response: Admitted
5 137. The model involves injecting batches of zebrafish with the gene-editing system and then
6 examining the fish at various points after injection for specific phenotypes that might be
7 indicative of a mutation, and appropriate and sufficient molecular testing must be
8 employed to determine whether or not targeted cleavage actually occurred. Mourrain
9 Dec. ¶¶24-74.
10 CVC Response: Denied
11 138. In accordance with this standard procedure, Dr. Raible advised the CVC inventors at the
12 outset of the zebrafish experiments that if they were a success, “[a]t least, one would
13 expect to generate mutations that could be screened by a PCR approach.” Ex. 4799; see
14 also Ex. 6204, Doudna Tr. 156:17-21.
15 CVC Response: Denied but acknowledged that the exhibit contains the partially
16 quoted text.
17 139. Phenotypic changes to a zebrafish alone do not suffice to show gene editing because non-
18 specific effects, such as delayed growth or cell toxicity, that are unrelated to targeted
19 DNA cleavage can cause apparent morphological changes. See, e.g., Mourrain Dec. ¶¶9,
20 15, 42.
21 CVC Response: Denied
22 140. CVC’s expert Dr. Moens agrees that “[n]onspecific toxicity” can “cause a spectrum of
23 defects.” Ex. 4343, Moens Dec. ¶53.
-2-30 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Denied but acknowledged that the exhibit contains the partially
2 quoted text
3 141. As of 2012, those of skill in the zebrafish art recognized the need for appropriate and
4 sufficient molecular testing to determine whether there was successful targeted cleavage.
5 Mourrain Dec. ¶¶24-30.
6 CVC Response: Denied
7 142. Dr. Raible testified that he followed “established strategy employed in the 2008 Doyon et
8 al. paper for ZFN-mediated mutagenesis.” Ex. 4294, Raible Dec. ¶23.
9 CVC Response: Admitted that the partially quoted text appears in Ex. 4294,
10 otherwise denied.
11 143. The 2008 Doyon paper used PCR to determine whether or not observed phenotypic
12 changes resulted from editing at target sites. Ex. 4424 at 706; Ex 3447, Mourrain Dec.
13 ¶25.
14 CVC Response: Denied
15 144. Dr. Raible adopted the same protocol for his CRISPR-Cas9 zebrafish experiments,
16 requiring at least positive PCR results to show success. Ex. 4799.
17 CVC Response: Denied
18 145. Dr. Raible followed the established protocol to see whether a “putative mutant,”
19 identified as an “eyeless fish among ~ 30 sibs” (Ex. 4338 at 80) was caused by targeted
20 gene editing or other factors. See, Ex. 4294, Raible Dec. ¶¶64-68.
21 CVC Response: Denied but acknowledged that the exhibit contains the partially
22 quoted text.
-2-31 - Interference No. 106,115 CVC Reply 2
1 146. The PCR results were negative as Dr. Chylinski reported in his August 31 presentation.
2 Ex. 4916 at 10. As reported, the PCR tests had “no effect visible.”
3 CVC Response: Denied but acknowledged that the exhibit contains the partially
4 quoted text.
5 147. Chylinski’s August 21 slide states that the putative mutant “might be unspecific,” i.e., not
6 the result of targeted gene cleavage by CRISPR-Cas9, and that PCR was negative. Ex.
7 4916 at 10.
8 CVC Response: Denied
9 148. On August 22, Raible injected more batches of fish, many of which died due to cell
10 toxicity. Exs, 4294 ¶70; 4337.
11 CVC Response: Admitted that Raible observed that some of the fish died but did
12 not determine cause of death.
13 149. On August 23 Raible observed “1 animal without eyes, but looks also retarded.” Exs.
14 4337; 4338 at 83.
15 CVC Response: Admitted.
16 150. Raible injected more batches of zebrafish on August 29 followed by PCR in September
17 2012, again with no success. Ex. 4294 ¶73.
18 CVC Response: Denied
19 151. With the images of the alleged chokh mutant provided by CVC, a POSA cannot conclude
20 the fish is a chokh mutant using the generally accepted criteria. Mourrain Dec. ¶¶101-10.
21 CVC Response: Denied
22 152. On or around September 14, 2012, in an email titled “no good news”, Cheng wrote to
23 Doudna and Jinek and said,
-2-32 - Interference No. 106,115 CVC Reply 2
1 Unfortunately, no cleavage for any RNA chimeras, despite using
2 the codon-optimized Cas9 constructs this time. See attached.
3 Quick thoughts: Martin - were you ever able to do some northerns
4 to see if the RNAs are being expressed robustly and intact?
5 Jennifer/Doudna - do we know for sure that the RNAs are
6 localized to the nucleus? if not, is there a motif we can append to
7 make sure that it does? :(
8 Ex. 4988 at 1-2.
9 CVC Response: Admitted.
10 153. On or around September 14, 2012, Doudna responded to Cheng suggesting exploring the
11 Cas9 construct stating,
12 Hi Aaron - taking a step back, have you tried simply repeating the
13 original experiment using increasing amounts of the plasmids
14 encoding the chimeric RNA and the original Cas9 construct? We
15 discussed this when we met in David’s office a few weeks ago, and
16 I think this is important to do, just to show reproducibility and rule
17 out any possible ZFN contamination, etc. Once you have done this
18 and assuming you see the same result as before, you can then do a
19 side by side expt with the original and new versions of the Cas9
20 construct. Since there are so many variables in these experiments, I
21 think we have to try to move forward in a stepwise fashion as
22 much as possible.
