Professional Documents
Culture Documents
By:
INTRODUCTION ...............................................................................................................1
II.
III.
IV.
ARGUMENT .......................................................................................................................1
V.
A.
B.
2.
C.
Broad Ignores UCs Extensive Support for the Embodiments of Count 1 ............. 5
D.
2.
3.
4.
Broads Remaining Attacks Are Not Relevant to the Benefit Inquiry ..... 14
CONCLUSION ..................................................................................................................16
-i-
Page(s)
- ii -
I.
INTRODUCTION
Senior Party (UC) Motion 4 points to several embodiments within the scope of Count 1
that are both described and enabled. Contrary to Broads assertions, nothing more is required
not working examples and not an actual reduction to practice. To obtain benefit, UC only
needed to show that its Provisionals evidence possession of at least one embodiment and that one
of ordinary skill in the art could carry out that embodiment. UC has satisfied that standard.
Apparently aware that it cannot attack the merits of UCs showing, Broad misstates UCs relied-
upon disclosures, attempts to add non-existent elements to Count 1, and offers immaterial
arguments and scientific theories. Such straw men warrant little attention.
10
II.
11
12
THE EVIDENCE
A list of exhibits upon which this Reply relies is set forth in Appendix 1.
III.
13
14
15
IV.
16
17
ARGUMENT
Broads Opposition 4 (BO4) fails to rebut UCs showing that each of its Provisionals
18
19
A.
20
At page 2, lines 19-24; page 4, lines 14-17; page 5, line 25 to page 6, line 1; page 14,
21
lines 18-20; page 15, lines 9-10; page 16, lines 4-5; and page 23, lines 9-11 of BO4, it is argued
22
that UC does not deserve benefit to its First and Second Provisionals because they do not
23
disclose a working example or an actual reduction to practice in eukaryotic cells. The response
24
-1-
Broad fails to cite any legal source requiring a working example or actual reduction to
practice. BO4, at 4:19-5:18. And for good reason there is no such requirement under the law
of written description or enablement. See, e.g., In re Borkowski, 422 F.2d 904, 908 (C.C.P.A.
1970) ([A] specification need not contain a working example if the invention is otherwise
disclosed in such a manner that one skilled in the art will be able to practice it without an undue
amount of experimentation.); Falko-Gunter Falkner v. Inglis, 448 F.3d 1357, 1366 (Fed. Cir.
2006) ([E]xamples are not necessary to support the adequacy of a written description . . . [and]
the written description standard may be met . . . even where actual reduction to practice of an
10
actual reduction to practice in eukaryotic cells are contrary to the law and should be disregarded.
11
B.
12
Broads arguments rely upon an alleged unpredictability in the art, which is based on an
13
incorrect reading and application of the law and an improper analysis of the art. When a proper
14
analysis is performed, it is clear that the art relating to the transfer of prokaryotic systems to
15
16
17
18
27, line 1 of BO4, it is argued that the relevant field of art was nascent and unpredictable.
19
The response is that, while the Type II CRISPR-Cas cleavage system was newly elucidated, the
20
relevant aspect for the inquiry at hand transitioning a prokaryotic system to a eukaryotic cell
21
was not a nascent and unpredictable field, but rather had been successfully accomplished for
22
decades using techniques that were so well known as to be commonplace as of the First
23
24
Predictability in the art is one of the Wands factors that is taken into account when
-2-
analyzing enablement. In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988). Similarly, the level of
detail required to satisfy the written description requirement varies depending on the nature and
scope of the claims and on the complexity and predictability of the relevant technology. Ariad
Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1351 (Fed. Cir. 2010). BO4 fails to analyze the
appropriate field of art for this inquiry. For biological subject matter, the Federal Circuit has
stated that the inquiry must be made not into the art in general, but rather the question is specific
for the predictability of the aspect at issue. Ariad, 598 F.3d at 1351 (emphasis added). The
aspect at issue in this benefit inquiry is not elucidation of the necessary and sufficient
components of the Type II CRISPR-Cas cleavage system, but rather application of that fully-
10
11
elucidated system to eukaryotic cells. See, e.g., BO4, at 15:9-13, 16:4-6; 16:27-17:6.
