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Filed on behalf of: Junior Party, Broad

By:
Steven R. Trybus
Harry J. Roper
Paul D. Margolis
Jenner & Block LLP
353 North Clark Street, Chicago, IL 60654
Telephone: 312-222-9350
Facsimile: 312-527-0484
strybus@jenner.com

Paper No. ____

UNITED STATES PATENT AND TRADEMARK OFFICE


____________________
BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________________
THE BROAD INSTITUTE, INC., MASSACHUSETTS INSTITUTE OF
TECHNOLOGY, and PRESIDENT AND FELLOWS OF HARVARD COLLEGE,
(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 and Application 14/704,551),
Junior Party,
v.
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, UNIVERSITY
OF VIENNA, and EMMANUELLE CHARPENTIER
(Application 13/842,859),
Senior Party.

Patent Interference No. 106,048 (DK)

BROAD et al. OPPOSITION 4

TABLE OF CONTENTS
I.

PRECISE RELIEF REQUESTED.......................................................................................1

II.

DESCRIPTION OF APPENDICES ....................................................................................1

III.

REASONS WHY THE MOTION SHOULD BE DENIED ................................................1


A.

Summary Of The Argument ....................................................................................1


1.

Doudna P1s Failure to Discuss Any Role of PAM Sequences


Would Have Conveyed a Lack of Possession for Non-Natural
Targets..........................................................................................................1

2.

Doudna P1 and P2 Do Not Reasonably Convey Possession of a


CRISPR-Cas9 System Adapted to Function in Eukaryotic Cells ................2

3.

The Guidance Set Forth in Doudna P1 and P2 Did Not Enable


Ordinarily Skilled Persons to Practice the Methods of Count 1 ..................3

4.

Doudna P3 Fails to Show Possession of the Invention of Count 1..............4

B.

Legal Standards ........................................................................................................4

C.

Senior Party Mischaracterizes The Factual Background Relating To


Allegedly Successful CRISPR Systems for Eukaryotic Cells To
Compensate for the Lack of Sufficient Disclosure in Its Provisional
Applications .............................................................................................................5

D.

1.

The Junior Partys Scientists Commenced CRISPR-Cas9 Work in


Eukaryotic Cells in 2011 and Achieved Success Prior to Jinek
2012..............................................................................................................6

2.

The Senior Party Inventors Approach Led to Many Frustrations


When Adapting Their System to a Eukaryotic Environment ......................8

3.

The Industry Recognized Dr. Zhangs Eukaryotic Contribution ...............10

Senior Party is Not Entitled to the Benefit of its Doudna P1, Doudna P2,
or Doudna P3 Applications ....................................................................................11
1.

Persons Of Ordinary Skill Would Have Concluded That Doudna


P1 Lacks Written Description Support For A CRISPR-Cas9
System For Use With Non-Natural Targets, Including Eukaryotic
DNA ...........................................................................................................11

2.

IV.

Persons Of Ordinary Skill Would Have Concluded That Doudna


P1 And P2 Do Not Reasonably Convey Possession Of A CRISPRCas9 System Adapted to Function In Eukaryotic Cells .............................14
a.

The Combination of Example 1, Paragraph 00165 and


Various Claims With Eukaryotic Limitations From Doudna
P1 and Doudna P2 Did Not Convey Possession ............................14

b.

Contemporaneous And Pre-Interference Statements


Confirm The Lack of Possession Based on In Vitro Tests ............17

c.

Persons of Ordinary Skill Would Not Have Known


Whether A CRISPR-Cas9 System That Functioned In Vitro
Would Function in a Eukaryotic Cell ............................................20

d.

Prior Difficulties Experienced in Attempting to Adapt


Other Prokaryotic Systems to Function in Eukaryotic Cells .........23

e.

The Later Work of Other Laboratories Does Not Support


Senior Partys Written Description Argument ...............................24

3.

The Doudna P1 And P2 Applications Do Not Enable A Skilled


Artisan To Practice An Embodiment Of Count 1 ......................................26

4.

The Doudna P3 Application Does not Provide Written Description


Support For An Embodiment Of Count 1 ..................................................28

CONCLUSION ..................................................................................................................30

APPENDIX 1: LIST OF EXHIBITS ..............................................................................................1


APPENDIX 2: STATEMENT OF MATERIAL FACTS................................................................1

ii

TABLE OF AUTHORITIES
Page(s)
CASES
Ariad Pharm., Inc. v. Eli Lilly & Co.,
598 F.3d 1336 (Fed. Cir. 2010)......................................................................................5, 15, 16
Chiron Corp. v. Genentech, Inc.,
363 F.3d 1247 (Fed. Cir. 2004)..................................................................................................5
Genentech Inc. v. Novo Nordisk AS,
108 F.3d 1361 (Fed. Cir. 1997)............................................................................................5, 27
Hunt v. Treppschuh,
523 F.2d 1386 (C.C.P.A. 1975) .................................................................................................5
In re Kaslow,
707 F.2d 1366 (Fed. Cir. 1983)..................................................................................................5
In re Wands,
858 F.2d 731 (Fed. Cir. 1988)..............................................................................................5, 27
Manning v. Paradis,
296 F.3d 1098 (Fed. Cir. 2002)................................................................................................21
Univ. of Rochester v. G.D. Searle & Co.,
358 F.3d 916 (Fed. Cir. 2004)....................................................................................................5
STATUTES
35 U.S.C. 112(a) ...........................................................................................................................5
OTHER AUTHORITIES
37 C.F.R. 41.202 ...........................................................................................................................5
MPEP 2163 ...................................................................................................................................5

iii

I.

PRECISE RELIEF REQUESTED


Junior Party Broad requests that the Board deny Senior Party Motion 4, which requests

that it be accorded the benefit of the filing date of each of its three Provisional Applications

(Doudna P1, P2 and P3). Senior Party should be denied the benefit of each of those applications.

II.

DESCRIPTION OF APPENDICES

Appendix 1 is a list of Exhibits cited in this motion. Appendix 2 is the Statement of

Material Facts, including a listing of UCs statement of facts, which are admitted, denied or

which cannot be admitted or denied, and Broads statement of facts in support of the opposition.

III.

REASONS WHY THE MOTION SHOULD BE DENIED

10

A.

11

Senior Party does not dispute that neither Doudna P1 nor Doudna P2 includes an

12

experiment that falls within the scope of Count 1, which is directed to a method of using

13

CRISPR-Cas9 systems to target DNA in eukaryotic cells. Instead, Senior Party relies on an in

14

vitro experiment in combination with the general disclosure that the system could be used in any

15

cells of interest, including eukaryotic cells. However, given the nascent state of the art, and

16

the uncertainties and unknowns associated with adapting the prokaryotic CRISPR-Cas9 system

17

to eukaryotic cells, as well as the failure to describe eukaryotic-specific considerations such as

18

use of a target sequence comprising a protospacer adjacent motif (PAM), the disclosures of

19

Doudna P1 and P2 fail to provide adequate written description and are non-enabling. Doudna P3

20

includes eukaryotic experiments, but the results are inconclusive at best and do not demonstrate

21

possession.

22
23
24

Summary Of The Argument

1.

Doudna P1s Failure to Discuss Any Role of PAM Sequences Would


Have Conveyed a Lack of Possession for Non-Natural Targets

Based on Doudna P1s lack of any discussion of the role, if any, of PAM sequences for
1

BROAD et al. OPPOSITION 4

non-natural targets, persons of ordinary skill would not have understood that the inventors were

in possession of an invention within the scope of Count 1. Count 1 is directed to a method, in a

eukaryotic cell, of cleaving or editing a target DNA molecule or modulating transcription of at

least one gene encoded thereon. Thus, the method of Count 1 is directed to a target DNA

molecule in a eukaryotic cell, which would not be a natural target of the prokaryotic CRIPSR-

Cas9 system.

By Doudna P1s May 2012 filing date, publications had already disclosed that PAM

sequences played a role in the ability of a CRISPR system in bacteria to recognize natural DNA

targets. The complete lack of any discussion of PAM would have indicated to persons of

10

ordinary skill that the inventors failed to describe an invention configured for use against non-

11

natural targets in a eukaryotic cell. In other words, the inventors failed to describe information

12

that persons of ordinary skill would have expected and required for making and using a CRISPR-

13

Cas9 system capable of functioning against non-natural DNA targets, such as in eukaryotic cells.

14

Thus, from an objective point of view, the failure to mention any role of PAM would have led

15

one of ordinary skill in the art to conclude that the inventors did not have possession of an

16

invention that falls within the scope of Count 1.

17
18
19

2.

Doudna P1 and P2 Do Not Reasonably Convey Possession of a


CRISPR-Cas9 System Adapted to Function in Eukaryotic Cells

Doudna P1 lacks sufficient written disclosure for the additional reason that it fails to

20

show objectively that the inventors had overcome the other unknowns and uncertainties relating

21

to transferring the prokaryotic CRISPR-Cas9 system to eukaryotic cells. Doudna P2 fails for the

22

same reason. Given the nascent state of the art and the unpredictable nature of this field, persons

23

skilled in the art in 2012 would have required actual experiments in eukaryotic cells to

24

demonstrate possession. In fact, contemporaneous statements of Senior Party UCs expert Dr.
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BROAD et al. OPPOSITION 4

Carroll in 2012 confirm that skilled persons would not have considered in vitro tests and the

general disclosure of eukaryotic cells sufficient to demonstrate possession. In a September 2012

article, Dr. Carroll commented on the Jinek 2012 paper, noting the in vitro experiments and

expressing doubt about whether the system would work in eukaryotic cells. Dr. Carroll stated

that [o]nly attempts to apply the system in eukaryotes i.e., actual eukaryotic testswould

address his concerns. Ex. 1152, Carroll 2012 at 1660. Dr. Carrolls statements confirm what

skilled persons would have concluded upon reading Doudna P1 and P2that the inventors were

not yet in possession of an invention of a CRISPR-Cas system adapted to function in eukaryotes.

Senior Party attempts to compensate for the deficiencies in Doudna P1 and P2 by

10

focusing on the timing of alleged success by others and linking that to publication of its Jinek

11

2012 paper, which included the same in vitro tests as Doudna P1 and P2. However, the test for

12

possession depends on what skilled persons would have understood based on a reading of the

13

application as of its filing date, not later publications or whether a system later turned out to be

14

easy to implement in the hands of others. As Dr. Carrolls article confirms, skilled persons

15

reading Jinek 2012 (and thus Doudna P1 and P2) would not have concluded that the inventors

16

possessed an invention of a CRISPR-Cas9 embodiment of Count 1.

17

Moreover, Senior Partys attempt to attribute the later alleged success of other groups to

18

Jinek 2012 is also wrong on the facts. For example, Senior Party asserts that Jinek 2012

19

triggered and guided the work of the Junior Party at the Broad. However, the Broad scientists

20

commenced their eukaryotic work in 2011 and were successful prior to Jinek 2012s publication.

21

Senior Partys reliance on other groups is also flawed.

22
23
24

3.

The Guidance Set Forth in Doudna P1 and P2 Did Not Enable


Ordinarily Skilled Persons to Practice the Methods of Count 1

Doudna P1 and P2 also do not enable embodiments within Count 1. Senior Party asserts
3

BROAD et al. OPPOSITION 4

that skilled persons could have adapted the chimeric RNA system disclosed in Example 1 to

eukaryotic cells using only routine techniques. Senior Party relies on its own alleged success in

eukaryotic cells as reported in Doudna P3, asserting that Doudna P3 simply followed the

teachings of Doudna P1 and P2 using routine techniques. The actual facts, however, show that

after its in vitro tests, Senior Party experienced many frustrations in eukaryotic cells.

Moreover, the Senior Party inventors received unpublished data from another group, as detailed

below, and then published their own results in eukaryotic cells, further evidencing both a lack of

possession and enablement in Doudna P1 and P2.


4.

9
10

Doudna P3 Fails to Show Possession of the Invention of Count 1

Finally, while Doudna P3 reports experiments involving CRISPR-Cas9 systems in

11

eukaryotic cells, the results are inconclusive at best. Ordinarily skilled persons reading Doudna

12

P3 would have concluded that the inventors still did not describe information sufficient to

13

objectively show possession of the invention of Count 1.

14

Moreover, the disclosure present in Doudna P3, but missing in Doudna P1 and P2, such

15

as actual eukaryotic testing (albeit failed in P3) and specific techniques for CRISPR-Cas systems

16

for eukaryotic cells, illustrate the type of information that would have been expected in Doudna

17

P1 and P2 to demonstrate actual possession of a CRISPR-Cas9 system for eukaryotic cells.

18

B.

Legal Standards

19

A party seeking the benefit of an earlier application in an interference must establish that

20

the earlier application provided a constructive reduction to practice of an embodiment within

21

the scope of the count, which must satisfy both the written description and the enablement

22

requirements. 37 C.F.R. 41.202; Hunt v. Treppschuh, 523 F.2d 1386, 1389 (C.C.P.A. 1975).

23
24

The test for determining compliance with the written description requirement is whether
the disclosure of the application as originally filed reasonably conveys to the artisan that the
4

BROAD et al. OPPOSITION 4

inventor had possession at that time of the later claimed subject matter, rather than the presence

or absence of literal support in the specification for the claim language. In re Kaslow, 707 F.2d

1366, 1375 (Fed. Cir. 1983). The application must show more than a wish or plan for

obtaining the claimed invention. Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927

(Fed. Cir. 2004). Rather, the description must clearly allow persons of ordinary skill in the art

to recognize that the inventor invented what is claimed. Ariad Pharm., Inc. v. Eli Lilly & Co.,

598 F.3d 1336, 1351 (Fed. Cir. 2010). When a technology is in an early stage of development,

the application typically must disclose more information to provide written description support.

Id. at 1358; see also MPEP 2163 ([F]or inventions in emerging and unpredictable

10

technologies, or for inventions characterized by factors not reasonably predictable which are

11

known to one of ordinary skill in the art, more evidence is required to show possession.).

12

A specification also must provide an enabling disclosure. 35 U.S.C. 112(a). A claim

13

can be sufficiently enabled even if experimentation is necessary, so long as the experimentation

14

is merely routine. If undue experimentation is needed, however, the claims are not enabled.

15

In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988). Moreover, when the invention arises out of

16

nascent technology, enablement requires a specific and useful teaching of how to practice the

17

invention. Genentech Inc. v. Novo Nordisk AS, 108 F.3d 1361, 1367-68 (Fed. Cir. 1997); see

18

also Chiron Corp. v. Genentech, Inc., 363 F.3d 1247, (Fed. Cir. 2004).

19
20
21

C.

Senior Party Mischaracterizes The Factual Background Relating To


Allegedly Successful CRISPR Systems for Eukaryotic Cells To Compensate
for the Lack of Sufficient Disclosure in Its Provisional Applications

22

Senior Partys Motion 4 is based on a flawed characterization of the factual background

23

of events leading to the development of CRISPR-Cas9 systems for eukaryotic cells. It is

24

indisputable that CRISPR-Cas9 was a nascent technology in an unpredictable field when Senior

25

Partys provisional applications were filed; yet, Senior Party has no eukaryotic experiments to
BROAD et al. OPPOSITION 4
5

demonstrate possession or provide an enabling disclosure in Doudna P1 or P2. Facts 81, 85; Ex.