-2-33 - Interference No. 106,115 CVC Reply 2
1 As for RNA localization [to the nucleus], I think we’re hoping that
2 the Cas9 protein binds the RNA such that the RNP is transported
3 into the nucleus. I wonder if having a too-efficient NLS on Cas9 is
4 actually counterproductive, if it means that Cas9 is transported
5 before it has a chance to find and bind the guide RNA…
6 Thoughts?
7 Ex. 4988 at 1.
8 CVC Response: Admitted.
9 154. On October 10, 2012, Cheng wrote Doudna regarding the October 3 surveyor and said,
10 “[t]he ZFN control worked, but no observed cleavage for the Cas-RNA complexes.” Ex.
11 5044.
12 CVC Response: Admitted.
13 155. Regarding the October 10, 2012 surveyor, Cheng said, “[a]s can be seen in the gels below
14 and on pages 68 and 69 of my lab notebook, there was no evidence of genomic cleavage
15 in any of the samples.” Ex. 4352, Cheng Dec. ¶130; see also Ex. 4365 at, 68-69.
16 CVC Response: Admitted.
17 156. On or around October 17, 2012, Doudna wrote an email to East, copying Ma and Jinek,
18 and said, “Hi Alex – I think that doing the experiment with cell extracts to test whether
19 the transfected Cas9 is active is a critical control. We should perhaps also be preparing
20 some of the other Cas9’s for mammalian expression, in case they work better for some
21 reason (i.e. folding or faster/better RNP assembly). Let’s discuss either later today or
22 tomorrow. Best – Jennifer.” Ex. 5167.
23 CVC Response: Admitted.
-2-34 - Interference No. 106,115 CVC Reply 2
1 157. Prior to October 24, 2012, Jinek admitted in his testimony that “I expected and believed
2 that the - that the sgRNAs would - would be functional, but up to that point we could not
3 detect DNA cleavage.” Jinek Interview Day 1, Ex. 6207 at 190:2-5.
4 CVC Response: Denied but acknowledged that the exhibit contains the partially
5 quoted text.
6 158. Prior to October, CVC had used a lysing method that immediately deactivated Cas9 so
7 that no cleavage could occur in the lysate and a positive PCR result would indicate
8 cutting in the cell. Ex. 3448, Breaker 3rd Dec. ¶¶ 254-261, 380-395.
9 CVC Response: Denied.
10 159. For all of CVC’s alleged ARTPs, Jinek used the lysing procedure shown in Exhibit 4382
11 at 26-27. Id. at ¶¶ 254-261, 313-375.
12 CVC Response: Denied
13 160. The “1x lysis buffer” on page 26-27 of Exhibit 4382 includes Roche protease inhibitors
14 and glycerol.
15 CVC Response: Admitted that this is a partial list of components of the 1x lysis
16 buffer.
17 161. No conditions for storage or preservation of the lysates produced by the lysing procedure
18 shown in Exhibit 4382 at 26-27 are documented by Jinek or East. Id. at ¶¶ 287-296.
19 CVC Response: Denied.
20 162. After months of failures, however, Jinek decided to run in vitro assays on the cell lysate
21 to determine if Cas9 expressed in human cells could complex with sgRNA and then cut
22 an exogenous DNA target. Ex. 4382 at 26; Ex. 4349, Jinek Tr. ¶ 238.
23 CVC Response: Denied.
-2-35 - Interference No. 106,115 CVC Reply 2
1 163. To conduct this in vitro assay experiment, he changed to a gentle lysate method designed
2 to keep proteins active in the lysate. Ex. 3448, Breaker 3rd Dec. ¶¶ 254-261, 380-395.
3 CVC Response: Denied.
4 164. Gentle lysing keeps the Cas9 and the DNA repair-mechanism proteins active, meaning
5 that it is possible for cleavage to occur outside the cell during and after the lysing step. Id.
6 at ¶¶ 254-309.
7 CVC Response: Denied
8 165. For the gentle lysing, Jinek eliminated any proteinase enzyme from the lysing process,
9 added a Roche protease inhibitor, and used a buffer that approximates the pH and ionic
10 conditions present in cells, suitable for maintaining enzymes and other protein factors
11 intact. Ex. 4382 at 26-27; Ex. 3448, Breaker 3rd Dec. ¶¶ 299-304.
12 CVC Response: Denied.
13 166. With a gentle lysing process, one cannot know whether any positive PCR resulted from
14 cleavage and NHEJ that occurred in the cell. Ex. 3448, Breaker 3rd Dec. ¶¶ 254-309.
15 CVC Response: Denied
16 167. A typical lysing procedure for genomic DNA testing includes deactivating the Cas9
17 protein as the cells are lysed, which precludes Cas9-mediated cleavage in the lysate. Id. at
18 268-286.
19 CVC Response: Denied
20 168. DNA released from the cell by lysing can be cleaved in the lysate thereby producing a
21 “positive” in the Surveyor assay that cannot distinguish between cleavage that happened
22 in the cell and cleavage that occurred in the lysate. Id.
23 CVC Response: Denied
-2-36 - Interference No. 106,115 CVC Reply 2
1 169. On October 19, Dr. Doudna advised Jinek and East that “doing the experiment with cell
2 extracts to test whether the transfected Cas9 is active is a critical control.” Ex. 5053.
3 CVC Response: Denied but acknowledged that the exhibit contains the partially
4 quoted text.
5 170. Jinek gave a portion of the gentle lysate to East to conduct PCR Surveyor assays to
6 determine if cleavage of the target DNA had occurred in the cell.
7 CVC Response: Unable to admit or deny due to lack of evidence cited in the
8 statement.
9 171. Dr. Jinek explained that he gave East a portion of the lysate for her analysis of genomic
10 DNA cleavage in part “because I had more lysate than I needed….” Ex. 4349, Jinek Dec.
11 ¶¶238-39.
12 CVC Response: Denied but acknowledged that the exhibit contains the partially
13 quoted text.