By the time UCs First Provisional was filed, persons of ordinary skill in the art had been
12
13
cells for decades using known techniques. See Facts 42, 48, 50; Ex. 1022, 135-150; Ex.
14
1024, 128-142; Exs. 1161, 1302, 1327, 1329, 1335, 1336, 1502. There was nothing nascent
15
and unpredictable about the aspect at issue it was conventional and commonplace. Id.
16
Further, given this extensive knowledge, the Federal Circuits other factors for evaluating the
17
adequacy of the disclosure also weigh in favor of granting UCs motion. Ariad, 598 F.3d at
18
1351 ([W]e have set forth a number of factors for evaluating the adequacy of the disclosure,
19
including the existing knowledge in the particular field, the extent and content of the prior art,
20
the maturity of the science or technology, and the predictability of the aspect at issue.) (internal
21
22
23
This wealth of existing knowledge and maturity of the technology is precisely why at
least seven groups were able to successfully use the Type II CRISPR-Cas system in eukaryotic
-3-
cells, draft manuscripts, and have them peer reviewed and published all within a year of the
publication of Jinek 2012. See Facts 60-64, 76; Ex. 1022, 158-162; Ex. 1024, 150-154;
Exs. 1055-1060, 1371, 1372. The only aspect of Count 1 that was not previously known was the
Type II CRISPR-Cas cleavage system, but the UC inventors fully described and utilized that
system in working examples in each of UCs Provisionals. See, e.g., Ex. 1003, 00248-00252;
At page 20, line 17-page 23, line 11 of BO4, it is argued that skilled persons would have
recognized that an in vitro environment is very different from the environment of eukaryotic
10
cells, and that various theoretical impediments (molecular crowding; temperature, pH, and ion
11
12
13
prokaryotic components to the eukaryotic cell; and possible issues arising from the increased
14
complexity of eukaryotic cells) exist that could negatively impact the ability to move a
15
prokaryotic system into eukaryotic cells. The response is that neither Broad nor its witnesses
16
points to even one example of a prokaryotic system that could not be utilized in a eukaryotic cell
17
due to any of these theoretical impediments. See BO4, at 21:3-22:19; Facts 135-149; Ex. 2009,
18
19
Drs. Breaker and Simons admitted that they could not identify any prokaryotic system
20
that could not be applied to eukaryotic cells due to these theoretical impediments. See Ex. 1555,
21
22
in the art would not have considered these theoretical impediments to be practical impediments,
23
and the rapid success of other independent research groups is objective evidence that these
24
theoretical impediments: (1) did not hinder transitioning the Type II CRISPR-Cas system to
-4-
eukaryotic cells, and (2) were not considered sufficiently likely to cause persons of ordinary skill
in the art to lack a reasonable expectation of success. Ex. Facts 63, 64, 76; 1534, 75-77, 93-
At page 23, line 12 to page 24, line 9 of BO4, it is argued that targetrons, also known as
Group II introns, provide an example of a prokaryotic system that was difficult to apply to
eukaryotic cells and that [t]he 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. The response is that targetrons were, as admitted by
Drs. Simons and Breaker, successfully utilized in eukaryotic cells many years before UCs First
10
Provisional was filed. See, e.g., Ex. 1534, 83; Ex. 1535, 83; Ex. 1556, at 217:15-218:2; Ex.
11
12
C.
13
At page 1, lines 13-15 of BO4, it is argued that Senior Party relies [only] on an in vitro
14
experiment in combination with the general disclosure that the system could be used in any cells
15
of interest, including eukaryotic cells. The response is that UC provided ample support to
16
17
In addition to the successful in vitro experiment and a discussion of the cell types in
18
which the Type II CRISPR-Cas system could be used, which is alone sufficient, UC also cited
19
disclosure in its Provisionals explaining how to provide the Cas9 protein and/or DNA-targeting
20
RNA in the form of a nucleic acid encoding those components, which can be included in a
21
vector, and which can be operably linked to a control element, such as a promoter functional in a
22
eukaryotic cell. See UC Motion 4, at 15:5-21 (citing, inter alia, Ex. 1003, 00120-00123,
23
00126, 00127); Facts 37-40. UC also cited disclosures in its Provisionals explaining how one
24
can introduce those vectors into eukaryotic cells using techniques that had been well-known for
-5-
decades. See UC Motion 4, at 15:22- 16:7 (citing, inter alia, Ex. 1003, 00121, 00129); Facts
41, 42. UC also cited additional disclosures in its Provisionals of conventional molecular
biology techniques that can be used in applying the Type II CRISPR-Cas system in eukaryotic
cells, such as the attachment of protein transduction domains or nuclear localization signals
(NLSs) to the Cas9 protein and the use of codon optimization. See UC Motion 4, at 16:8-17:6
(citing, inter alia, Ex. 1003, 0033, 00115, 00124, 00179); Facts 43-47.