2009 11.117. Senior Party attempts to compensate for this lack of specific teachings by

focusing on the alleged impact of Jinek 2012 and timing of alleged success by others. According

to Senior Party at page 18, lines 17-19 and page 19, lines 15-24 of its Motion 4, the Jinek 2012

paper taught the necessary and sufficient components for a CRISPR-Cas9 complex, which

triggered four other independent groups to successfully use CRISPR-Cas9 systems in

eukaryotic cells. Senior Party states at page 18, lines 4-6, that the rapid success by other

groups after Jinek 2012 constitutes independent, empirical evidence that the First Provisional

describes and enables embodiments within the scope of Count 1. See also UC Motion 4 at

10

1:24-2:3 (arguing that success of others after Jinek 2012 evidences the sufficiency of the

11

guidance provided by the First Provisional as well as the ease and predictability of applying the

12

methods to eukaryotic cells.).

13

Junior Partys response is that Senior Partys characterization is flawed in three important

14

respects: (1) it assumes that Jinek 2012 triggered and guided the work of the other groups; (2) it

15

suggests that CRISPR-Cas9 work in eukaryotic cells did not occur prior to Jinek 2012 because

16

such work could not even proceed, or certainly be successful, until Jinek 2012 identified the

17

three minimal components of the CRISPR-Cas9 interference complex; and (3) it asserts that the

18

other groups implemented the Jinek system to achieve success.

19

Junior Party provides a more complete and accurate factual background below. The facts

20

show that the Junior Partys scientists work using CRISPR-Cas9 systems in eukaryotic cells

21

commenced long before the June 2012 publication of the Jinek 2012 paper.

22
23
24

1.

The Junior Partys Scientists Commenced CRISPR-Cas9 Work in


Eukaryotic Cells in 2011 and Achieved Success Prior to Jinek 2012

By early 2011, Junior Partys scientist Feng Zhang had conceived of the idea of, and
6

BROAD et al. OPPOSITION 4

began experiments, adapting CRISPR-Cas9 systems to function in eukaryotic cells. See Ex.

2412; Paper 53; Ex. 2009 11.102. Fact 88. Prior publications had identified components as

playing a potential role in the natural CRISPR-Cas9 system. Fact 89; Ex. 1032; Ex. 1153; Ex.

1227; Ex. 2009 11.98. Dr. Zhangs group used those components for their eukaryotic

experiments, understanding that they were sufficient, without first determining the minimum

components that were necessary.

Consistent with that approach, Dr. Zhang filed a grant proposal with the NIH on January

12, 2012 showing his development of single vector delivery of a multiplexed system with dual

molecule guide RNAs and Cas9. Fact 90; Ex. 2411; Ex. 2009 11.97. Dr. Zhang identified the

10

design of a mammalian CRISPR expression system, using four componentsCas9 nuclease

11

and bacterial RNase III genes from Streptococcus thermophilus LMD-9, a tracrRNA element to

12

facilitate the processing of guide RNAs, and the guide RNA array. Ex. 2411 at 16 (caption for

13

Figure 4)(emphasis added). With those components, Dr. Zhang adapted a CRISPR-Cas9 system

14

to function in eukaryotic cells using a dual molecule guide RNA. Fact 91.

15

The Cong et al. 2013 article reported Dr. Zhangs success with a dual molecule guide

16

RNA, stating that Cotransfection of all four required CRISPR components resulted in efficient

17

cleavage of the protospacer in eukaryotic cells. Ex. 1055 at 2; Fact 92. Thus, Senior Partys

18

contentions that Jinek 2012 spurred DNA cleavage and gene editing eukaryotes using the

19

CRISPR/Cas9 system by the Junior Party and that knowledge of the minimal basic

20

components was required for success in eukaryotes are false. UC Motion 4 at 19:10-14.

21

Dr. Zhangs group at the Broad also later conducted tests relating to a single molecule

22

chimeric RNA, using a modified version of the Chimera A disclosed in Jinek 2012. Fact 96; Ex.

23

1055 at 2; Ex. 2009 11.11.106. The Broad scientists used special techniques that they
7

BROAD et al. OPPOSITION 4

developed in their earlier eukaryotic work to adapt a modified Chimera A to function in

eukaryotic cells, including the use of two NLSs to target the Cas9 protein to the nucleus, a vector

to drive expression of the Cas9 and the chimeric guide, and a slightly longer tracrRNA segment

that included four extra nucleotides as compared to Chimera A. Fact 97; Ex. 1055 at 2; Ex. 2009

11.107. Even with those improvements, eight of the 14 modified Chimera A-type samples

tested by the Broad scientists did not work at all in a eukaryotic cell. Fact 98; Ex. 1055 at 20;

Ex. 2009 11.130. The tests showed that Chimera A with its short tracrRNA component was

inferior to the previously developed system with a longer tracrRNA component. Id.; Fact 163.

Other research groups also used a longer tracrRNA component in the eukaryotic

10

environment, as the Broad had. For example, the Church group at Harvard published the Mali

11

2013 article using a longer tracrRNA component. Fact 99; Ex. 1056 at 824 ; Ex. 2009 11.109.

12

The Junior Party includes both Harvard and the Broad. Fact 100. Harvard is affiliated with the

13

Broad and some Broad scientists have advisors at both institutions. Fact 100. Dr. George

14

Church was co-advisor to Dr. Cong, who conducted the tests at the Broad showing that the

15

longer tracrRNA functioned better in the eukaryotic environment than Chimera A. Fact 101;

16

Simons Tr. 89:4-15. Dr. Hwangs Harvard group thanked Church for sharing unpublished

17

results. Ex. 1058 at 6; Ex. 2009 11.109; Fact 102. None of these groups indicated Jinek 2012

18

triggered their initial work. Ex. 1055; Ex. 1056; Ex. 1058; Ex. 1059.

19
20
21

2.

The Senior Party Inventors Approach Led to Many Frustrations


When Adapting Their System to a Eukaryotic Environment

While Dr. Zhangs laboratory at the Broad worked at adapting CRISPR for eukaryotes

22

starting in 2011, the Senior Partys inventors took a different approach that led to many

23

frustrations in a eukaryotic environment. Ex. 2230, Ex. 2009 11.100. The Senior Partys UC

24

and University of Vienna inventors assert a conception date for Count 1, including the eukaryotic
8

BROAD et al. OPPOSITION 4

cell claim limitations, no later than October 18, 2011. Fact 121; Senior Party Priority Statement

at 1. Senior Party Dr. Charpentier asserts an even earlier conception date of July 30, 2010. Fact

122; id. Yet, Senior Party does not assert that there was any actual reduction to practice until

October 29, 2012. Fact 103. Thus, Senior Party failed to make the claimed invention for over a

year (over two years for Dr. Charpentier), despite its contention that one would have been

immediately motivated to develop gene-editing tools for eukaryotic cells. Facts 121-122.

And, as described below, they did not report any success until after receiving unpublished data.

8
9

In 2011, the Senior Partys inventors took the approach of conducting in vitro
experiments to determine the minimal necessary and sufficient components, including a

10

minimal tracrRNA component length. Ex. 2009 11.99. The Senior Partys inventors created

11

two versions of a chimeric RNA with a covalent linker between the crRNA component and the

12

tracrRNA component, both with substantially shorter tracrRNA components than wild-type

13

tracrRNA. Ex. 1155, Jinek 2012 at 818, 820; Ex. 1003 at 2, 0006; Fact 93. The Jinek 2012

14

paper reported that Chimera A version worked efficiently in vitro, while the even shorter

15

Chimera B version did not. Fact 94; Ex. 1155 at 820. The Jinek 2012 paper did not teach any

16

longer tracrRNA components for use as a part of a chimeric RNA, but instead suggested to those

17

of ordinary skill the use of Chimera A. Ex. 1155; Ex. 2009 11.66; Fact 95.

18

However, Chimera A did not translate well to the eukaryotic environment. Fact 105.

19

Senior Party asserts in its Motion 4 at page 5:21-6:23 that one could adapt Chimera A for

20

eukaryotes using routine techniques. Junior Partys response is that Dr. Doudnas groupthose

21

who were best positioned to do what Senior Party now arguesactually experienced many

22

frustrations trying to make it work in eukaryotes, as Dr. Doudna publicly acknowledged:

23
24

Doudna experienced many frustrations getting CRISPR to work in human


cells. But she knew if she succeeded, CRISPR would be a profound discovery
9

BROAD et al. OPPOSITION 4

1
2
3

and maybe even a powerful gene therapy technique.


Ex. 2230 (emphasis added); Fact 87.
The timeline of events corroborates Dr. Doudnas many frustrations. The Doudna P1

application, which was filed on May 25, 2012, set forth in vitro experiments with Chimera A and

expressed the hope that the system could be applied to eukaryotic cells. As Senior Party stated,

after publication of those results, persons of ordinary skill would have been immediately

motivated to apply the Type II CRISPR-Cas system to Eukaryotic cells. UC Motion 3 at 9

(emphasis added). Yet, the Doudna P2 application, filed four months later on October 19, 2012,

reported no such eukaryotic experiments despite such immediate motivation. Ex. 1004.

10

Indeed, consistent with Dr. Doudnas frustrations trying to adapt the Jinek system to

11

eukaryotes, Dr. Doudnas laboratory was surprised when they learned that Dr. Churchs

12

laboratory had achieved success in eukaryotes. Ex. 2230; Fact 106. It was only after the Church

13

laboratory shared unpublished data that Dr. Doudnas laboratory reported they were able to adapt

14

a CRISPR-Cas9 system for eukaryotic cells in the Jinek 2013 paper. Fact 107; Ex. 2009

15

11.141. The Senior Party inventors then filed a third provisional patent application (Doudna

16

P3) containing the same eukaryotic test results as in Jinek 2013. Ex. 1005 at Figures 36-38; Ex.

17

1055 at Figures 3-5; Fact 108. Those tests included the use of a chimeric RNA with longer

18

tracrRNA components than what was used in Chimera A. Fact 108. However, as discussed in

19

more detail below in Section III.D.4, the eukaryotic test results in the Jinek 2013 paper and

20

Doudna P3 are inconclusive at best, consistent with their many frustrations.

21
22

3.

The Industry Recognized Dr. Zhangs Eukaryotic Contribution

Thus, the Senior Party inventors focus on in vitro experiments led them to develop

23

Chimera A, but appears to have misguided their efforts to adapt a CRISPR-Cas9 system for use

24

in eukaryotic cells. The short tracrRNA component of Chimera A worked well in in vitro
10

BROAD et al. OPPOSITION 4

environments, but not in the more complex eukaryotic environment. In contrast, Dr. Zhangs

approach resulted in the first CRISPR-Cas9 system adapted to function in eukaryotic cells.

The industry has recognized the pioneering nature of Dr. Zhangs work, differentiating it

from the biochemical work and in vitro data by the Senior Partys inventors. Recently, Drs.

Zhang, Horvath, Doudna, Charpentier and Barrangou jointly received the 2016 Gairdner Award

for their work with CRISPR-Cas systems. Ex. 2419; Fact 110. The Gairdner Foundations

announcement identified the respective contributions of each of the award recipients. For Dr.

Zhang, they noted that he pioneered the development of the microbial CRISPR-Cas system as a

[sic] genome editing tools for function in eukaryotic cells. See Fact 111; Ex. 2419 at 2. This

10

award counters Senior Partys pre-interference claim of the alleged ease and predictability of

11

applying the methods [of Jinek 2012] to eukaryotic cells. UC Motion 4 at 1:24-2:3. Indeed, the

12

actual timeline of events belies the entirety of Senior Partys benefit arguments.

13
14

D.

Senior Party is Not Entitled to the Benefit of its Doudna P1, Doudna P2, or
Doudna P3 Applications

15

Senior Party asserts that it is entitled to the benefit of each of its earlier applications,

16

Doudna P1, Doudna P2, and/or Doudna P3. To obtain benefit, Senior Party must show that those

17

earlier applications meet two requirements: (a) that each application conveyed to one of ordinary

18

skill in the art as of its filing date that the inventors were in possession of at least one

19

embodiment within the scope of Count 1, and (b) that each application enabled one of ordinary

20

skill in the art to practice that embodiment of Count 1 without undue experimentation. Senior

21

Party has not met either the written description or enablement requirements for Doudna P1 and

22

P2, and has not established written description in Doudna P3.

23
24
25

1.

Persons Of Ordinary Skill Would Have Concluded That Doudna P1


Lacks Written Description Support For A CRISPR-Cas9 System For
Use With Non-Natural Targets, Including Eukaryotic DNA
11

BROAD et al. OPPOSITION 4

Persons of ordinary skill reviewing the Doudna P1 application in May 2012 would have

concluded that the Senior Party inventors were not in possession of an invention of a CRISPR-

Cas9 System that falls within the scope of Count 1 for multiple reasons. First, Doudna P1

includes no discussion of the role of PAM sequences for non-natural targets. Fact 82; Ex. 2009

11.43.

DNA molecule or modulating transcription of at least one gene encoded thereon. Thus, Count 1

is directed to a target DNA molecule in a eukaryotic cell, not a natural target of the prokaryotic

CRIPSR-Cas9 system. Fact 112; Ex. 2009 11.17. Based on the lack of any PAM discussion,

skilled persons would have concluded that the inventors had not begun to provide the

10

Count 1 is directed to a method, in a eukaryotic cell, of cleaving or editing a target

information needed to use CRISPR systems with non-natural targets. Ex. 2009 11.43.

11

Prior to Doudna P1s May 2012 filing date, persons of ordinary skill would have been

12

aware of publications disclosing that PAM sequences played a role in the ability of a CRISPR

13

system in bacteria to target natural DNA targets. Fact 84; Ex. 2246 (Deveau); Ex. 1298

14

(Mojica); Ex. 2009 11.12-11.13. In light of these disclosures, persons of ordinary skill would

15

have expected that if an application described a CRISPR-Cas9 invention directed to work on

16

non-natural targets, that it would address whether or not PAM played a role with respect to non-

17

natural targets, or at least mentioned PAM sequences. Fact 84; Ex. 2009 11.43.

18

Doudna P1, however, says nothing about PAM sequences. Ex. 2009 11.43. Indeed,

19

Figure 5 of Doudna P1 is particularly telling, showing cleavage assay results conducted using six

20

Cas9 orthologs from different species of bacteria, of which three did not show any cleavage.

21

Fact 113; Ex. 1003 at 95; Ex. 2009 11.49. To persons of ordinary skill, these poor results

22

would have indicated that the Doudna inventors did not appreciate the need to match the

23

different Cas9 proteins for the different species to different PAM sequences. Fact 114. The
12

BROAD et al. OPPOSITION 4

absence of information on PAM sequences in P1, and the failure to appreciate the significance of

PAM in the experiments in Figure 5, would have indicated to skilled persons that the inventors

had not yet begun to provide the type of information required for possession of a CRISPR-Cas9

invention applied to non-natural targets. Ex. 2009 11.52-53.