14 172. The potential cleavage activity in lysate created confounding problems for determining
15 whether targeted cleavage had occurred in the cell prior to lysing. Ex. 3448, Breaker 3rd
16 Dec. ¶¶ 287-312.
17 CVC Response: Denied
18 173. Jinek showed that CRISPR-Cas9 systems expressed in the transfected human cells were
19 able to cleave genomic DNA after the cells were lysed. Ex 4349, Jinek Dec. ¶253.
20 CVC Response: Denied
21 174. The gentle lysing protocol also preserves the activity of the enzymes that are responsible
22 for NHEJ and so those repairs/edits can likewise occur in the active lysate. Id. at ¶¶ 267-
23 277.
-2-37- Interference No. 106,115 CVC Reply 2
1 CVC Response: Denied
2 175. Examples of these lysis and extract preparation methods are described in numerous
3 papers cited in a 2010 review entitled “Nonhomologous DNA End Joining in Cell-Free
4 Extracts.” Ex. 6312.
5 CVC Response: Denied
6 176. Dr. Breaker explains that a cell lysis and extract preparation protocol similar to that
7 prepared by Jinek detailed below demonstrated NHEJ activity in cell free systems. Ex.
8 3448, Breaker 3rd Dec. ¶¶ 299-304.
9 CVC Response: Denied
10 177. CVC’s first alleged ARTP on October 31 was based on a PCR test of sample where Jinek
11 had used the gentle lysing method. Id. at ¶¶ 313-379.
12 CVC Response: Admitted that CVC achieved a successful ARTP on October 31,
13 otherwise denied.
14 178. All of the cell extracts given to East for PCR Surveyor assay analysis for CVC’s alleged
15 ARTPs retained active Cas9 protein and sgRNA for some period of time after the cells
16 were gently lysed. Id. ¶¶ 313-379.
17 CVC Response: Denied
18 179. On September 14, Cheng used a chimeric RNA with U6 promoter to attempt to cleave the
19 CLTA6 target in U2-OS cells but reported, after following standard lysing protocols, that
20 “there was no evidence of genomic cleavage.” Ex. 4352, Cheng Dec. ¶102; Ex. 6201,
21 Cheng Tr. 92:1-10 (confirming his use of a U6 promoter in his U2-OS experiments).
22 CVC Response: Denied but acknowledged that the exhibit contains the partially
23 quoted text.
- 2-38 - Interference No. 106,115 CVC Reply 2
1 180. On November 1, 2012 East used a system with chimeric RNA with U6 promoter to target
2 CLTA6 in U2-OS cell) and reports a success after using the gentle lysing method. Ex,
3 4353, East Dec. ¶102.
4 CVC Response: Admitted that East used a system with chimeric RNA with U6
5 promoter to target CLTA6 in U2-OS cell and achieved a successful ARTP on
6 November 1.
7 181. CVC’s earliest alleged ARTP in human cells is based on an October 29 email from East
8 reporting an “unexpected singleton band,” and stating that she needed to “repeat both the
9 PCR and nuclease cleavage steps to confirm this isn’t a weird PCR byproduct.” Ex. 4488,
10 see also Ex. 5242.
11 CVC Response: Admitted that CVC’s October 31 ARTP is based in part on the
12 October 29 email Ex. 4488.
13 182. Doudna’s recent biography states as follows:
14 “When [East] showed me the data, it was immediately clear to me
15 that she had beautiful evidence of genome editing by Cas9 in the
16 human cells,” Doudna says. “This is a classic difference between a
17 student who is in training and someone like me who’s been doing
18 this for a while. I knew what I was looking for, and when I saw the
19 data she had, it just clicked and I thought, ‘Yes, she’s got it.’
20 Whereas she was unsure and thought she might have to do the
21 experiments again, I was saying, ‘Oh my gosh, this is huge! This is
22 so exciting!’”
23 Ex. 3681 at 189.
- 2-39 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Admitted.
2 183. In an October 29 email, Doudna posed a question to East regarding whether the band was
3 due to Cas9-cleaved DNA:
4 Hi Alex - this looks interesting! Is it possible that you would get a
5 singleton band due to the way the Cas9-cleaved DNA is repaired?
6 The +/- nuclease control will be good to see.
7 Ex. 5072.
8 CVC Response: Admitted.
9 184. On Tuesday October, 30, at Dr. Doudna’s request, Ms. East performed a further Surveyor
10 assay with a new PCR product that she made using her gDNA from Dr. Jinek’s lystates
11 (she also reran the sample from Monday October 29). Ex. 4366 at 34-35.
12 CVC Response: Admitted.
13 185. East summarized her results to Doudna and Jinek, the same day:
14 Here are the results from this afternoon, which are not particularly
15 conclusive. The singleton band was reproducible from the PCR
16 reaction I performed yesterday, but only a smear appears in the
17 PCR I performed today.
18 Ex. 5083.
19 CVC Response: Admitted.
20 186. Dr. Doudna’s response was that the PCR results “might be consistent with the position of
21 the Cas9 cleavage site being shifted relative to the ZFN cleavage site.” Id.
22 CVC Response: Admitted.
- 2-40 - Interference No. 106,115 CVC Reply 2
1 187. Ms. East ran a subsequent experiment showing signs of a weak band, which led Dr.
2 Doudna to respond on November 5: “Woo hoo, looks good Alex! Can’t wait to see RFP
3 and protein transfection data.” Ex. 5171.
4 CVC Response: Admitted that CVC achieved a successful ARTP on November
5 5, and that the partially quoted text appears in Ex. 5171; otherwise denied.
6 188. On November 7, Doudna advised Charpentier:
7 We are making some progress here with human cells, and should
8 have more data to discuss soon. I’d like to get a couple more
9 replicate experiments done to confirm what we are seeing, and
10 then will be in touch to set up a phonecall or Skype.