If the Doudna inventors had understood the significance of PAM when the Doudna P1

application was filed in May 2012, then they chose to conceal that information for some reason.

It is notable that one of the Senior Party inventors, Dr. Charpentier, was a co-author on

Makarova 2010, which stated that the CRISPR-Cas9 system probably involves a process that

requires the PAM, and was aware of the relationship between PAM sequences and CRISPR for

10

natural targets. Ex. 2009 11.45. It also notable that Figure 1A from Doudna P1 appears to

11

have deleted a marker indicating the PAM sequence on the DNA target. A comparison of Figure

12

1A from Doudna P1 and Figure 5A from the Jinek 2012 article is telling on this point:

13
14

Figure 5A from Jinek identifies PAM as part of the CRISPR system, with a diagonal

15

line extending from the upper right portion of the figure. Figure 1A from Doudna P1 includes a

16

similar line extending up and to the right, shown in the red box, but with no label identifying
13

BROAD et al. OPPOSITION 4

PAM. Figure 1A of Doudna evidently had been scrubbed to remove any reference to PAM

sequences. Ex. 2009 11.45. It is unclear why reference to PAM was not included in Figure 1A

of Doudna P1, but it is clear that without it, Doudna P1 would have conveyed to persons of

ordinary skill in the art at the time it was filed that the inventors were not in possession of a

CRISPR-Cas embodiment adapted to function on non-natural targets.

Moreover, as discussed below, separate and apart from the PAM issue, skilled persons

would have concluded that the inventors had done nothing to overcome the other unknowns and

uncertainties relating to transferring a protein/RNA-based prokaryotic system to eukaryotic cells.

9
10
11
12

2.

Persons Of Ordinary Skill Would Have Concluded That Doudna P1


And P2 Do Not Reasonably Convey Possession Of A CRISPR-Cas9
System Adapted to Function In Eukaryotic Cells

Senior Party asserts at page 13, lines 2-7 of Motion 4 that Doudna P1 and Doudna P2

13

each provide written description support for an embodiment of Count 1 that functions in a

14

eukaryotic cell. Junior partys response is that persons skilled in the art reading Doudna P1 and

15

P2 would not have recognized that the inventors possessed as their invention an embodiment of a

16

CRISPR-Cas9 system adapted to function in eukaryotic cells. The evidence shows that skilled

17

persons would not predict a CRISPR-Cas system would work in a eukaryotic cell based on in

18

vitro tests. Fact 119; Ex. 2009 11.6, 11.92. Rather, skilled persons in 2012 would have

19

required positive eukaryotic tests results to recognize that the inventor invented what is

20

claimed in Count 1, a CRISPR-Cas system for eukaryotic cells. Fact 120; Ex. 2009 11.32.

21
22
23

a.

The Combination of Example 1, Paragraph 00165 and Various


Claims With Eukaryotic Limitations From Doudna P1 and
Doudna P2 Did Not Convey Possession

24

For written description with respect to Doudna P1 and Doudna P2, the Senior Party relies

25

primarily on the combination of two disclosures: Example 1 and Paragraph 00165. UC Motion 4

26

at 4 line 11-12. Junior Partys response is that those portions of the applications would not have
14

BROAD et al. OPPOSITION 4

conveyed to persons of ordinary skill that the inventors had invented a CRISPR-Cas9 system

capable of functioning in eukaryotic cells. Example 1 describes only in vitro testing in a cell-

free environment, not testing in eukaryotic cells. Fact 115; Ex. 1003 at 248-253; Ex. 1004; Ex.

2009 11.5. Paragraph 00165 broadly states that cells of interest include cells of any

organism, including eukaryotic cells. Ex. 1003 at 00165. However, the mere recitation of

eukaryotic cells is not sufficient for written description. Ariad, 598 F.3d at 1350 (Generic

claim language appearing in ipsis verbis in the original specification does not satisfy the written

description requirement if it fails to support the scope of the genus claimed.).

Example 1 and Paragraph 00165 do not provide any testing or other evidence that a

10

CRISPR-Cas9 system actually works in eukaryotic cells. Fact 117; Ex. 1003 at 165, 248-253;

11

Ex. 1004; Ex. 2009 11.22. Nor do Example 1 and Paragraph 00165 identify any structural

12

aspect of the CRISPR-Cas9 system of Example 1 that purportedly makes it adapted to function

13

in eukaryotic cells. Id. Thus, Paragraph 00165 is nothing more than a wish or a plan for future

14

experiments, which legally does not provide written description support. Eli Lilly, 119 F.3d at

15

1566.

16

Senior Party also points to other portions of Doudna P1 and P2, including claims and

17

other portions of the specification, which it says provide written description support. From

18

Doudna P1, Senior Partys Motion 4 (at 13:20-14:18, 15:22-16:4,) cites paragraphs 121, 129,

19

167, 177, 178, 201, 215, and 216, as well as claims 54, 58, 61, 66 and 72-75 as allegedly

20

providing written description support. In Doudna P2, Senior Partys Motion 4 (at 21:15-22:2)

21

relies on paragraphs 00114, 00116, and 00120 as well as claims 60, 65, 66, 69-72, and 98-100.

22

Junior Partys response is that none of these excerpts cure the deficiencies in Doudna P1

23

and P2. Fact 118. Paragraph 00216 of Doudna P1 is duplicative of paragraph 00165. Ex. 1003;
15

BROAD et al. OPPOSITION 4

Ex. 2004; Ex. 2009 11.25. The other paragraphs Senior Party cites state broadly the hope of

genetically modifying cells, of expressing nucleotides or polypeptides in cells or of injecting

nucleotides or polypeptides into cells. Id. Likewise, the claims only recite some aspect of a

eukaryotic cell. Ex. 1003; Ex. 2004; Ex. 2009 11.25. None of these disclosures provide

evidence that the CRISPR system would actually work in eukaryotes, and none of them identify

a structural aspect that would make the system adapted for eukaryotic cells. Ex. 2009 11.25.

Senior Partys reliance on a 2009 patent application by Sontheimer supports the lack of

written description in the Doudna P1 and P2 applications. See UC Motion 3 at 10. The

Sontheimer inventors, like the Senior Party inventors, stated that their CRISPR system could be

10

used in eukaryotic cells and also taught routine techniques for eukaryotic experiments. Fact 123;

11

Ex. 1161 at 0054-0060; Ex. 2009 11.33. The Sontheimer inventors conducted experiments in

12

prokaryotic cells using the Type III CRISPR system. Their application discussed possible

13

techniques and tests for potentially establishing that the CRISPR system functioned in eukaryotic

14

cells, but they included no actual eukaryotic experiments. Fact 124; Ex. 2009 11.33

15

The Sontheimer inventors nevertheless presented claims to the use of the CRISPR system

16

in eukaryotic cells. Ex. 2413 at 9. The Patent Office, however, rejected those claims for lack of

17

written description and for non-enablement, stating for example:

18
19
20
21
22
23
24
25
26
27

Further, the disclosure of the instant specification is entirely prophetic such that
"CRISPR RNA-directed DNA targeting is tested for efficacy in animals by
exploiting the genetic and phenotypic tools that are available for the Drosophila
melanogaster." ... There is not a single working example that demonstrates even a
slightest possibility that such method steps would inhibit a broad spectrum of
eukaryotic DNA sequences in a subject, nor is there sufficient guidance that
would lead one of ordinary skill in the art to perform the claimed methods without
undue experimentation at the time of filing.
Ex. 2413 at 9 (emphasis added); Fact 125.
Similarly, the Doudna P1 and P2 applications are entirely prophetic and they do not
16

BROAD et al. OPPOSITION 4

contain a single working example that demonstrates to persons of ordinary skill that such an

embodiment would function in eukaryotic cells. Ex. 2009 11.117; Fact 126. Due to the

uncertainties and unknowns in adapting prokaryotic CRISPR systems to eukaryotic cells, persons

of ordinary skill in the art would not have recognized that the Senior Party inventors possessed

the claimed invention in the absence of actual eukaryotic test results demonstrating successful

adaptation of the system. Id.

Moreover, skilled persons reviewing Doudna P2 would have had an additional reason to

conclude that the Senior Party inventors lacked possession. 1 According to Senior Party, after the

publication of Jinek 2012, persons of ordinary skill would have been immediately motivated to

10

apply the Type II CRISPR-Cas system to eukaryotic cells. UC Motion 3 at 9:1-3. Based on

11

that motivation, skilled persons who read Jinek 2012 in June 2012 would have expected to see

12

eukaryotic experiments in Doudna P2 if the inventors could adapt the system for eukaryotic cells

13

using routine techniques. Ex. 2009 11.74. The absence of such tests four months later would

14

have confirmed that Doudna P2 did not convey the information needed to show possession of an

15

invention of a CRISPR-Cas9 system adapted to function in a eukaryotic cell. Ex. 2009 11.74.

16
17
18

b.

Contemporaneous And Pre-Interference Statements Confirm


The Lack of Possession Based on In Vitro Tests

This interference presents the unusual situation where there is pre-interference evidence

1 A person of ordinary skill would also note that the results in Doudna P2, Figure 5, are
inconsistent and that the inventors did not explain whether it was due to a lack of a PAM
sequence, inappropriate tracr length, or some other factor. From that, persons of ordinary
skill would have concluded that the inventors lacked possession of an embodiment of Count
1, which is directed to non-natural targets in eukaryotic cells. Ex. 2009 11.76.
17

BROAD et al. OPPOSITION 4

on whether skilled persons reading a patent application would have concluded that the inventors

possessed the claimed invention. The test for written description relates to how persons of

ordinary skill would have read the disclosure of a patent application at the time it was filed. This

is a hypothetical inquiry because skilled persons typically cannot access and read the application

until its publication months later. Here, however, the Senior Party asserts that the Jinek 2012

paper is a proxy for Doudna P1 and P2, because it contains the same in vitro tests set forth in

Doudna P1 and P2 with the same general disclosure of eukaryotic cells. Fact 104; UC Motion 4

at 18:14-19:5. Senior Party relies on the alleged ability of skilled workers to practice the Jinek

2012 system in eukaryotic cells as evidence that the Doudna P1 and P2 applications provide a

10
11

sufficient disclosure.
In fact, Senior Party UCs expert, Dr. Carroll, admitted that all of the information in

12

Doudna P1 and P2 either appears in Jinek 2012 or were well-known routine techniques, thereby

13

making Jinek 2012 an appropriate proxy for how persons of ordinary skill would have read

14

Doudna P1 and Doudna P2. Fact 127; Carroll Tr. at 255:4-258:17; 305:6-338:10; Ex. 2009

15

11.86-87. Accordingly, contemporaneous statements regarding Jinek 2012 address the

16

hypothetical inquiry of whether Doudna P1 or P2 would have conveyed possession of the

17

invention of an embodiment of a CRISPR-Cas9 system that functioned in a eukaryotic cell.

18

Dr. Carrolls 2012 statements show that skilled persons would not have believed that the

19

inventors possessed such an invention. Ex. 2009 11.92. In his September 2012 article,

20

Molecular Therapy 20(9): 1659-60 (September 2012) (Ex. 1152), Dr. Carroll observed that [a]ll

21

the experiments described in Jinek 2012 were performed in vitro with purified components

22

and expressed doubt about whether the CRISPR-Cas system would function in eukaryotic cells:

23
24

What about activity of the system in eukaryotic cells? Both zinc fingers and TALE
modules come from natural transcription factors that bind their targets in a
18

BROAD et al. OPPOSITION 4

1
2
3
4

chromatin context. This is not true of the CRISPR components. There is no


guarantee that Cas9 will work effectively on a chromatin target or that the
required DNARNA hybrid can be stabilized in that context Only attempts to
apply the system in eukaryotes will address these concerns.

Ex. 1152 at 1660 (with emphasis added); Fact 128. Dr. Carroll purports to have the knowledge

of one of ordinary skill in the art. Fact 129; Ex. 1024 at 19 (my knowledge as one of

ordinary skill in the art and as an expert in the relevant field). Dr. Carrolls statement confirms

that skilled persons, even with expert knowledge, reading the in vitro experiments and with

knowledge of the existing techniques in the art, questioned whether the inventors provided

10

sufficient information to demonstrate possession of a eukaryotic invention. Ex. 2009 11.85.

11

Dr. Carroll insisted at this deposition that it was actually his strong expectation at the

12

time that the CRISPR-Cas9 system would work in eukaryotic cells. Carroll Tr. at 117:5-118:2.

13

Dr. Carroll, however, did not dispute that his 2012 article does not state any expectation of

14

success. Id.; Fact 130. Instead, he stated that if he had 20 pages instead of two-and-a-half

15

pages to talk about the subject, he would have explained that other prokaryotic systems had

16

been found to work in a eukaryotic context. Carroll Tr. at 117:5-118:2. But, Dr. Carroll could

17

have said that in one sentence. Instead, he only expressed concerns, stating that [o]nly attempts

18

to apply the system in eukaryote will address these concerns. Ex. 1152 at 1660; Fact 131. Dr.

19

Carrolls attempt now to revise his 2012 opinion only highlights the extent to which his 2012

20

article is dispositive on a central issue in this interference: how skilled persons actually would

21

have interpreted Jinek 2012 and, by extension, the disclosure in Doudna P1 and P2.

22

Dr. Doudna also made statements confirming that the information in Doudna P1 and P2

23

did not convince her that they possessed an invention that would work in eukaryotic cells. Fact

24

86. In a July 2014 article, Dr. Doudna confirmed her view that even after the publication of

25

Jinek 2012, they did not know if the CRISPR-Cas9 system would work in eukaryotic cells:
19

BROAD et al. OPPOSITION 4

1
2
3
4

Says Doudna, Our 2012 paper was a big success, but there was a problem. We
werent sure if CRISPR/Cas9 would work in eukaryotesplant and animal
cells. Unlike bacteria, plant and animal cells have a cell nucleus, and inside, DNA
is stored in a tightly wound form, bound in a structure called chromatin.

Ex. 2207; Fact 132; Fact 86. Likewise, an article from July 28, 2012, reported the views of Drs.

Charpentier and Doudna that even after Jinek 2012, they did not know whether the CRISPR

system would work in eukaryotes: [t]he next steps, Charpentier and Doudna say, are to test the

single-RNA construct along with Cas9 to find out whether the RNA-programmed enzyme works

in eukaryotic organisms. Ex. 2290; Ex. 2009 11.89; Fact 133.

10

For the same reasons of uncertainty that Drs. Carroll, Doudna, and Charpentier expressed

11

with respect to whether a CRISPR-Cas9 system would work in eukaryotic cells based on in vitro

12

tests in Jinek 2012, skilled persons reading Doudna P1 and P2 would have likewise concluded that

13

the Senior Party inventors did not yet provide sufficient information to show possession of a

14

CRISPR-Cas9 system adapted to function in eukaryotes. Ex. 2009 11.92; see Manning v. Paradis,

15

296 F.3d 1098, 1104 (Fed. Cir. 2002) (contemporaneous statements by inventor outweigh later

16

statements made for purposes of interference).

17
18
19

c.

20

Persons of Ordinary Skill Would Not Have Known Whether A


CRISPR-Cas9 System That Functioned In Vitro Would
Function in a Eukaryotic Cell

Senior Party asserts at page 20, lines 18-19 of Motion 4 that using the CRISPR-Cas9 of

21

Example 1 in eukaryotic cells required only well-known, routine experiments with a new

22

technology that had been fully described in the First Provisional. Junior Partys response is

23

that, just as Dr. Carroll stated, skilled persons would have recognized an in vitro environment is

24

very different from the environment of eukaryotic cells. Fact 134; Ex. 2009 11.114. The in

25

vitro testing in Example 1 was conducted with a limited set of synthetic components in a test

26

tube under artificial conditions. Ex. 2009 11.28. In contrast, it was known that the contents of
20

BROAD et al. OPPOSITION 4

eukaryotic cells cause molecular crowding which can impact the folding of proteins like Cas9.

Fact 135; Ex. 2009 11.29. In addition, temperature, ion concentration, pH, and cellular milieu

in a eukaryotic cell differ from that of a cell-free in vitro test. Fact 135; Ex. 2009 11.30. It

would have been known in the art that those differences could have unpredictable effects on

Cas9 and RNA expression, Cas9 folding, and CRISPR-Cas9 formation and function, which

could prevent a CRISPR-Cas9 system from functioning even if it worked in an in vitro

environment. Id.

8
9

In addition, as of May 2012, persons of ordinary skill recognized that other features of a
eukaryotic environment made it so that an in vitro test did not convey that the CRISPR-Cas9

10

would work in eukaryotic cells. Persons of ordinary skill knew: (1) that it may not be possible to

11

deliver the necessary components of adapted CRISPR-Cas9 systems to eukaryotic cells so they

12

would be properly transcribed and translated in eukaryotic cells, (Fact 136; Ex. 2009 11.31); (2)

13

that proteins or nucleic acids in eukaryotic cells could interfere with the various components of

14

CRISPR-Cas9 and eukaryotic cells, (Fact 137; id.); and (3) that systems in eukaryotic cells

15

developed to attack foreign invaders may degrade the RNA of the CRISPR-Cas9 system (Fact

16

138 id.). Doudna P1 and P2 did not account for any of the unique features of eukaryotic cells,

17

which might prevent CRISPR-Cas9 from functioning in eukaryotic cells. Fact 139.

18

Furthermore, it was known that CRISPR-Cas9 evolved in nature in prokaryotic cells,

19

which differ greatly from eukaryotic cells. Fact 140; Ex. 2009 11.88. Thus, Count 1 requires

20

engineering a CRISPR-Cas9 system to function in a manner that is antithetical to its natural

21

function in prokaryotic cells, and to function in a vastly more complex and different

22

environment. Fact 141; Ex. 2009 10.5. In particular, skilled persons understood that the size of

23

the human genome is at least about a thousand-fold greater than that of bacteria. Fact 142; Ex.
21

BROAD et al. OPPOSITION 4

2009 10.7. Indeed, it was also understood that while CRISPR was found naturally in many

prokaryotic cells, it had never been found (and still has not been found) in any eukaryotic cells in

nature. Fact 143; id. Thus, persons of ordinary skill would have concluded that the Doudna

inventors actually lacked possession in the absence of successful testing in eukaryotic cells. Id.

It was also known that the proteins and/or RNA molecules of a CRISP-Cas9 system,

which originated in prokaryotic cells, may have toxic effects on eukaryotic cells that must be

overcome in order for CRISPR-Cas9 to function properly. Fact 144; Ex. 2009 11.31. Toxic

effects include genome editing in locations other than the target DNA, which are called off-

target effects. Fact 145; id. UCs expert, Dr. Carroll, agreed that off-target effects are an

10

important concern for genome editing and that off-target effects could, in fact, be lethal.

11

Carroll Tr. at 227:3-6, 229:2-12; Fact 146. UCs expert, Dr. Greider, testified that skilled

12

persons would have had no expectation regarding possible off-target effects from using CRISPR-

13

Cas9 in eukaryotic cells without doing experiments. Greider Tr. at 405:14-22; Fact 147.

14

In addition, it was known that it may not be possible to localize the CRISPR-Cas9 system

15

with target DNA at the same time and place in the crowded and compartmentalized environment

16

of eukaryotic cells. Fact 148. It was known at the times that Doudna P1 and P2 were filed that a

17

nuclear localization signal (NLS) can sometimes be used to direct components to the nucleus

18

of a eukaryotic cell. Ex. 2009 11.31. Doudna P1 and Doudna P2 do not teach to use an NLS in

19

eukaryotic cells, much less how many NLSs to use or in what locations. Fact 149; id.

20

Based on deposition questioning, it appears that Senior Party might argue that some

21

obstacles in dealing with eukaryotic cells are irrelevant because they relate to the nucleus. At her

22

deposition, UCs expert Dr. Greider testified that the DNA target of interest resides in the

23

nucleus. Greider Tr. 285:20-286:1; Fact 150. But, when questioned by UCs counsel on
22

BROAD et al. OPPOSITION 4

redirect, she testified that eukaryotic cells contain other DNA targets of interest outside the

nucleus, including mitochondrial DNA and chloroplast DNA. Greider Tr. 422:4-18. However,

other than a conclusory statement that it was possible to inject CRISPR-Cas9 into mitochondria

and chloroplast, (id.), Dr. Greider provided no opinion that Doudna P1 or P2 provide sufficient

information to show possession an embodiment of Count 1 for delivering a functioning CRISPR-

Cas9 system into a mitochondria or chloroplast. Fact 151; Ex. 2009 11.31.

Moreover, arguments relating to DNA targets outside the nucleus do not deal with the

numerous other scientific uncertainties stated above that do not relate to the nucleus. Thus,

persons of ordinary skill would have required actual test results in eukaryotic cells to conclude

10

that the inventors had overcome the uncertainties relating to a CRISPR-Cas9 system adapted to

11

function anywhere in eukaryotic cells. Ex. 2009 11.31.

12
13

d.

Prior Difficulties Experienced in Attempting to Adapt Other


Prokaryotic Systems to Function in Eukaryotic Cells

14

Persons of ordinary skill reading Doudna P1 and Doudna P2 also would have been aware

15

of prior difficulties adapting systems that originated in prokaryotic cells to eukaryotic cells. Fact

16

152. The well-known history of targetrons, which are gene-editing tools based on group II

17

self-splicing ribozymes, served as the most reasonable comparator for CRISPR researchers

18

because targetrons originated in prokaryotes and include both protein and RNA components, like

19

the CRISPR system. Facts 153-155; Ex. 2010 1.45-1.48. Group II self-splicing ribozymes

20

were initially described in 1986, and targetrons were developed that acted on prokaryotic genes

21

by 2003. Fact 156; Ex. 2010 1.46. But even after nine more years of research by multiple

22

laboratories, researchers had achieved little or no targetron functioning in eukaryotic cells. Fact

23

157; Ex. 2010 1.47-1.54. Persons of ordinary skill reading the Doudna P1 and P2 applications

24

would have understood that similar challenges could face researchers attempting to adapt the
23

BROAD et al. OPPOSITION 4

1
2

CRISPR-Cas9 system to function in eukaryotic cells. Fact 158; Ex. 2010 1.54.
The targetron experience taught that simply because a prokaryotic system could edit

genes in other environments did not mean that it could be adapted to function in eukaryotic cells.

Fact 159. Indeed, skilled persons would have known that one problem that frustrated research on

targetrons, the level of Mg2+ ions, might also pose a problem for CRISPR, which also evolved

to function in the Mg2+ rich environment of prokaryotic cells. Fact 160; Ex. 2010 1.51-1.53.

The history of prior failed attempts to transfer prokaryotic systems to eukaryotes would

have increased the insistence of persons of ordinary skill to see actual experimental results in

eukaryotic cells to show possession of a CRISPR-Cas9 invention for eukaryotic cells. Fact 161.

10

Indeed, statements from other researchers confirm the unpredictability and difficulty of

11

transferring the bacterial CRISPR-Cas system to eukaryotes. Dr. Luciano Marraffini, who

12

studies CRISPR/Cas systems at Rockefeller University, stated that Its not trivial to make

13

CRISPR/Cas systems work in eukaryotic cells, and that [o]ne thing is to have them in silico

14

and have a sequence and realize theyre smaller, and another thing is to do the [eukaryotic]

15

experiments and make it work. Fact 109; Ex. 2213. Similarly, Dr. Churchwho provided

16

unpublished data to the Doudna groupcharacterized the move of CRISPR-Cas from bacteria to

17

eukaryotic cells as a huge jump. Fact 116; Ex. 2423.

18
19
20

e.

The Later Work of Other Laboratories Does Not Support


Senior Partys Written Description Argument

Senior Party contends that the rapid success of numerous other research groups

21

in applying the [CRISPR-Cas9 system of Doudna P1 and Doudna P2] to eukaryotic cells

22

immediately after the invention was published is evidence that Doudna P1 describes and

23

enables embodiments within the scope of Count 1. UC Motion 4 at 18. Senior Party contends

24

that, after Jinek 2012 published information from Doudna P1 and P2, four other laboratories
24

BROAD et al. OPPOSITION 4

published articles showing the use of the CRISPR-Cas9 systems in eukaryotic cells. The

response is that this does not support Senior Partys argument for multiple reasons.

First, the publications by the four other research groups occurred after the filing dates of

Doudna P1 and P2. Therefore, these publications are legally irrelevant because a person of

ordinary skill in the art could not be aware of them as of the filing dates of the applications.

Second, Senior Party is also wrong when it suggests that Jinek 2012 inspired Dr. Zhang

to start his eukaryotic work. As shown in Section III.C.1 above, Dr. Zhang started his work on

eukaryotic targets in 2011 and succeeded prior to the publication of Jinek 2012. Fact 162.

Third, Senior Party cites no support for its assertion that the articles came from

10

independent research groups. Fact 164; Ex. 1024 at 151. Dr. Carroll indicated that by

11

independent he included groups sharing data. Carroll Tr. 102:1-7. As noted above, Dr.

12

Church, the senior author of the Mali 2013 article at Harvard, was co-advisor to Dr. Zhangs

13

colleague Dr. Cong. Fact 101; Ex. 2009, Simons.3d at 11.111. Dr. Church and Keith Joung

14

(the senior author on the Hwang article) are reported to be members of the Broad, which is a

15

community including scientists from Harvard, Harvard Hospitals and MIT founded to accelerate

16

breakthroughs and to impact human health by encouraging collaboration. Dr. Church

17

acknowledged that his graduate student Dr. Cong worked with Dr. Zhang on the eukaryotic

18

experiments at the Broad. Fact 170; Ex. 2423. Moreover, the Hwang 2013 paper acknowledges

19

the assistance of George Church. Ex. 1058 at 6. These are hardly what most would consider

20

entirely independent research groups.

21

Fourth, the actions of these research groups do not indicate the understanding of persons

22

of ordinary skill. Many of these research groups include individuals with significantly more than

23

the ordinary level of skill, including Drs. Church and Joung and Jin-Soo Kim of the Cho 2013
25

BROAD et al. OPPOSITION 4

paper, who have significant history working with mammalian cells using both zinc fingers and

TALENs. Fact 165; Ex. 2009 11.112. Dr. Carroll confirmed that Dr. Kim and Dr. Doudna had

higher than the ordinary level of skill. Carroll Tr. 263:18-264:20; Fact 166.

4
5
6

3.

The Doudna P1 And P2 Applications Do Not Enable A Skilled Artisan


To Practice An Embodiment Of Count 1

Senior Party asserts in Motion 4 that Doudna P1 and P2 enable embodiments falling

within Count 1. UC Motion 4 at 14:19, 22:3. Junior Partys response is that nothing in Doudna

P1 and P2 enable a person of ordinary skill to use a CRISPR-Cas9 system of Count 1 in

eukaryotic cells. Fact 167. Senior Party relies on the alleged ability of skilled persons to use

10

Chimera A of Example 1 in eukaryotic cells. However, Doudna P1 and P2 do not teach a person

11

of ordinary skill how to adapt a CRISPR-Cas9 system with Chimera A or any guide RNA to

12

function in eukaryotic cells. Those applications certainly do not provide the specific and useful

13

teaching of how to practice the invention required for technology like CRISPR, which was in a

14

nascent stage of development when the applications were filed. Genentech, 108 F.3d at 1367-68.

15

Consideration of the Wands factors emphasizes that practicing the method of Count 1

16

based on Doudna P1 or P2 would have required undue experimentation. Wands, 858 F.2d at

17

737. For Factor 1, Doudna P1 and P2 each provide essentially no useful guidance for practicing

18

the method of the count in eukaryotic cells. Ex. 2009 11.117. For Factor 2, adapting Chimera

19

A to function in eukaryotic cells required extensive experimentation, as shown by Dr. Doudnas

20

own many frustrations. Id. For Factor 3, the applications contained no working examples of

21

CRISPR-Cas9 functioning in eukaryotic cells. Id. For Factors 4, 5, and 6, the nature of the

22

technology requires more disclosure than may be needed in other instances, given the nascent

23

state of CRISPR-Cas9 art. Id. For Factor 7, the technology here is highly unpredictable, with

24

multiple examples where others tried and failed to use prokaryotic systems in eukaryotic cells.
26

BROAD et al. OPPOSITION 4

Ex. 2009 11.32; Ex. 2010 1.45-1.54. As Dr. Carroll observed in 2012, the only way to

establish what would happen if one used CRISPR-Cas9 in eukaryotic cells was to conduct the

experiment. Ex. 1152 at 1660; Ex. 2009 11.32. In the absence of such tests, the result was

completely unpredictable. Id.

The lack of enablement is also demonstrated by the failures of other researchers using the

CRISPR-Cas9 system with Chimera A using routine techniques. Fact 105. The Chen US

application No. 61/734,256, filed December 6, 2012 (Chen P1), included unsuccessful testing

of Chimera A and a modified Chimera A. Fact 168; Ex. 2125 at 32, 34; Carroll Tr. at 178:6-18.

Chen P1 presents evidence from three tests in eukaryotic cells with Chimera A or modified

10

Chimera A type RNA, all three of which of generated negative results, indicating that Chimera A

11

did not function in eukaryotic cells. Fact 168; Ex. 2009 11.123-11.129. Altogether, the results

12

in Chen P1 are at best inconclusive, if not outright failures. Fact 169; Ex. 2009 11.123-11.129.

13

On the other hand, Dr. Zhangs group at the Broad used non-routine techniques to adapt

14

CRISPR-Cas9 with a modified Chimera A-type RNA for eukaryotic cells. They used two NLS

15

groups (one on each end) on the Cas9 protein, expressed the Chimera A using a vector, and

16

added four nucleotides to the end of the tracr component that were not present in Jinek 2012s

17

Chimera A. Ex. 1055 at 2; Ex. 2009 11.130. Even with those improvements, eight of the 14

18

samples tested reported no activity at all. Ex. 1055 at 21; Ex. 2009 11.130.