11 Ex. 5092 at 1; see also Ex. 4351, Charpentier Dec. ¶75.
12 CVC Response: Admitted.
13 189. On November 11, 2012 after further tests, Dr. Doudna inquired on the status of further
14 testing. Ex. 5096.
15 CVC Response: Unable to admit or deny.
16 190. Dr. Jinek responded to Dr. Doudna as follows:
17 I have processed the lysates over the weekend but have not had
18 time to run the final gel. I will do first thing tomorrow morning.
19 Alex has done the Surveyor assay over the weekend and should
20 have sent you her results by now. Basically, it seems that the
21 original U6-promoter constructs give us some hints of activity
22 while the new CMV constructs did not really work.
23 Id.
- 2-41 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Admitted that the partially quoted text appears in Ex. 5096.
2 191. On November 17, 2012 Dr. Doudna stated in Ex. 5106:
3 Hi Martin - thanks for the update, although too bad about the
4 assay. Hopefully the next round and/or next transfections work out
5 better. Seems like the cells are particularly sensitive to these
6 constructs, perhaps? Something to investigate in future
7 experiments…
8 Ex. 5106.
9 CVC Response: Admitted that the partially quoted text appears in Ex. 5106.
10 192. As of mid-November, Doudna, heard from a colleague, that CRISPR-Cas9 could function
11 in eukaryotic cells:
12 “I hope you’re sitting down,” an excited colleague told Doudna in
13 an unexpected phone call. “CRISPR is turning out to be
14 absolutely spectacular in [Harvard geneticist] George Church’s
15 hands.” He had even gotten it to work in human cells. Thrilled,
16 Doudna immediately contacted Church.
17 Ex. 3203 at 3.
18 CVC Response: Denied but acknowledged that the exhibit contains the partially
19 quoted text.
20 193. On November 14, Dr. Doudna wrote to Dr. Church stating “We will be very interested to
21 hear how your experiments progress. And yes, there is a lot of interest in Cas9 at the
22 moment - we are hopeful that it will turn out to be useful for genome editing and
23 regulation in various cell types.” Ex. 5235.
- 2-42 - Interference No. 106,115 CVC Reply 2
1 CVC Response: Admitted that the partially quoted text appears in Ex. 5235.
2 194. When Dr. Doudna learned of Church’s progress, “she gave him a call.” Ex. 3681 at 197,
3 ¶2 and “He was gracious and explained the experiments he had done and the paper he had
4 submitted” and he also shared that Zhang also had submitted a manuscript. Id.
5 CVC Response: Denied but acknowledged that the exhibit contains the partially
6 quoted text.
7 195. On December 8, Dr. Church sent Dr. Doudna a draft of Mali et al., stating “I look
8 forward to exchanging ideas about possible practical applications.” Ex. 6060 at 1. When
9 Doudna reviewed the manuscript that Church had sent, Doudna “was deflated.” Ex. 3681
10 at 197, ¶3.
11 CVC Response: Denied but acknowledged that the exhibit contains the partially
12 quoted text.
13 196. On December 10, Dr. Doudna wrote to Dr. Jinek about submitting a short paper to eLife
14 “focused on the results in extracts.” Ex. 5132.
15 CVC Response: Denied but acknowledged that the exhibit contains the partially
16 quoted text.
17 197. Dr. Jinek responded:
18 We could show all this and perhaps say that this prompted us to
19 redesign the RNAs. As a result, we see now detectable DSB
20 formation in vivo using 2nd generation RNAs with the Surveyor
21 assay as our final figure/panel.
22 Ex. 5132.
23 CVC Response: Admitted that the partially quoted text appears in Ex. 5132.
- 2-43 - Interference No. 106,115 CVC Reply 2
1 198. Doudna states her biography regarding the eLife experiments that Jinek “felt what we had
2 wasn’t worth publishing.” Ex. 3681 at 198, ¶4.
3 CVC Response: Admitted that the partially quoted text appears in Ex. 3681.
4 199. Dr. Jinek stated “If we publish this work, we’re going to look like amateurs in the
5 genome editing field.” Id. ¶6.
6 CVC Response: Admitted that the partially quoted text appears in Ex. 3681.
7 200. Dr. Doudna, however, “put her foot down,” rushing experiments with 2nd generation
8 RNA—not relied on here—to eLife, and daily hounding the main scholar who was doing
9 the peer review for her paper, Detlef Weigel at the Max Planck Institute, to prompt
10 acceptance on January 3, 2013, coinciding with online publication in Science of the Cong
11 and Mali articles. Id. at 198-202.
12 CVC Response: Denied.
- 2-44 - Interference No. 106,115 CVC Reply 2
1 CVC Additional Facts 201 - 276
2 201. The earliest date Broad alleges for Zhang’s conception of Count 1 is June 26, 2012. Paper
3 2118, 42:9-10; Ex. 3424, ¶¶126, 168.
4 202. Before June 26, 2012, Jinek had already constructed the pMJ874 vector. See e.g., Ex.
5 4382, 4-7; Ex. 4349, ¶¶124-128; Ex. 4603; Ex. 4345, ¶¶167-172; Ex. 3004, ¶¶408-450.
6 203. The pMJ874 vector uses the U6 promoter to drive expression of the CLTA6 sgRNA
7 targeting a specific CLTA target gene sequence next to a PAM. See e.g., Ex. 4382, 4-7;
8 Ex. 4349, ¶¶124-128; Ex. 4603; Ex. 4345, ¶¶167-172; Ex. 3004, ¶¶408-450.
9 204. Before June 26, 2012, Jinek had constructed a S. pyogenes Cas9 expression vector with a
10 strong CMV promoter, NLSs, and a GFP tag, and had successfully expressed the Cas9
11 protein in human HEK293T cells and visualized nuclear localization of Cas9. See e.g.,
12 Ex. 4349, ¶¶109, 123-127, 155; Ex. 4444; Ex. 4484; Ex. 4536; Ex. 4345, ¶173.
13 205. Before June 26, 2012, the CVC inventors were codon optimizing the Cas9 gene. Paper
14 1579, 18:17-19:4; Ex. 4349, ¶¶109, 123-127, 155; Ex. 4444; Ex. 4484; Ex. 4536; Ex.
15 4345, ¶173.
16 206. The pMJ874 vector that CVC constructed by May 28 is the same vector Jinek and East
17 used in their October and November 2012 experiments in HEK293T cells. See e.g., Paper
18 1579, 17:11-19:9; Ex. 4382, 4-7; Ex. 4349, ¶¶124-128; Ex. 4603; Ex. 4345, ¶¶167-172;
19 Ex. 3004, ¶¶408-450.
20 207. CVC’s S. pyogenes Cas9 vector constructed by May 28 uses the same CMV promoter,
21 encodes the same Cas9 amino acid sequence, and the same NLS that Jinek and East used
22 when reducing the invention to practice in October 2012. Ex. 4349, ¶¶109, 123-127, 155;
23 Ex. 4444; Ex. 4484; Ex. 4536; Ex. 4345, ¶173.
- 2-45 - Interference No. 106,115 CVC Reply 2
1 208. CVC’s vector transfection embodiment comprises a eukaryotic cell (HEK293T cells) and
2 a Type II CRISPR-Cas system comprising a Cas9 protein (S. pyogenes Cas9) and sgRNA
3 (CLTA6 sgRNA). See Paper 1579, 7:17-19:9; Ex. 4345, ¶¶167-174.
4 209. The experiments East and Jinek performed using CLTA sgRNA CRISPR-Cas9 in
5 HEK293T cells in October and November 2012 are the same experiments described in
6 Example 2 of CVC’s P3 application. Ex. 3004, ¶¶408-450; Ex. 4382, 4-7; Ex. 4349,
7 ¶¶124-128; Ex. 4603; Ex. 4345, ¶¶167-172.
8 210. The PTAB stated in its Decision on Motions that CVC’s P3 “is a constructive reduction
9 to practice of Count 1,” “[b]ecause Example 2 provides the protocols necessary and
10 results of a CRISPR-Cas9 system in eukaryotic, human cells.” Paper 877, 106:1-14.
11 211. Before June 26, 2012, CVC contemplated delivering a pre-formed sgRNA-Cas9
12 ribonucleoprotein (RNP) complex to zebrafish embryos using nuclear microinjection.
13 Paper 1579, 14:2-18:4; Ex. 4382, 7; Ex. 4349, ¶¶124-128; Ex. 4603; Ex. 5105, 16-29.
14 212. Before June 26, 2012. CVC constructed a working sgRNA and Cas9 system as a pre-
15 formed RNP complex and confirmed that it was capable of targeting and cleaving at least
16 five different eukaryotic target DNA sequences (within the jellyfish GFP gene) in vitro.
17 Paper 1579, 17:11-19:9; Ex. 4382, 2, 7; Ex. 4349, ¶¶124-128; Ex. 4345, ¶¶167-172.
18 213. Before June 26, 2012, CVC described using microinjection for introducing their sgRNA
19 CRISPR-Cas9 system into eukaryotic cells, citing multiple articles published for ZFN
20 and TALENs that describe nuclear microinjection into zebrafish embryos. Ex. 5105, 2-3,
21 8; Ex. 4349, ¶¶72-74; Ex. 4350, ¶¶57-61; Ex. 4345, ¶¶168-164.
22 214. The pre-formed RNP complex CVC demonstrated to target and cleave eukaryotic gene
23 sequences (GFP) in vitro before June 26, 2012, comprised a chimera A sgRNA and a S.
- 2-46 - Interference No. 106,115 CVC Reply 2
1 pyogenes Cas9 protein. Paper 1579, 17:11-19:9; Ex. 4382, 2, 7; Ex. 4349, ¶¶124-128; Ex.
2 4345, ¶¶167-172.
3 215. Raible’s first impression upon discussing the project with Chylinski was that the
4 reduction to practice “should be a quite rapid procedure, to be done within the next 1-2
5 months” based on the “stunning efficiency of the CRISPR/CAS system … in vitro.” Ex.
6 4799; Ex. 4294, ¶¶14-16.
7 216. Chylinski, Charpentier and Raible all “expected” that injecting CVC’s sgRNA CRISPR-
8 Cas9 system as a pre-formed RNP complex into zebrafish embryos would result in
9 successful target DNA cleavage. Ex. 4348, ¶¶11, 25, 115; Ex. 4351, ¶¶23, 26, 56, 60; Ex.
10 4294, ¶¶10, 13, 14, 74.
11 217. Raible prepared the sgRNA and Cas9 components for his August 9, 2012 experiment in
12 zebrafish as a pre-formed RNP complex comprising a chimera A sgRNA and S. pyogenes
13 Cas9 protein. Ex. 4294, ¶¶8, 54; Ex. 4382, 1-7; Ex. 4349, ¶¶124-128; Ex. 4603; Paper
14 1579, 14:2-17:10.
15 218. Microinjecting a pre-formed sgRNA-Cas9 RNP complex directly to the nucleus of a
16 eukaryotic cell avoids any issues with RNA degradation, complex localization, complex
17 formation, and chromatin. Ex. 4036, ¶¶136-137; Ex. 4343, ¶¶93-106; Ex. 4345, ¶87.
18 219. Microinjection of zebrafish embryos with a pre-formed RNP complex negates any
19 potential impact of chromatin because the zebrafish cells are rapidly dividing. Ex. 4345,
20 ¶113; Ex. 4343, ¶103.