19

Senior Party relies on Dr. Kims work with Chimera A (Cho 2013), where CRISPR-

20

Cas9 systems with Chimera A were introduced to eukaryotic cells using a technique called

21

nucleofection. Ex. 1059 at 6; Ex. 2009 11.132. Rather than routine research, those researchers

22

added 2-15 times more plasmids and RNA to the eukaryotic cells than was recommended by the

23

manufacturer of their nucleofection equipment. Fact 172; id. Overloading cells in that manner
27

BROAD et al. OPPOSITION 4

was not routine and would not work for real world applications. Fact 93; Ex. 2009 11.133. This

confirms that persons of ordinary skill would have needed undue experimentation to make the

Chimera A function in eukaryotic cells. Facts 173, 176. Indeed, in subsequent papers from the

same laboratory, the researchers abandoned Chimera A and used a longer tracr. Fact 174; Ex.

1509; Ex. 1508; Carroll Tr. at 279:20-281:8, 288:10-289:12, 289:13-291:7; Ex. 2009 11.134.

Also, the Kim group did not attribute their alleged success in eukaryotic cells to Jinek

2012 or the Senior Party. To the contrary, the Kim group filed a patent application and recently

submitted arguments, and a supporting declaration of Bryan Cullen, stating that their eukaryotic

work was not obvious over Jinek 2012. Fact 171; Ex. 2422 at 11, 20-24.

10

It is also significant to note that the journal SCIENCE has a large reach in the scientific

11

community, with 570,400 readers each week and over five million monthly visits to the Science

12

website. Fact 175; Ex. 2420 at 5; Ex. 2009 11.113. Given this reach, the fact that only one

13

highly specialized group, using non-routine techniques, was able to use a CRISPR-Cas9 system

14

with unmodified Chimera A in eukaryotic cells, highlights the shortcomings of the description in

15

Jinek 2012 and the Doudna applications. Fact 176; Ex. 2009 11.113.

16
17
18
19
20

Thus, the record demonstrates that Doudna P1 and P2 did not enable a person of ordinary
skill to practice an embodiment of Count 1 without undue experimentation.
4.

The Doudna P3 Application Does not Provide Written Description


Support For An Embodiment Of Count 1

Senior Party asserts in Motion 4 that Doudna P3 contains a working example of the

21

methods of Count 1. UC Motion 4 at 23:3. Junior Partys response is that the working

22

example in Doudna P3 was in fact a failure it did not show that the CRISPR-Cas9 systems of

23

Doudna P3 were adapted to function in eukaryotic cells. The Doudna P3 application, filed

24

January 29, 2013 (Fact 177), includes for the first time testing in eukaryotic cells, and also adds a
28

BROAD et al. OPPOSITION 4

description of new CRISPR-Cas9 systems with tracrRNA components that differ from Chimera

A. Fact 178; Ex. 2009 11.137. While Doudna P3 asserts that its tests in eukaryotic cells were

successful, the results in the application show otherwise. Persons skilled in the art reading

Doudna P3 in January 2013 would have concluded that the eukaryotic test results reported in

Doudna P3 are inconclusive at best. Thus, persons of ordinary skill would have concluded from

reading Doudna P3 that the inventors did not provide information showing possession of an

invention of a CRISPR-Cas9 system that functioned in eukaryotic cells.

8
9

Only two experiments in Doudna P3 purport to show cleavage in living eukaryotic cells.
Fact 179. According to the methods section in Doudna P3, the experiment reported in Figure

10

38B of Doudna P3 should show bands of 360 bp for PCR product with no cleavage. Ex. 1005 at

11

412; Fact 180; Ex. 2009 11.140. The gel should show bands between 160 and 200 bp if the

12

CRISPR-Cas9 successfully cleaved the target DNA. Fact 181; Ex. 2009 11.140. But the

13

samples containing Cas9 and sgRNA do not show any bands between 160 and 200 bp. Fact 182;

14

Ex. 1005 at 38B; Ex. 2009 11.140. Persons skilled in the art would have concluded that the

15

named inventors failed to obtain cleavage in eukaryotic cells. Fact 183; Ex. 2009 11.140.

16

The only data in Doudna P3 from a eukaryotic cells experiment is in Figure 36E, which

17

shows a single lane suggested to show cleavage. The authors claim that a very faint band in lane

18

5 from the left represents cleavage of the target sequence resulting in editing of the sequence.

19

Ex. 1005 at 418, Figure 36E. Persons of ordinary skill reviewing Fig. 36E would have

20

concluded that the data was inconclusive. Fact 184; Ex. 2009 11.151-11.159. It is clear from

21

the figure that the authors did not purify their PCR product as evidenced by the additional bands

22

visible in lanes 2 and 4. Fact 185; Ex. 2009 11.151-11.159

23

In addition, experiments in Figure 36B of Doudna P3 would have confirmed that the P3
29

BROAD et al. OPPOSITION 4

experiments in eukaryotic cells were unsuccessful. Figure 36B according to Doudna P3,

revealed abundant Cas9 expression and nuclear localization. Fact 186; Ex. 1005 at 416. To

persons of ordinary skill, however, the test results did not indicate nuclear localization. Fact 187;

Ex. 2009 11.154. The failure of the Senior Party inventors to localize the Cas9 protein in the

nucleus could account for the poor results shown in Figures 36E and 38B. Fact 188; Ex. 2009

11.162.

In view of the poor experimental results in Doudna P3, Doudna P3 did not provide

sufficient information to demonstrate possession. Moreover, Senior Party contends that Jinek

2013, a proxy for Doudna P3, is pertinent to written description because it allegedly showed that

10

the system published in Jinek 2012 (disclosed in the First Provisional) worked in eukaryotic

11

cells. UC Motion 4 at 19:15-19. Junior Partys response is that because the eukaryotic tests in

12

Doudna P3 were unsuccessful, if anything, they would demonstrate that the inventors also did

13

not possess a CRISPR-Cas9 system that could function in eukaryotic cells the time of filing of

14

Doudna P1 and P2. In addition, to the extent that Doudna P3 discloses information such as such

15

as actual eukaryotic testing (albeit failed in P3) and specific techniques for CRISPR-Cas systems

16

for eukaryotic cells, it only highlights the absence of that information in Doudna P1 and P2.

17

IV.

18
19

CONCLUSION
For the reasons set forth herein, Senior Partys Motion 4 should be denied.

Dated: August 15, 2016

Respectfully submitted,

20
21
22
23
24
25
26
27

/s/Steven R. Trybus
Steven R. Trybus
Reg. No. 32,760
Counsel for Broad
Jenner & Block LLP
353 North Clark Street
Chicago, IL 60654
Telephone: (312) 222-9350
strybus@jenner.com
30

BROAD et al. OPPOSITION 4

APPENDIX 1: LIST OF EXHIBITS

EXHIBIT
NUMBER

DESCRIPTION

1003

U.S. Provisional Patent Application No. 61/652,086, filed on


May 25, 2012 (Doudna P1 or P1).

1004

U.S. Provisional Patent Application No. 61/716,256, filed on


October 19, 2012 (Doudna P2 or P2)

1005

U.S. Provisional Patent Application No. 61/757,640, filed on


January 28, 2013 (Doudna P1 or P1).

1024

Declaration of Dr. Dana Carroll.

1055

Cong et al., Multiplex Genome Engineering Using


CRISPR/Cas Systems, 339 Science 819-823 (2013).

1056

Mali, et al., RNA-Guided Human Genome Engineering via


Cas9, 339 Science 823-826 (2013).

1058

Hwang et al., Efficient genome editing in zebrafish using a


CRISPR-Cas system, 31 Nature Biotechnology 227-29 (2013).

1059

Cho et al., Targeted genome engineering in human cells with


the Cas9 RNA-guided endonuclease, 31 Nature Biotechnology
230-232 (2013).

1152

Carroll, A CRISPR Approach to Gene Targeting, 20 Molecular


Therapy 1659-1660 (2012).

1155

Jinek et al., A programmable dual-RNA-guided DNA


endonuclease in adaptive bacterial immunity, 337 Science 816821 (2012).

1161

Sontheimer et al., U.S. Patent Publication No. 2010/0076057,


published on March 25, 2010.

1508

Kim et al., Highly efficient RNA-guided genome editing in


human cells via delivery of purified Cas9 ribonucleoproteins,
24 Genome Research 1012-1019 (2014) with Supplemental
Information.

A1-1

1509

Cho et al., Heritable Gene Knockout in Caenorhabditis elegans


by Direct Injection of Cas9-sgRNA Ribonucleoproteins, 195
Genetics 1177-1180 (2013) with Supplemental Materials and
Methods.

2009

Third Declaration of Dr. Paul Simons, executed August 15,


2016.

2010

Declaration of Dr. Ronald Breaker, executed August 15, 2016.

2125

U.S. Patent Application No. 61/734,256, filed December 18,


2013.

2207

Chen et al., U.S. Patent Application No. 61/734,256, filed


December 18, 2013.

2230

Pandika, Rising Stars: Jennifer Doudna, CRISPR Code Killer,


OZY (Jan. 7, 2014), http://www.ozy.com/rising-stars/jenniferdoudna-crispr-code-killer/4690.

2246

Deveau et al., Phage Response to CRISPR-Encoded Resistance


in Streptococcus thermophiles, 190 J. Bacteriology 1390-1400
(2008).

2290

Max E. Perutz Laboratories, Programmable RNA Complex


Could Speed Genome Editing in the Lab (June 28, 2012),
https://www.mfpl.ac.at/about-us/news/article/newsdetail/programmable-rna-complex-could-speed-genomeediting-in-the-lab.html.

2411

SN 14/704,551 Excerpt, NIH grant application.

2413

SN 12/565,589 Excerpt.

BROAD et al. OPPOSITION 4

APPENDIX 2: STATEMENT OF MATERIAL FACTS

UCs Alleged Facts with Broads Responses

1. The U.S. Provisional Application No. 61/652,086 (the First Provisional) titled

Methods and Compositions for RNA-Directed Site-Specific DNA Modification, was filed on

May 25, 2012, and lists Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, and Krzysztof

Chylinski as co-inventors. See Ex. 1003.

7
8

Broads response: ADMIT

9
10

2. U.S. Provisional Application No. 61/716,256 (the Second Provisional), titled,

11

Methods and Compositions for RNA-Directed Site-Specific DNA Modification, was filed on

12

October 19, 2012, and lists Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, Krzysztof

13

Chylinski, and James Harrison Doudna Cate as co-inventors. See Ex. 1004.

14
15

Broads response: ADMIT

16
17

3. U.S. Provisional Application No. 61/757,640 (the Third Provisional), titled, Methods

18

and Compositions for RNA-Directed Site-Specific DNA Modification, was filed on January 28,

19

2013, and lists Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, Krzysztof Chylinski,

20

and James Harrison Doudna Cate as co-inventors. See Ex. 1005.

21
22

Broads response: ADMIT

23
24

4. The technology behind Count 1 is explained in the Declarations of Drs. Carroll and
A2-1

Greider. See Ex. 1022, 36-67, 332-337, Ex. 1024, 30-61, 323-328 (both citing Exs. 1007,

1020, 1032, 1033, 1125, 1126, 1153, 1154, 1155, 1156, 1199, 1208, 1211, 1214, 1225, 1227,

1254, 1255, 1261, 1262, 1270, 1271, 1288, 1298, 1299, 1301, 1304, 1322, 1370).

4
5

Broads response: DENY

6
7

5. Each of the First Provisional, Second Provisional, and Third Provisional describes and

enables an anticipation of Count 1. See Exs. 1003-1005; see also Ex. 1022, 35, 329, 332-363,

373-448, Ex. 1024, 29, 320, 323-354, 3640439.

10
11

Broads response: DENY

12
13

6. The First Provisional describes and enables methods within the scope of Count 1. See

14

Ex. 1003; see also Ex. 1022, 341-363, 373-431, Ex. 1024, 332-354, 364-422.

15
16

Broads response: DENY

17
18

7. Example 1 of the First Provisional sets forth all elements of Count 1 other than the

19

eukaryotic cell element. See Ex. 1003, 00248-00252, Figures 3, 5; see also Ex. 1022, 341-

20

349, Ex. 1024, 332-340.

21
22

Broads response: DENY

23

A2-2

8. The eukaryotic cell element of Count 1 is described and enabled throughout the First

Provisional, including at Paragraph 00165. See Ex. 1003, 00165; see also 00167, 00177,

00178, 00201, 00215, 00216; see also Ex. 1022, 341-363, 373-431, Ex. 1024, 332-354,

364-422.

5
6

Broads response: DENY

7
8

9. Example 1 in combination with Paragraph 00165 describes and enables an anticipation of

Count 1 because one of ordinary skill in the art as of May 25, 2012, would have recognized that

10

the listed inventors were in possession of the methods of Count 1 and could have performed

11

those methods using only routine and predictable techniques. See Ex. 1003, 00165, 00248-

12

00252; see also Ex. 1022, 350-351, Ex. 1024, 341-342.

13
14

Broads response: DENY

15
16

10. Example 1 in the First Provisional is an actual working example of a method of cleaving

17

target DNA, albeit not in a eukaryotic cell. See Ex. 1003, 00248-00252, Figs. 3, 5; see also

18

see also Ex. 1022, 350-352, Ex. 1024, 341-343.

19
20

Broads response: DENY

21
22

11. Example 1 of the First Provisional satisfies all aspects of the second element of Count 1,

23

except for contacting in a eukaryotic cell, because it employs Cas9, which is necessarily a Type

A2-3

II CRISPR-associated Cas protein, and specifies that the [t]arget DNAs were obtained by

chemical synthesis and that the target DNA was contacted with a Type II CRISPR-Cas system:

[t]he DNA-targeting RNA/polypeptide complexes were assembled . . . . [and] the assembled

complex was added to target DNA and incubated. See Ex. 1003, 00248-00249; see also Ex.

1156, pp. 4-6; see also Ex. 1022, 350-353, Ex. 1024, 341-344.

6
7

Broads response: DENY

8
9
10

12. Included in the Type II CRISPR-Cas system of Example 1 of the First Provisional were

11

engineered and non-naturally-occurring single-molecule DNA-targeting RNAs. See Ex. 1003,

12

00248; see also Ex. 1022, 353, Ex. 1024, 344.

13
14

Broads response: ADMIT

15
16

13. Contacting a target DNA with the system of Example 1 of the First Provisional in

17

eukaryotic cells is taught throughout the First Provisional. See Ex. 1003, 00165; see also Ex.

18

1003, 00167, 00177, 00178, 00201, 00215, 00216; see also Ex. 1022, 352-353, 376, Ex.

19

1024, 343-344, 367.

20
21

Broads response: DENY

22
23

14. Example 1 of the First Provisional satisfies all aspects of the third, fourth, fifth, and sixth

A2-4

elements of Count 1 because it uses the required system and specifies that the DNA-targeting

polypeptide was based on the sequence of Streptococcus pyogenes Cas9 and that a complex

was assembled from [t]he DNA-targeting RNA and the [Cas9] polypeptide. See Ex. 1003,

00248-00249; see also Appendix 3; see also Ex. 1022, 354, Ex. 1024, 345.