21 220. Breaker testified that, if making recombinant protein outside of a eukaryotic cell
22 (“premade” protein), “then codon optimization would have no effect.” Ex. 5264, 88:15-
23 89:1.
- 2-47 - Interference No. 106,115 CVC Reply 2
1 221. In Chylinski’s June 28 email to Charpentier, he stated that injecting RNPs into zebrafish
2 meant there was “no need for in vivo expression of neither RNA nor protein, codon bias
3 optimization and so on.” Ex. 4796; Ex. 4348, ¶116; Ex. 4351, ¶¶58-61.
4 222. “[D]esigner zinc fingers (ZFs), transcription activator–like effectors (TALEs), and
5 homing meganucleases” are considered “genome-editing technologies.” Ex. 3201, 819.
6 223. The meganuclease, I-SceI, is a gene-editing protein. See Ex. 3201, 819; Ex. 4328; Ex.
7 4622; Ex. 5344; Ex. 4343, ¶87.
8 224. Before 2012, artisans routinely injected I-SceI meganucleases into fish embryos. See Ex.
9 4328; Ex. 4622; Ex. 5344; Ex. 4343, ¶87.
10 225. Luciano Marraffini is a third party not involved in this patent interference proceeding.
11 Ex. 5240.
12 226. Marraffini and Zhang worked together as collaborators on applying CRISPR in
13 prokaryotes and eukaryotes beginning in January 2012 and ending in 2013. Ex. 5265,
14 15:8-15, 17:11-17; Ex. 5262, 179:10-22.
15 227. Broad had the opportunity to, but did not cross-examine or otherwise challenge
16 Marraffini’s deposition testimony in this proceeding. Ex. 5265, 72:4-9.
17 228. Marraffini testified that, on June 26, 2012, he told Zhang “about the role of tracr in the
18 [DNA] cutting complex in a CRISPR-Cas9 system.” Ex. 5265, 29:20-30:3; Ex. 3713, 27-
19 29.
20 229. Marraffini testified that, prior to June 26, 2012, he “never had any communications with
21 Dr. Zhang about single-guide RNA in a CRISPR-Cas9 system.” Ex. 5265, 23:19-24:9;
22 Ex. 3713, 27-29.
23 230. On June 26, Marraffini communicated to Zhang CVC’s design of a single molecule guide
- 2-48 - Interference No. 106,115 CVC Reply 2
1 RNA (sgRNA) for CRISPR-Cas9. Ex. 5265, 64:9-18, 66:7-67:7, 68:13-21; Ex. 3713, 27-
2 29.
3 231. Marraffini testified that, on June 26, 2012, he informed Zhang that CVC’s sgRNA design
4 “would be an important tool for genome editing in eukaryotes specifically.” Ex. 5265,
5 68:13-21.
6 232. After learning of CVC’s sgRNA CRISPR-Cas9 system from Marraffini, Zhang applied
7 CVC’s sgRNA CRISPR-Cas9 system in his own experiments in July and August, 2012.
8 Ex. 4768, 26; Ex. 3713, 29; Ex., 3751, 1; Ex. 3424, ¶124; Ex. 5013, ¶¶59-63. Ex. 3537, 3;
9 Ex. 3829; Ex. 3201, Fig. 2B; Ex. 3425, ¶24.
10 233. All of the experimental data reported in the Cong 2013 paper were obtained from
11 experiments performed after June 26, 2012. Ex. 5262, 228:2-8, 229:21-230:7; Ex. 3564;
12 Ex. 3201; Ex. 5013, ¶A75. Ex. 3777; Ex. 3580; Ex. 3577; Ex. 3752, 2.
13 234. The Mali 2013 manuscript was submitted to Science before Cong 2013 published in
14 January 2013. Ex. 3623, 826.
15 235. The Hwang 2013 manuscript was submitted to Nature Biotechnology before Cong 2013
16 published in January 2013. Ex. 4233, 227.
17 236. The Cho 2013 manuscript was submitted to Nature Biotechnology before Cong 2013
18 published in January 2013. Ex. 4076, 230.
19 237. The Chen provisional application no. 61/734,256 was filed before Cong 2013 published
20 in January 2013. Ex. 5020.
21 238. Group II introns, ribozymes, and riboswitches are all catalytic RNAs—i.e., the RNA is the
22 catalytic component that cuts the DNA. Ex. 4345, ¶114; Ex. 5349, 193:3-194:18.
- 2-49 - Interference No. 106,115 CVC Reply 2
1 239. Group II introns are different in nucleotide length and structure than a chimera A sgRNA.
2 Ex. 5347, 177:22-178:9; Ex. 5349, 193:3-194:18; Ex. 5318.
3 240. CVC successfully detected rx3/chokh mutant phenotype in zebrafish by August 9, 2012.
4 Paper 1579, 23:8-26:24; Ex. 4294, ¶¶55-58; Ex. 4338, 79-80; Exs. 4912-4915; Ex. 4916,
5 10; Ex. 4343, ¶¶45-58; Ex. 4351, ¶70; Ex. 4348, ¶127.
6 241. CVC successfully detected CRISPR-Cas9-mediated target DNA cleavage in mammalian
7 cells by October 31, 2012. Paper 1579, 27:17-35:9; Ex. 4345, ¶¶87-101, 201-218; Ex.
8 4349, ¶¶245-251; Ex. 4353, ¶¶87-142; Ex. 4366, 31-53.
9 242. Before 2012, prokaryotic RNA-protein complexes had been used successfully in
10 eukaryotic cells. Ex. 5316; Ex. 5317; Ex. 5347, 158:5-165:7.