5
6

Broads response: DENY

7
8

15. Figure 3B of the First Provisional illustrates the structure of the engineered and non-naturally

occurring single-molecule DNA-targeting RNAs, which combined the targeter-RNA

10

and activator-RNA into a single molecule. See Ex. 1003, 00251, Figs. 1, 3B; see also

11

Appendix 3; see also Ex. 1022, 354, Ex. 1024, 345.

12
13

Broads response: DENY

14
15

16. Example 1 of the First Provisional satisfies all aspects of element 7 of Count 1 because

16

the DNA-targeting RNA formed a complex with the Cas9 protein during incubation [with each

17

other] in the cleavage buffer for 15 min[utes] at room temperature. See Ex. 1003, 00249; see

18

also Ex. 1022, 355, Ex. 1024, 346.

19
20

Broads response: DENY

21
22

17. Example 1 of the First Provisional explains how the Cas9 protein was targeted to the

23

target DNA by adding the assembled DNA-targeting RNA/Cas9 complex to target DNA and

A2-5

incubated for 1 h[ou]r at 37C. See Ex. 1003, 00249; see also Ex. 1022, 355, Ex. 1024,

346.

3
4

Broads response: DENY

5
6

18. Figure 1 of the First Provisional supports the seventh element of Count 1 because it

depicts the DNA-targeting RNA forming a complex with the site-directed modifying polypeptide

(such as Cas9) and that cleavage (depicted by scissors) occurs because the Cas9 protein was

targeted to the target DNA. See Ex. 1003, Fig. 1; see also Appendix 3; see also Ex. 1022, 355,

10

Ex. 1024, 346.

11
12

Broads response: DENY

13
14

19. Figures 3A and 5A of the First Provisional show that cleavage of the target DNA

15

molecules did occur, i.e., the methods worked. See Ex. 1003, 00248-00252, Figs. 3A, 5A; see

16

also Ex. 1022, 355, Ex. 1024, 346.

17
18

Broads response: DENY

19
20

20. The First Provisional states the tested single-molecule DNA-targeting RNAs (RNA

21

chimera A) supported efficient target DNA cleavage. See Ex. 1003, 00251, Fig. 3B.

22
23

Broads response: ADMIT

A2-6

1
2

21. Example 1 of the First Provisional demonstrates the necessary and sufficient components

of the CRISPR-Cas9 system. See Ex. 1003, 00248-00252; see also Ex. 1022, 342-349, Ex.

1024, 333-340.

5
6

Broads response: DENY

7
8

22. Extensive guidance in the First Provisional, such as that provided in Paragraph 00165,

explained that the methods of Example 1 could be used in host cells, including eukaryotic cells.

10

See Ex. 1003; see also Ex. 1022, 341-358, 373-407, Ex. 1024, 332-349, 364-398.

11
12

Broads response: DENY

13
14

23. Claims 54, 58, 61, 66, and 72-75 of the First Provisional describe and enable all elements

15

of Count 1, including the eukaryotic limitation. See Ex. 1003, pp. 76-78; see also Ex. 1022,

16

359-363, 380-381, Ex. 1024, 350-354, 371-372.

17
18

Broads response: DENY

19
20

24. Claim 54, whose DNA-targeting RNA can be a single-molecule DNA-targeting RNA as

21

illustrated in Figure 1B, provides the framework of Count 1s second, third, fourth, and fifth

22

elements of relying upon a DNA-targeting RNA comprising i) a targeter-RNA or guide

23

sequence that hybridizes with the target sequence, and ii) an activator-RNA, or a trans-activating

A2-7

CRISPR RNA (tracrRNA), that hybridizes with the targeter-RNA to form a double-stranded

RNA duplex of a protein binding segment. See Ex. 1003, p. 76, Fig. 1B; see also Ex. 1022,

360, Ex. 1024, 351.

4
5

Broads response: DENY

6
7

25. Claims 58, 61, and 66 of the First Provisional make clear that the methods for cleaving

target DNA set forth in Example 1 may take place in a eukaryotic cell, such as in animal cells.

See Ex. 1003, pp. 76-77; see also Ex. 1022, 361, Ex. 1024, 352.

10
11

Broads response: DENY

12
13

26. Claim 72 of the First Provisional satisfies the second element of Count 1 because it

14

specifies that the DNA-modifying polypeptide used in the method of Claim 54 comprises an

15

amino acid sequence having at least about 75% amino acid sequence identity to amino acids 7-

16

166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted in Figure 2, or to the

17

corresponding domains in any of the amino acid sequences depicted in Figure 12. See Ex.

18

1003, p. 78; see also Ex. 1022, 362, Ex. 1024, 353.

19
20

Broads response: DENY

21
22

27. Because Cas9 proteins are necessarily part of a Type II CRISPR system and because the

23

Cas9 protein may be mutated up to 25% from a naturally-occurring Cas9, the second and sixth

A2-8

elements of Count 1 are satisfied by Claim 72 of the First Provisional. See Ex. 1003, pp. 76-78;

see Ex. 1156, pp. 4-6; see also Ex. 1022, 362, Ex. 1024, 353.

3
4

Broads response: DENY

5
6

28. As used in Claim 75 of the First Provisional, nuclease activity is synonymous with

cleavage. See Ex. 1003, p. 78; see also Ex. 1022, 363, Ex. 1024, 354.

8
9

Broads response: DENY

10
11

29. Claims 54 and 73-75 of the First Provisional, especially in view of Figure 1, satisfy

12

element seven of Count 1. See Ex. 1003, p.78; see also Ex. 1022, 363, Ex. 1024, 354.

13
14

Broads response: DENY

15
16

30. From at least Claims 54, 58, 61, 66, and 72-75 of the First Provisional, one of ordinary

17

skill in the art would have recognized that the inventors were in possession of the methods of

18

Count 1 and would have been able to perform those methods from the enabling disclosures

19

throughout the First Provisional. Ex. 1003, pp. 76-78; see also Appendix 3; see also Ex. 1022,

20

380, Ex. 1024, 371.

21
22

Broads response: DENY

23

A2-9

31. Many disclosures in the First Provisional other than Example 1 in combination with

Paragraph 00165 and Claims 54, 58, 61, 66, and 72-75 demonstrate that by May 25, 2012, the

inventors were in possession of methods that include each and every element of Count 1. See

Ex. 1003; see also Ex. 1022, 356-358, Ex. 1024, 347-349.

5
6

Broads response: DENY

7
8

32. The First Provisional uses Cas9, formerly known as Csn1, as an example of a site-directed

modifying polypeptide. See Ex. 1003, 0006, 0096; see also Ex. 1022, 356, Ex.

10

1024, 347.

11
12

Broads response: ADMIT

13
14

33. The First Provisional explains that the target DNA used in the disclosed methods may

15

include a cell from any organism and specifically identifies, among others, a cell of a single-

16

cell eukaryotic organism. See Ex. 1003, 0165; see also Ex. 1022, 356, Ex. 1024, 347.

17
18

Broads response: DENY

19
20

34. Figure 1B of the First Provisional illustrates a single-molecule DNA-targeting RNA,

21

demonstrating that the inventors were in possession of an engineered and non-naturally occurring

22

Type II CRISPR-Cas system, because the natural system uses a DNA-targeting RNA comprising

23

two RNA molecules (illustrated in Figure 1A). See Ex. 1003, 0004, Fig. 1; see also Ex. 1022,

A2-10

357, Ex. 1024, 348.

2
3

Broads response: DENY

4
5

35. The First Provisional makes clear that the inventors possessed the methods of Count 1 by

explaining that crRNA, tracrRNA, and the Cas9 protein were both necessary and sufficient for

Cas9 cleavage of target DNA and that such cleavage would occur in a eukaryotic cell. See Ex.

1003, 0002-0004, 0006, 0096, 0165, Fig. 1A; see also Ex. 1022, 358, Ex. 1024, 349.

9
10

Broads response: DENY

11
12

36. Guidance provided in Example 1 and Paragraph 00165 of the First Provisional, Claims

13

54, 58, 61, 66, and 72-75, and numerous passages in the Specification demonstrate that one of

14

ordinary skill in the art as of May 25, 2012, would have readily performed the methods of Count

15

1 without undue experimentation. See Ex. 1003, 00248-00252; see also Ex. 1022, 373-

16

407, Ex. 1024, 364-398.

17
18

Broads response: DENY

19
20

37. The First Provisional teaches that the Cas9 protein and/or the DNA-targeting RNAs can

21

be provided in the form of a nucleic acid containing a nucleotide sequence encoding those

22

components, and further explains that the nucleotide sequences can be contained on an

23

expression vector, such as a viral vector, as would be commonly done when performing the

A2-11

method in a eukaryotic cell. See Ex. 1003, 00120-00123; see also Ex. 1022, 387, Ex. 1024,

378.

3
4

Broads response: DENY

5
6

38. Suitable expression vectors are specifically identified in the First Provisional and it was

known as of May 25, 2012, that vectors, such as viral vectors, were used to deliver prokaryotic

polynucleotides into eukaryotic cells. See Ex. 1003, 00120-00123; see Exs. 1031, 1039,

1194, 1203, 1218, 1283, 1285, 1334, 1353; see also Ex. 1022, 388-389, Ex. 1024, 379-

10

380.

11
12

Broads response: DENY

13
14

39. The First Provisional explains that [i]n some embodiments, a nucleotide sequence

15

encoding a DNA-targeting RNA and/or a site-directed modifying polypeptide is operably linked

16

to a control element, e.g., a transcriptional control element, such as a promoter, and that the

17

transcriptional control element may be functional in [] a eukaryotic cell. See Ex. 1003,

18

00126; see also Ex. 1022, 390, Ex. 1024, 381.

19
20

Broads response: ADMIT

21
22

40. The First Provisional provides numerous examples of promoters that were known by May

23

25, 2012, to be functional in eukaryotic cells. See Ex. 1003, 00127; see also Exs. 1229, 1237,

A2-12

1310, 1332, 1337; see also Ex. 1003, 00127; see also Ex. 1022, 390-392, Ex. 1024, 381-

383.

3
4

Broads response: DENY

5
6

41. The First Provisional guides one to carry out the methods in eukaryotic cells by stating

that the methods can involve introducing into a cell (or a population of cells) one or more

nucleic acids comprising nucleotide sequences encoding a DNA-targeting RNA and/or a site-

directed modifying polypeptide, and explains that there are numerous well-known methods that

10

can be used to accomplish this introduction. See Ex. 1003, 00121, 00129; see also Ex. 1022,

11

393, Ex. 1024, 384.

12
13

Broads response: DENY

14
15

42. Methods for introducing a nucleic acid into a eukaryotic cell were known in the art for

16

over 30 years before the First Provisional was filed. See Exs. 1040, 1200, 1248, 1260, 1307; see

17

also Ex. 1022, 393-394, Ex. 1024, 384-385.

18
19

Broads response: DENY

20
21

43. The First Provisional explains the use of elements to target the Cas9 protein to a desired

22

cellular compartment, such as Protein Transduction Domains (PTDs) or nuclear localization

23

signals (NLSs). See Ex. 1528; see also Ex. 1022, 395, Ex. 1024, 386.

A2-13

1
2

Broads response: DENY

3
4

44. PTDs and NLSs are helpful, but not always necessary, to target eukaryotic genomic DNA

(residing in the nucleus of eukaryotic cells). See Ex. 1022, 395, Ex. 1024, 386.

6
7

Broads response: DENY

8
9

45. The First Provisional discloses that the Cas9 protein can include a conjugate to facilitate

10

traversing a cell or organelle membrane (a PTD). See Ex. 1003, 00115; see also Ex. 1022,

11

395, Ex. 1024, 386.

12
13

Broads response: ADMIT

14
15

46. Some of the PTDs discussed in the First Provisional were well known NLSs, including

16

the sequence RKKRRQRRR. See Ex. 1003, 00115, 00179; see also Ex. 1022, 395, Ex.

17

1024, 386.

18
19

Broads response: DENY

20
21

47. The First Provisional also provides examples of expression vectors that contain an NLS,

22

such as the vector pSVK3. See Ex. 1003, 00124; see also Ex. 1022, 395, Ex. 1024, 386.

23

A2-14

Broads response: DENY

2
3

48. For decades prior to the filing of the First Provisional, researchers had used nuclear

localization signals to target prokaryotic peptides expressed in eukaryotic cells. See Ex. 1528;

see also Ex. 1022, 395, Ex. 1024, 386.

6
7

Broads response: DENY

8
9

49. The First Provisional explains that standard recombinant nucleic acid manipulation

10

techniques, such as codon optimization, can be used to practice the methods in eukaryotic cells.

11

See Ex. 1003, 0033; see also Exs. 1029, 1030, 1035, 1213, 1217, 1235, 1236, 1293, 1302,

12

1319, 1327, 1329, 1335, 1336, 1502; see also Ex. 1003, 0033; see also Ex. 1022, 396-404,

13

Ex. 1024, 387-395.

14
15

Broads response: DENY

16
17

50. For many years prior to the filing of the First Provisional, researchers had used codon

18

optimization to enhance expression of prokaryotic polypeptides in eukaryotic cells. See Ex.

19

1003, 0033; see also Ex. 1022, 397, Ex. 1024, 388.

20
21

Broads response: DENY

22
23

51. The First Provisional would have allowed persons of ordinary skill in the art to carry out

A2-15

the methods of Count 1 without undue experimentation. See Ex. 1003; see also Ex. 1022,

405-407, 431, Ex. 1024, 396-398, 422.

3
4

Broads response: DENY

5
6

52. In terms of adapting in vitro results to a eukaryotic environment, the relative skill of those

in the art as of May 25, 2012, was high and the art associated with biotechnology was vast and

highly developed. See Ex. 1022, 406, Ex. 1024, 397.

9
10

Broads response: DENY

11
12

53. Numerous passages throughout the First Provisional set forth each and every element of

13

Count 1 and the application explains how to perform the methods. See Ex. 1003; see also Ex.

14

1022, 406, Ex. 1024, 397.

15
16

Broads response: DENY

17
18

54. Because the First Provisional provides detailed descriptions and the reagents and

19

techniques necessary to perform the methods were well-known, minimal experimentation was

20

needed to perform the methods of Count 1. See Ex. 1003; see also Ex. 1022, 406, Ex. 1024,

21

397.

22
23

Broads response: DENY

A2-16

1
2

55. That the First Provisional describes and enables embodiments within the scope of Count

1 is further evidenced by the U.S. Patent and Trademark Office rejecting Junior Partys claims as

anticipated by the First Provisional. See Exs. 1003, 1512-1527; see also Ex. 1022, 430a-430f,

Ex. 1024, 421a-421f.

6
7

Broads response: DENY

8
9

56. Example 1 in the First Provisional is an actual, working example of all but the eukaryotic

10

element of Count 1, and one of ordinary skill in the art could readily and predictably have

11

transitioned Example 1 to eukaryotic cells based upon the guidance provided in the application.

12

See Ex. 1003; see also Ex. 1022, 406, Ex. 1024, 397.

13
14

Broads response: DENY

15
16

57. The rapid success of numerous other research groups in applying the system of Example

17

1 of the First Provisional to eukaryotic cells immediately after the invention was published is

18

empirical evidence that the First Provisional describes and enables embodiments within the

19

scope of Count 1. See Exs. 1003, 1055, 1056, 1058, 1059, 1508-1510; see also Ex. 1022,

20

408-430, Ex. 1024, 399-421.

21
22

Broads response: DENY

23

A2-17

58. Jinek 2012 publicly disclosed the components that are necessary and sufficient for

cleavage by a Type-II CRISPR-Cas system (crRNA, tracrRNA, and Cas9) and demonstrated

successful reconstitution of the system outside of its natural bacterial environment. See Ex.

1155; see also Ex. 1022, 410, Ex. 1024, 401.

5
6

Broads response: DENY

7
8
9

59. Jinek 2012 explained the basic components required for the CRISPR system to operate,

10

demonstrated its effectiveness outside of a cell, and discussed its usefulness within a eukaryotic

11

cell. See Ex. 1155; see also Ex. 1022, 413, Ex. 1024, 404.

12
13

Broads response: DENY

14
15

60. Within just seven months after Jinek 2012 published, the Cong (Ex. 1055), Mali (Ex.

16

1056), Cho (Ex. 1059), and Hwang (Ex. 1058) groups had successfully performed experiments,

17

prepared and submitted manuscripts for review, and published papers on their results showing

18

that the system published in Jinek 2012 (disclosed in the First Provisional) worked in eukaryotic

19

cells. See, e.g., Ex. 1055, p. 819, middle col.; Ex. 1056, p. 1, Ex. 1059, p. 230, left col, and Ex.

20

1058, pp. 1-2; see Exs. 1002, 1011-1013, 1057, 1371, 1372; see also Ex. 1022, 414-423, Ex.

21

1024, 405-414.

22
23

Broads response: DENY

A2-18

1
2

61. Cong, Mali, Cho, and Hwang used the single molecule DNA-targeting RNA from the

First Provisional and Jinek 2012 and attribute Jinek 2012 as the inspiration that led to the rapid

and successful demonstration of DNA cleavage and gene editing in eukaryotes. See Ex. 1155,

Fig. 5; Ex. 1055, p. 819 and Fig. 2; Ex. 1056, p. 1, and Fig. 1; Ex. 1059, p. 230, left col, and Ex.

1058, pp. 1-2; see also Ex. 1022, 416-418, Ex. 1024, 407-409.

7
8

Broads response: DENY

9
10

62. The ordinary, well-known, and routine techniques (including particular promoters,

11

codon-optimization, and nuclear localization signals) employed by Cong, Mali, Cho, and Hwang

12

to successfully move the system of Jinek 2012 into eukaryotic cells are disclosed in the First

13

Provisional. See Exs. 1003, 1055, 1056, 1058, 1059; see also Ex. 1022, 423, Ex. 1024, 414.

14
15

Broads response: DENY

16
17

63. Less than two years after Jinek 2012 published, at least three more groups Kim (Ex.

18

1508), Cho (Ex. 1509), and Sung (Ex. 1510) successfully transitioned the methods of the First

19

Provisional to eukaryotic cells. See Exs. 1508-1510, 1531, 1532; see also Ex. 1022, 424-430,

20

Ex. 1024, 415-421.

21
22

Broads response: DENY

23

A2-19

64. The Cong, Mali, Cho, Hwang, Kim, Cho, and Sung groups were not conducting

extensive research, nor were they engaged in undue experimentation but were instead performing

well-known, routine experiments with a new technology that had been fully described in the First

Provisional. See Exs. 1003, 1055, 1056, 1058, 1059, 1508-1510; see also Ex. 1022, 430, Ex.

1024, 421.

6
7

Broads response: DENY

8
9
10

65. The Second Provisional provides at least one constructive reduction to practice of Count
1. See Ex. 1004; see also Ex. 1022, 432-439, Ex. 1024, 423-430.

11
12

Broads response: DENY

13
14

66. The Second Provisional includes all relevant content from the First Provisional to show at

15

least one constructive reduction to practice of Count 1. See Ex. 1004; see also Ex. 1022, 434-

16

436, Ex. 1024, 425-427.

17
18

Broads response: DENY

19
20

67. Claims 60, 65, 66, 69-72, and 98-100 of the Second Provisional disclose embodiments

21

within the scope of Count 1. See Ex. 1004, pp. 111, 112, 115-117; see also Appendices 6-8; see

22

also Ex. 1022, 435, Ex. 1024, 426.

23

A2-20

Broads response: DENY

2
3

68. All of the pertinent disclosure found to describe and enable an embodiment within the

scope of Count 1 in the First Provisional is carried through and included in the Second

Provisional. Compare Ex. 1003 with Ex. 1004; see also Ex. 1022, 437-439, Ex. 1024,

428-430.

7
8

Broads response: DENY

9
10

69. That in vitro working Example 1 of the First Provisional could have readily been

11

transitioned to a eukaryotic cell is evidenced by Cho, Mali, Cong, and Hwang and their reliance

12

on Jinek 2012. See Exs. 1003, 1004, 1055, 1056, 1058, 1059; see also Ex. 1022, 437, Ex.

13

1024, 428.

14
15

Broads response: DENY

16
17

70. The information in the Second Provisional that is not in the First Provisional provides

18

more guidance to one of ordinary skill in the art for carrying out the methods of Count 1. See Ex.

19

1004, 0016-0018, 0053-0061, 0084-0089, 00178, 00255-00256, 00289-00359, Figs. 13-35;

20

see also Ex. 1022, 437-439, Ex. 1024, 428-430.

21
22

Broads response: DENY

23

A2-21

71. The Third Provisional contains a working example of the methods of Count 1, which is

one of several constructive reductions to practice of Count 1 in that application. See Ex. 1005;

see also Ex. 1022, 440-448, Ex. 1024, 431-439.

4
5

Broads response: DENY

6
7

72. The Third Provisional includes all relevant content from the First Provisional to describe

and enable an embodiment within the scope of Count 1. Compare Ex. 1003 to Ex. 1005; see also

Ex. 1022, 442, Ex. 1024, 433.

10
11

Broads response: DENY

12
13

73. Claims 64, 69, 70, 73-76, and 102-104 of the Third Provisional disclose embodiments

14

within the scope of Count 1. See Ex. 1005, pp. 152, 153, 155-160; see also Ex. 1022, 443, Ex.

15

1024, 434.

16
17

Broads response: ADENY

18
19

74. All of the pertinent disclosure found in the First Provisional and the Second Provisional

20

to describe and enable an embodiment within the scope of Count 1 is carried through and

21

included in the Third Provisional. Compare Ex. 1003 with Ex. 1004 and Ex. 1005; see also Ex.

22

1022, 445-448, Ex. 1024, 436-439.

23

A2-22

Broads response: DENY

2
3

75. Example 1 of the First Provisional could have readily been trasitioned to a eukaryotic

cell even as early as May 25, 2012, and certainly by the Third Provisionals filing date of

January 28, 2013. See Exs. 1003, 1005; see also Ex. 1022, 445, Ex. 1024, 436.

6
7

Broads response: DENY

8
9

76. The rapid successes at performing the methods of Example 1 in eukaryotic cells by Mali,

10

Cho, Cong, Hwang, Kim, Cho, and Sung further evidence that one of ordinary skill in the art was

11

readily and predictably able to transition those methods to eukaryotic cells. See Exs. 1055, 1056,

12

1058, 1059, 1508-1510; see also Ex. 1022, 445, Ex. 1024, 436.

13
14

Broads response: DENY

15
16

77. Additional information included in the Third Provisional not included in the First

17

Provisional provides guidance to one of ordinary skill in the art for carrying out the methods of

18

Count 1. See Ex. 1005, 0005-0028, 0064-0074, 00119-00120, 00240-00244, Figs. 36-46; see

19

also Ex. 1022, 445, Ex. 1024, 436.

20
21

Broads response: DENY

22
23

78. The Third Provisional includes working Examples 2 and 3. See Ex. 1005, 00408-

A2-23

00450; see also Ex. 1022, 446, Ex. 1024, 437.

2
3

Broads response: DENY

4
5

79. Not only does Example 2 of the Third Provisional describe and enable embodiments

within Count 1, it further demonstrates that one of ordinary skill in the art would have applied

the methods disclosed in the First Provisional in eukaryotic cells without undue experimentation.

See Ex. 1005, 00408-00423; see also Ex. 1022, 447, Ex. 1024, 438.

9
10

Broads response: DENY

11
12

80. U.S. Patent Application Serial No. 13/842,859 currently shares four inventors from each

13

of the Provisional Applications, i.e., Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier,

14

and Krzysztof Chylinski. See Ex. 1001, pp. 346-347; see also Exs. 1003-1005, 1533.

15

Broads response: ADMIT

A2-24

Broads Additional Facts:

81. Neither Doudna P1 nor P2 includes an experiment that falls within the scope of Count 1. Ex.

1003; Ex. 1004; Ex. 2009 11.22.

82.

P1 lacks any discussion of PAM sequences. Ex. 1003; Ex. 2009 11.43.

83.

Prior to P1s May 2012 filing date, the role of PAM sequences in CRISPR system

targeting of natural DNA targets in bacteria was known. Ex. 2009 11.12-11.13.

84.

be addressed when adapting a CRISPR-Cas9 system for non-natural target DNA. Ex. 2009

11.43.

Persons of ordinary skill (POSITA) in 2012 would have expected the role of PAM to

10

85.

The state of the art in May 2012 relating to CRISPR-Cas9 systems was nascent and

11

unpredictable. Ex. 2009 11.117.

12

86.

13

would work in a eukaryotic cell. Ex. 2230.

14

87.

15

Ex. 2009 11.100.

16

88.

17

Cas9 systems to function in eukaryotic cells. See Ex. 2412; Paper 53; Ex. 2009 11.102.

18

89.

19

CRISPR-Cas9 system in prokaryotes. Ex. 1032; Ex. 1153; Ex. 1227; Ex. 2009 11.98-11.99.

20

90.

21

development of a CRISPR-Cas9 system showing his development of a single vector delivery of a

22

multiplexed system with dual molecule guide RNA and Cas9. Ex. 2411 at 16; Ex. 2009 11.97.

23

91.

Dr. Doudna conceded that even after Jinek 2012, she did not know if the Jinek system

After its in vitro tests, Senior Party experienced many frustrations in eukaryotic cells.

By early 2011, Junior Partys Feng Zhang had begun experiments adapting CRISPR-

By 2011, publications had identified certain components that potentially played role in

Dr. Zhang filed a grant application with the NIH on January 12, 2012, relating to his

Using four componentsCas9 nuclease and bacterial RNase III genes, a tracrRNA

A2-25

element, and the guide RNA arrayDr. Zhangs group successfully adapted a CRISPR-Cas9

system to function in eukaryotic cells using a dual molecule guide RNA. Ex. 1055 at 2.

92.

system to function in eukaryotic cells. Ex. 1055 at 2.

93.

B), with shorter tracrRNA components than the wild-type tracrRNA. Ex. 1003 at 2, 0006.

94.

Chimera B did not. Ex. 1155 at 820.

95.

The Cong et al. 2013 article reported Dr. Zhangs groups work adapting a CRISPR-Cas

Senior Partys inventors created two versions of chimeric RNA (Chimera A and Chimera

Jinek 2012 reported that Chimera A worked efficiently in vitro but the even shorter

The Jinek 2012 paper did not teach any longer tracrRNA components for their chimeric

10

RNA, but instead suggested to those of ordinary skill the use of Chimera A with its relatively

11

short tracrRNA component as compared to wild-type tracrRNA. Ex. 1155; Ex. 2009 11.66.

12

96.

13

to the CRISPR-Cas9 system previously developed in their laboratory. Ex. 1155 at 20;

14

97.

15

function in eukaryotic cells, using non-routine techniques, adding two NLSs to target the Cas9

16

protein to the nucleus, and a vector to express Cas9 and crRNA, with four nucleotides not

17

present on Chimera A. Ex. 1055 at 2.

18

98.

19

eukaryotic cell. Ex. 1055 at 20; Ex. 2009 11.130.

20

99.

21

longer tracrRNA. Ex. 1056 at 824; Ex. 2009 11.109.

22

100.

23

and some Broad scientists have advisors at both institutions. Ex. 2423; Ex. 2009 11.90.

Dr. Zhangs group at the Broad compared a modified version of Chimera A of Jinek 2012

Dr. Zhangs group at the Broad adapted CRISPR-Cas9 with Chimera A-type RNA to

Eight of the 14 Chimera A-type samples tested by the Broad scientists did not work in a

Dr. Churchs group at Harvard published the Mali 2013 article in January 2013 using

The Junior Party includes both the Broad and Harvard, which is affiliated with the Broad,

A2-26

101.

Dr. Zhangs group at the Broad included Dr. Cong and Dr. Church at Harvard was the co-

advisor to Dr. Cong. Simons Tr. 89:4-15.

102.

Dr. Hwangs group thanked Dr. Church for sharing unpublished results. Ex. 1058 at 6.

103.

Senior Party does not assert actual reduction to practice until October 29, 2012. Paper 58.

104.

The Senior Partys in vitro work published in Jinek 2012. UC Motion 4 at 18:14-19:5

105.

The Jinek system with Chimera A did not work in eukaryotic cells using routine

techniques. Ex. 2009 11.74.

106.

Harvard had achieved success in eukaryotes. Ex. 2230.

Dr. Doudnas laboratory was surprised when they learned that Dr. Churchs laboratory at

10

107.

After Dr. Churchs laboratory shared unpublished results, Dr. Doudnas laboratory

11

published Jinek 2013 paper with attempt to adapt CRISPR-Cas9 system to function in

12

eukaryotic cells. Ex. 2009 11.141.

13

108.

14

2013 using a chimeric RNA with longer tracrRNA components than Chimera A. Ex. 1005 at

15

Figures 36-38; Ex. 1055 at Figures 3-5.

16

109.

17

stated that Its not trivial to make CRISPR/Cas systems work in eukaryotic cells, and that

18

[o]ne thing is to have them in silico and have a sequence and realize theyre smaller, and

19

another thing is to do the [eukaryotic] experiments and make it work. Ex. 2213.

20

110.

21

Gairdner Award for their work with CRISPR-Cas systems. Ex. 2419.

22

111.

23

CRISPR-Cas system as a genome editing tools for function in eukaryotic cells. Ex. 2419 at 2.

Senior Partys inventors filed P3 containing the same eukaryotic test results as in Jinek

Dr. Luciano Marraffini, who studies CRISPR/Cas systems at Rockefeller University,

Drs. Zhang, Horvath, Doudna, Charpentier and Barrangou jointly received the 2016

The Gairdner Foundation noted Dr. Zhangs work in the development of the microbial

A2-27

112.

Count 1 recites a target DNA molecule in a eukaryotic cell that is not a natural target of

the prokaryotic CRIPSR-Cas9 system. Ex. 2009 11.17.

113.

bacteria, of which three did not show any cleavage. Ex. 1003 at 95; Ex. 2009 11.49.

114.

the correct PAM for all six orthologs in Figure 5 of Doudna P1. Ex. 2009 11.49.