11 243. siRNA and shRNA had been used successfully to modulate gene expression by 2002 and
12 were common tools by 2004. Exs. 5319-5322; Ex. 4097; Ex. 5347, 192:5-198:22;
13 244. Before 2012, shRNAs were routinely used without triggering any immune response to
14 foreign RNA. Ex. 5328; Ex. 5321; Ex. 5347, 242:17-245:5.
15 245. RNase A is an extracellular protein, meaning it naturally degrades RNA outside of the
16 cell. Ex. 5327, 2194; Ex. 5347, 233:9-238:7.
17 246. RNase T1 exists only in bacteria and fungi. Ex. 5327, 2194; Ex. 5347, 233:9-238:7.
18 247. Stigloher et al. discloses that lens-to-nose length in rx3/chokh mutant fish is shorter than
19 wild-type, not longer. Ex. 4291; Ex. 5348, 156:1-18; 157:4-159:17; 167:5-171:5.
20 248. Langenberg et al. (Ex 3693) and Bohnsack et al. (Ex. 3698) each discloses rx3/chokh
21 mutant fish embryos with no neural crest cell migration at 24-28 hours. Ex. 3693; Ex.
22 3698; Ex. 5348, 156:1-18; 157:4-159:17; 167:5-171:5.
- 2-50 - Interference No. 106,115 CVC Reply 2
1 249. Hoshijima et al. discloses scoring chohk phenotypes at 24 hours based solely on lack of
2 pigmented retina. Ex. 5342; Ex. 5348, 156:1-18; 157:4-159:17; 167:5-171:5, 179:17-21.
3 250. Mueller et al. discloses that hyperpigmentation related to eye mutations does not begin
4 until 3 days post fertilization. Ex. 5343; Ex. 5348, 156:1-18; 157:4-159:17; 167:5-171:5.
5 251. Rosen et al. discloses that developmental delays are a normal result from microinjection.
6 Ex. 5348, 40:2-11, 185:4-186:16; Ex. 5345.
7 252. U.C. hosted the 5th Annual CRISPR Research Conference at U.C. Berkeley from June
8 20–22, 2012. Ex. 4350, ¶82; Ex. 4351, ¶¶53-54; Ex. 4348, ¶¶112-113; Ex. 4349, ¶¶147-
9 148; Ex. 4768; Ex. 5266, 179:21-180:10; Ex. 5256.
10 253. On June 21, 2012, at the 5th Annual CRISPR Research Conference, Jinek and Chylinski
11 presented CVC’s discovery that Cas9, crRNA, and tracrRNA are the necessary and
12 sufficient components of the Type II CRISPR-Cas9 DNA-cleavage complex. Ex. 4350,
13 ¶82; Ex. 4351, ¶¶53-54; Ex. 4348, ¶¶112-113; Ex. 4349, ¶¶147-148; Ex. 4768, 17, 29;
14 Ex. 5266, 179:21-180:10; Ex. 5016, ¶¶11-13; Ex. 5018, ¶¶10-15; Ex. 5265, 23:6-18,
15 24:17-25:3, 29:20-30:3.
16 254. On June 21, 2012, at the 5th Annual CRISPR Research Conference, Jinek and Chylinski
17 presented CVC’s design of a sgRNA CRISPR-Cas9 system. Ex. 4350, ¶82; Ex. 4351,
18 ¶¶53-54; Ex. 4348, ¶¶112-113; Ex. 4349, ¶¶147-148; Ex. 4768, 25-26; Ex. 5266, 179:21-
19 180:10; Ex. 5016, ¶15; Ex. 5018, ¶17-18; Ex. 5265, 23:19-24:9.
20 255. The eLife reviewers of Jinek 2013 referred to the it as “an excellent paper” and did not
21 dispute that the data in the manuscript show gene editing in eukaryotic cells. Ex. 5329.
22 256. Cas9 is only active between 20 and 44°C and cannot cleave DNA at the cold temperatures
23 Jinek and East used, meaning that CVC’s DNA cleavage occurred in the cells before the
- 2-51 - Interference No. 106,115 CVC Reply 2
1 cells were lysed. See Ex. 5334, 4, 6; Ex. 5333, 23103.
2 257. Jinek and East kept their cell lysates at 4 °C or frozen (-20 °C). Ex. 4382, 26-27 (cells
3 were lysed “at 4°C” and separated using a “refrigerated centrifuge”); Ex. 4366, 26 (cells
4 lysed by “shak[ing] at 4°”); EX. 4366, 66 (lysate “place[d] @ -20 [°C]”).
5 258. After DNA cleavage, the NHEJ process requires the addition of ATP in order for indels
6 to be detectable in a Surveyor assay. Ex. 6335, 14067; Ex. 6336, 4; Ex. 6343, 850; Ex.
7 6347, 1.
8 259. Neither Jinek nor East added any ATP to their cell lysate preparations. Ex. 4382, 26-27;
9 Ex. 4366, 26, 66; Ex. 5347, 276:7-16.
10 260. ZFNs, TALENs and Cas9 are “fundamentally just nucleases.” Ex. 5314, 1.
11 261. CRISPR-Cas9’s rapid adoption “owes much to the groundwork laid by its predecessor
12 nucleases, [ZFNs], meganucleases, and [TALENs].” Ex. 5315, 412.
13 262. Exhibit 3286 discusses CRISPR-Cas9, ZFNs, and TALENs, but does not mention group
14 II introns, ribozymes, or riboswitches. Ex. 3286.
15 263. Marraffini testified that, prior to June 26, 2012, he never had “any communications with
16 Dr. Zhang about whether tracr was part of the DNA cutting complex in a CRISPR-Cas9
17 system.” Ex. 5265, 24:17-25:3, 23:6-18, 29:20-30:3, 36:17-37:10; Ex. 3713, 27-29.