115.

eukaryotic cells. Ex. 1003 at 248-253; Carroll Tr. 156:20-157:8; Ex. 2009 11.5.

116.

Figure 5 of Doudna P1 shows results using six Cas9 orthologs from different species of

Persons of ordinary skill would have understood that the Doudna P1 inventors did not use

Example 1 of Doudna P1 and P2 describes only in vitro testing and not testing in

Dr. Church characterized the move of CRISPR-Cas from bacteria to eukaryotic cells as a

10

huge jump. Ex. 2423.

11

117.

12

CRISPR-Cas9 system of Example 1 actually works in eukaryotic cells. Ex. 2009 11.22.

13

118.

14

CRISPR-Cas9 actually worked in eukaryotic cells, or identify a structural aspect of a CRISPR-

15

Cas9 system that would make it adapted for eukaryotic cells. Ex. 2009 11.25.

16

119.

17

CRISPR-Cas system in a eukaryotic cell based on in vitro experiments. Ex.2009 11.6, 11.92.

18

120.

19

that the Doudna inventors provided sufficient information to show possession of an invention of

20

an embodiment of a CRISPR-Cas system functioning in eukaryotic cells. Ex.2009 11.6, 11.92.

21

121.

22

Senior Partys UC and University of Vienna inventors asserted conception date for Count 1 of

23

October 18, 2011. Paper 58 at 1.

Example 1 and Paragraph 00165 of P1 and P2 do not provide any evidence that the

None of the portions of P1 or P2 cited by Senior Party provide evidence that the

Persons of ordinary skill in the art in 2012 would not predict the operability of a

Skilled persons in 2012 would have required positive eukaryotic tests results conclude

Senior Party did not make an embodiment of Count 1 until more than a year after the

A2-28

122.

Senior Party did not make an embodiment of Count 1 until more than two years after Dr.

Charpentiers asserted conception date for Count 1 of July 30, 2010. Paper 58 at 1.

123.

taught techniques for eukaryotic experiments. Ex. 1161 at 0054-0060; Ex. 2009 11.33.

124.

The Sontheimer application did not include actual eukaryotic experiments. Ex. 1161.

125.

The claims in the Sontheimer application to Type III CRISPR system for use in

eukaryotic cells were rejected by the USPTO. Ex. 2413 at 23.

126.

have conveyed to persons of ordinary skill lack of possession of an invention of a CRISPR-Cas9

Sontheimer application stated that its CRISPR system could be used in eukaryotes and

The lack of tests in eukaryotic cells in Doudna P2 four months after Jinek 2012 would

10

system adapted to function in a eukaryotic cell. Ex. 2009 11.74.

11

127.

12

or was known. Carroll Tr. at 255:4-258:17; 305:6-338:10; Ex. 2009 11.86-87.

13

128.

14

Jinek 2012 were performed in vitro with purified components and expressed doubt about

15

whether the CRISPR-Cas system would function in eukaryotic cells. Ex. 1152 at 1660.

16

129.

Dr. Carroll claims to have knowledge of one of ordinary skill in the art. Ex. 1024 at 19.

17

130.

Dr. Carroll did not dispute that his 2012 article does not state any expectation of success

18

for CRISPR-Cas9 systems in eukaryotic cells. Carroll Tr. at 117:5-118:2.

19

131.

20

system in eukaryotes will address concerns he had raised. Ex. 1152 at 1660.

21

132.

22

not know if the CRISPR-Cas9 system would work in eukaryotic cells. Ex. 2207.

23

133.

Dr. Carroll admitted that all of the information in P1 and P2 either appears in Jinek 2012

Dr. Carroll observed in a September 2012 article that [a]ll the experiments described in

In 2012, Dr. Carroll stated in reference to Jinek 2012 that [o]nly attempts to apply the

In a July 2014 article, Dr. Doudna confirmed that after publication of Jinek 2012, she did

An article from July 28, 2012 stated that [t]he next steps according to Dr. Charpentier

A2-29

and Dr. Doudna were to test the single-RNA construct with Cas9 to find out whether it works in

eukaryotic organisms. Ex. 2290.

134.

different from the environment of eukaryotic cells. Ex. 2009 11.114.

135.

Cas9 and RNA expression, Cas9 folding, and CRISPR-Cas9 complex formation and function,

even if it worked in an in vitro environment. Ex. 2009 11.30.

136.

systems to eukaryotic cells so as to be properly transcribed and translated. Ex. 2009 11.31.

A POSITA in May 2012 would have recognized that an in vitro environment is very

Differences between eukaryotic and prokaryotic cells could have unpredictable effects on

It was known in May 2012 that it may not be possible to deliver components of CRISPR

10

137.

It was known in May 2012 that proteins or nucleic acids present in eukaryotic cells could

11

interfere with interactions of components of CRISPR-Cas9. Ex. 2009 11.31.

12

138.

13

of CRISPR-Cas9 system. Ex. 2009 11.31.

14

139.

15

prevent CRISPR-Cas9 from functioning in eukaryotic cells. Ex. 2009 11.31.

16

140.

It was known in May 2012 that CRISPR-Cas9 evolved in prokaryotes. Ex. 2009 11.88.

17

141.

Count 1 requires engineering a CRISPR-Cas9 system to function in a manner that is

18

antithetical to its natural function in prokaryotic cells. Ex. 2009 10.5.

19

142.

20

larger than that of bacteria. Ex. 2009 10.7.

21

143.

22

nature. Ex. 2009 10.7.

23

144.

It was known in May 2012 that systems in eukaryotic cells developed may degrade RNA

P1 and P2 did not discuss any of the unique features of eukaryotic cells that might

A POSITA in May 2012 understood that the human genome is at least a thousand-fold

It was understood in May 2012 that CRISPR had never been found in eukaryotes in

It was known in May 2012 that the proteins and RNA molecules of a CRISPR-Cas9

A2-30

system may be toxic to eukaryotic cells. Ex. 2009 11.31.

145.

which are called off-target effects. Ex. 2009 11.31.

146.

and that off-target effects could, in fact, be lethal. Carroll Tr. at 227:3-6, 229:2-12.

147.

effects from CRISPR-Cas9 in eukaryotes without doing experiments. Greider Tr. at 405:14-22.

148.

CRISPR-Cas9 system to target DNA in the environment of a eukaryotic cell. Ex. 2009 11.31.

Toxic effects include the effect of genome editing in locations other than the target DNA,

Dr. Carroll testified that off-target effects are an important concern for genome editing

Dr. Greider testified that POSITA would have had no expectation regarding off-target

It was known in May 2012 that it may not be possible to localize the components of the

10

149.

P1 and P2 do not teach using an NLS in eukaryotic cells. Ex. 2009 11.31; Ex. 1003.

11

150.

Dr. Greider testified DNA target of interest is in nucleus. Greider Tr. at 285:20-286:1.

12

151.

Dr. Greider provided no opinion that the Senior Party inventors possessed an

13

embodiment of Count 1 for delivering a functioning CRISPR-Cas9 system, with the requisite

14

RNA, into a mitochondria or chloroplast. Greider Tr. (7/20/16) 422:4-18.

15

152.

16

difficulties adapting a prokaryotic system to eukaryotic cells. Ex. 2010 1.45-1.54.

17

153.

18

targetrons, gene editing tools based on group II self-splicing ribozymes. Ex. 2010 1.54

19

154.

20

to adapt them to function in eukaryotic cells. Ex. 2010 1.45-1.48

21

155.

Targetrons include protein and RNA components, like CRISPR systems. Ex. 2010 1.45.

22

156.

Group II self-splicing ribozymes were initially described in 1986, and targetrons were

23

developed that could act on bacterial genes by 2003. Ex. 2010 1.46.

A POSITA reading P1 and P2 at the times they were filed would have known of prior

A POSITA in May 2012 would have also been aware of the history of research relating to

Like CRISPR-Cas9, targetrons originated in prokaryotic cells, and researchers attempted

A2-31

157.

After nine more years of research, researchers had not been able to adapt targetrons to

function effectively in eukaryotic cells. Ex. 2010 1.47-1.54

158.

similar challenges could face researchers attempting to adapt the CRISPR-Cas9 system described

in Example 1 to function in eukaryotic cells. Ex. 2010 1.54.

159.

environments did not show adaptation to function in eukaryotic cells. Ex. 2010 1.45-1.54.

160.

problem for CRISPR-Cas9 as with targetrons, since both systems evolved to function in

A POSITA in May 2012 reading the P1 and P2 applications would have understood that

History of targetrons taught that ability of prokaryotic system to edit genes in other

A POSITA in May 2012 would have known that the level of Mg2+ ions could be a

10

prokaryotic cells. Ex. 2010 1.51-1.53.

11

161.

12

POSITA would have needed experimental results in eukaryotic cells to show possession of a

13

CRISPR-Cas9 invention for eukaryotic cells. Ex. 2009 11.35.

14

162.

15

publication of Jinek 2012. Ex. 1055 at 2; Ex. 2411; Ex. 2009 11.97.

16

163.

17

than modified Chimera A-type system for eukaryotic cells. Ex. 1055 at 20; Ex. 2009 11.130.

18

164.

19

2013, Cho 2013 and Hwang 2013 articles were inspired by Jinek 2012 or that those groups were

20

independent with respect to CRISPR research. Ex. 2009 11.6.

21

165.

22

2013 articles include individuals with more than the ordinary level of skill, including Drs.

23

George Church, Keith Joung and, Jin-Soo Kim. Ex. 2009 11.112.

Given the history of prior failed attempts to transfer prokaryotic systems to eukaryotes,

Dr. Zhang achieved success with a CRISPR-Cas9 system in eukaryotic cells prior to the

Dr. Zhang showed that his CRISPR-Cas9 system with longer tracrRNA worked better

Senior Party has not shown that the research groups responsible for the Cong 2013, Mali

The research groups responsible for the Cong 2013, Mali 2013, Cho 2013 and Hwang

A2-32

166.

Dr. Carroll confirmed that Dr. Kim and Dr. Doudna, one of the authors on Jinek 2013,

had higher than the ordinary level of skill. Carroll Tr. 263:18-264:20.

167.

P1 and P2 provide no guidance for practicing Count 1 in eukaryotes. Ex. 2009 11.114.

168.

Chen P1 reported testing of Chimera A and modified Chimera A, all of which generated

negative results. Ex. 2125 at 32, 34; Carroll Tr. at 178:6-18.

169.

Ex. 2009 11.123-11.129.

170.

the eukaryotic experiments at the Broad. Ex. 2423.

The testing in Chen P1 of Chimera A or modified Chimera A was inconclusive or failed.

Dr. Church acknowledged that his graduate student Dr. Cong worked with Dr. Zhang on

10

171.

The Kim group filed a patent application and recently submitted arguments, and a

11

supporting declaration of Bryan Cullen, stating that their eukaryotic work was not obvious over

12

Jinek 2012. Ex. 2422 at 11, 20-24.

13

172.

14

manufacturer of their nucleofection equipment, which was not routine. Ex. 2009 11.132.

15

173.

16

experimentation to make unmodified Chimera A function in eukaryotic cells. Ex. 2009 11.133.

17

174.

18

tracr. Ex. 1509; Ex. 1508; Carroll Tr. at 279:20-291:7; Ex. 2009 11.134.

19

175.

20

each week and over five million monthly visits to Science website. Ex. 2420; Ex. 2009 11.113.

21

176.

22

with unmodified chimera A could function in eukaryotic cells. Ex. 2009 11.113.

23

177.

Cho 2013 added more plasmids and RNA to eukaryotic cells than recommended by

The testing in Cho 2013 confirms that a POSITA would have needed extensive

In later papers from Cho laboratory, researchers abandoned Chimera A and used a longer

The journal SCIENCE has large reach in scientific community, with 570,400 readers

Only Dr. Chos group using non-routine techniques showed that a CRISPR-Cas9 system

The Doudna P3 application was filed January 29, 2013. Ex. 1005.

A2-33

178.

P3 includes testing in eukaryotic cells, and also adds a description of new CRISPR-Cas9

systems with tracrRNA components that differ from Chimera A. Ex. 1005.

179.

Two experiments in P3 purport to show cleavage in living eukaryotic cells. Ex. 1005.

180.

According to the methods section in P3, the experiment reported in Figure 38B of P3

should show bands of 360 bp for the PCR product in all samples. Ex. 1005 at 412.

181.

Cas9 successfully cleaved the target DNA. Ex. 2009 11.140.

182.

between 160 and 200 bp. Ex. 1005 at 36E; Ex. 2009 11.140.

In P3, Figure 38B, the gel should show bands between 160 and 200 bp if the CRISPR-

The samples in P3 containing Cas9 and sgRNA do not show any diagnostic bands

10

183.

A POSITA would have concluded from Figure 38B of P3 that there was no successful

11

cleavage in eukaryotic cells. Ex. 2009 11.140.

12

184.

13

details are missing. Ex. 2009 11.151-11.159.

14

185.

15

purify their PCR product as evidenced by the additional bands visible in lanes 2 and 4 and that

16

the test results were inconclusive at best. Ex. 2009 11.151-11.159.

17

186.

18

Cas9 expression and nuclear localization. Ex. 1005 at 416.

19

187.

20

Partys inventors obtained nuclear localization. Ex. 2009 11.154.

21

188.

22

the poor results shown in Figures 36E and 38B. Ex. 2009 11.162.

A POSITA reviewing Fig. 36E of P3 would have concluded that necessary experimental

A POSITA reviewing Fig. 36E of P3 would have concluded that the authors did not

Figure 36B reports results of an experiment that according to P3, revealed abundant

To a POSITA, the results in Fig. 36B of P3 would not have indicated that the Senior

The failure in Fig. 36B of P3 to localize the Cas9 protein in the nucleus could account for

A2-34

CERTIFICATE OF FILING AND SERVICE


I hereby certify that on the 15th day of August, 2016, a true and complete copy of the
foregoing BROAD et al. OPPOSITION 4 is being filed by 5:00 pm EST via the Interference
Web Portal. Pursuant to agreement of the parties, service copies are being sent by e-mail by 8:00
pm EST, to counsel for Senior Party as follows:
Todd R. Walters, Esq.
Erin M. Dunston, Esq.
Travis W. Bliss, Ph.D., Esq.
BUCHANNAN INGERSOLL & ROONEY PC
1737 King Street, Suite 500
Alexandria, Virginia 22314-2727
(703) 836-6620
todd.walters@bipc.com
erin.dunston@bipc.com
travis.bliss@bipc.com

Date: August 15, 2016

Li-Hsien Rin-Laures, M.D., Esq.


Sandip H. Patel, Esq.
Greta Noland
MARSHALL GERSTEIN & BORUN LLP
6300 Willis Tower
233 South Wacker Drive
Chicago, Illinois 60606
(312) 474-6300
lrinlaures@marshallip.com
spatel@marshallip.com
gnoland@marshallip.com

/s/ Steven R. Trybus


Steven R. Trybus
Reg. No. 32,760
Lead Counsel for Broad
Jenner & Block LLP
353 North Clark Street
Chicago, IL 60654
Telephone: (312) 222-9350
Facsimile: (312) 527-0484
strybus@jenner.com