18 264. Marraffini testified that, prior to June 26, 2012, he and Zhang had only discussed “the
19 importance of tracr for what [they] knew at the time was generation of RNA guides.” Ex.
20 5265, 24:17-25:3, 23:6-18, 29:20-30:3, 36:17-37:10; Ex. 3713, 27-29.
21 265. Marraffini testified that, on June 26, 2012, he told Zhang “about the role of tracr in the
22 [DNA] cutting complex in a CRISPR-Cas9 system.” Ex. 5265, 29:20-30:3; Ex. 3713, 27-
23 29.
- 2-52 - Interference No. 106,115 CVC Reply 2
1 266. Marraffini testified that, before June 26, 2012, he and Zhang only discussed “the
2 importance of tracr for what we knew at the time was generation of RNA guides,” and
3 that “the system, as we knew it, required RNase III to generate guides.” Ex. 5265, 24:17-
4 25:3, 37:2-10.
5 267. On June 26, 2012, Marraffini sent an email to Zhang, stating that he “checked” the Jinek
6 2012 draft manuscript and “[CVC] provide pre-processed crRNAs to the in vitro reaction,
7 so there is no need for RNAse III….” Ex. 3713, 29; Ex. 5265, 36:17-37:10.
8 268. Marraffini testified that, once he told Zhang about CVC’s sgRNA CRISPR-Cas9 system
9 for genome editing in eukaryotic cells, the next steps of applying CVC’s system were
10 “pretty straightforward.” Ex. 5265, 31:8-19.
11 269. Marraffini attended the 5th Annual CRISPR Research Conference at UC Berkeley,
12 including Jinek and Chylinski’s presentation on June 21, 2012. Ex. 5256, 4; Ex. 5266,
13 179:21-180:10; Ex. 3713, pp. 27-29; Ex. 5265, 18:20-19:3.
14 270. Marraffini served as a reviewer for Science on CVC’s Jinek 2012 draft manuscript (Ex.
15 5254). Ex. 3713, 62; Ex. 5265, 21:13-20, 64:19-22; Ex. 5255, 2.
16 271. Barrangou testified that he attended the June 21 CRISPR Conference and recalled “[a]fter
17 the Jinek-Chylinski presentation, …there was excitement in the room because the CVC
18 inventors’ CRISPR-Cas9 system was a game changer for genome editing in eukaryotes”
19 and further recalled “having a conversation with Dr. Doudna and Dr. Sontheimer the
20 same day as the Jinek-Chylinski presentation during which we discussed how quickly the
21 field would be moving from that point forward to apply the CVC inventors’ sgRNA
22 CRISPR-Cas9 system in eukaryotes, given its simplicity.” Ex. 5016, ¶16.
23 272. Barrangou testified, “[w]ith the Jinek-Chylinski presentation, CRISPR-Cas9-mediated
- 2-53 - Interference No. 106,115 CVC Reply 2
1 genome editing in eukaryotes (and other organisms) became feasible, as its
2 implementation now only required straightforward, routine techniques that were known
3 in the field (for example, techniques to deliver and express the three (mature crRNA,
4 mature tracrRNA, and Cas9) or two (sgRNA and Cas9) components of the system to the
5 eukaryotic cells).” Ex. 5016, ¶17.
6 273. Dr. Erik Sontheimer testified that he attended the June 21 CRISPR Conference and
7 recalled that “after the Chylinski and Jinek presentation, I and the other attendees
8 immediately recognized that the CRISPR-Cas9 system they disclosed was revolutionary
9 for genome editing in eukaryotes” and further recalled “speaking with Dr. Doudna,
10 separately and together with Dr. Barrangou, at the 2012 CRISPR meeting, and we
11 discussed that those in the field would rush to apply the CVC inventors’ sgRNA
12 CRISPR-Cas9 system in eukaryotes.” Ex. 5018, ¶19.
13 274. Sontheimer testified he “appreciated that after the Chylinski and Jinek presentation,
14 scientists in the field would be able to quickly apply the CVC inventors’ sgRNA
15 CRISPR-Cas9 system for genome editing in eukaryotic cells, because the process for
16 doing so was straightforward and required only routine genome-editing techniques...” Ex.
17 5018, ¶21.
18 275. Raible testified that “showing successful cleavage in a eukaryote" using CVC’s sgRNA
19 CRISPR-Cas9 system in zebrafish was an “expected result.” Ex. 4294, ¶74.
20 276. Raible testified that he “believed that other labs with more resources would likely
21 generate such data before I would be able to” and he stated “[t]he experiments I
22 performed for the CVC inventors were a side project that I took on in addition to my full-
23 time research and teaching obligations at the University.” Ex. 4294, ¶¶74-75.
- 2-54 - CERTIFICATE OF SERVICE
I hereby certify that the foregoing CVC’S REPLY 2 is being filed via the Interference
Web Portal by 8:00 PM Eastern Time on May 6, 2021, pursuant to an agreement between the
parties, and thereby served on the attorney of record for the Senior Party pursuant to ¶ 105.3 of
the Standing Order. Pursuant to the agreement between the parties, the foregoing was also served
via email by 11:00 pm ET on counsel for the Senior Party at:
Raymond N. Nimrod Quinn Emanuel Urquhart & Sullivan, LLP 51 Madison Avenue, 22nd Floor New York, NY 10010 (212) 849-7000 [email protected] [email protected] [email protected]
Steven R. Trybus Locke Lord LLP 353 North Clark Street Chicago, IL 60606 (312) 433-0699 [email protected] [email protected] [email protected]
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C
/Eldora L. Ellison/ Eldora L. Ellison, Ph.D., Esq. Lead Attorney for UC and UV Registration No. 39,967
Date: May 6, 2021
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C. 1100 New York Avenue, NW Washington, DC 20005-3934
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