At page 15, lines 11-13; page 16, lines 5-6; page 26, lines 10-13; and page 26, lines 17-18
necessary for practicing the Type II CRISPR-Cas system in eukaryotic cells. The response is
10
that Broad has not identified any structural modifications or adaptations that are necessary to
11
move the system into eukaryotic cells because no such necessary adaptations exist. As a further
12
response, Broad again ignores the numerous cited passages in UCs Provisionals that explain
13
which conventional techniques could be used and that indeed were used to apply the Type II
14
15
Broad fails to identify any structural modifications or other adaptations that are actually
16
required for the Type II CRISPR-Cas system to operate in a eukaryotic cell because there are
17
none. See, e.g., Ex. 1022, 161-162, 416, 417; Ex. 1024, 153-154, 407, 408; Exs. 1012,
18
1058-1060. None of codon optimization, vectors, NLSs, and a longer tracrRNA sequence is
19
required to achieve cleavage of target DNA in eukaryotic cells. Id. Nonetheless, the UC
20
Provisionals describe all of these optional features. Ex. 1022, 387-404, 422; Ex. 1024,
21
378-395, 413; facts 36-47; see also UC Motion 4, at 15:5-17:6. Broad again ignores the
22
numerous cited passages in UCs Provisionals that explain which conventional techniques could
23
be used and that indeed were used to apply the Type II CRISPR-Cas system to eukaryotic
-6-
cells. For example, UC cited to passages that discuss delivery of nucleic acid vectors encoding
the Cas9 protein and DNA-targeting RNA components, codon optimization of Cas9, and the
attachment of protein transduction domains to the Cas9 protein. See UC Motion 4, at 15:5-17:6
(citing, inter alia, Ex. 1003, 0033, 00115, 00120-00124, 00126, 00127, 00129, 00179); Facts
37-46. Not only are these structural aspect[s] that would make the system adapted for
eukaryotic cells, they are the same structural aspects that Broad and other research groups used
to apply the Type II CRISPR-Cas system to eukaryotic cells. UC Motion 4, at 20:2-5; Fact 62;
9
10
11
12
13
D.
14
also demonstrated by the failures of other researchers using the CRISPR-Cas9 system with
15
Chimera A using routine techniques, citing specifically to U.S. App. No. 61/734,256 (Chen).
16
The response is that Chen shows that researchers successfully used Chimera A and the Type II
17
18
including a DNA-targeting RNA based on the Chimera A sequence from Jinek 2012, was
19
introduced into eukaryotic cells. Ex. 2009, 11.126-11.128. The level of expression of green
20
fluorescent protein (GFP) within those eukaryotic cells was then used to determine whether the
21
Type II CRISPR-Cas system was functioning. Id. As Dr. Simons admitted, Chen specifically
22
indicates that [t]he percent GFP detected in each of the four experimental treatments (A-D) was
23
greater than in the control treatments (E,F). Ex. 2009, 11.128 (quoting Ex. 2125, 0066). Dr.
24
Carroll agrees. See Ex. 2007, at 183:1-11. The higher percentage of GFP shown in Chen is
-7-
objective evidence that Chen obtained positive results when applying a Type II CRISPR-Cas
At page 26, lines 18-20 of BO4, it is argued that adapting Chimera A to function in
eukaryotic cells required extensive experimentation. The response is that there is no credible
evidence to support Broads assertion. While Broad relies upon a quote from Dr. Doudna that
her group encountered many frustrations when moving forward with the technology, Broad
fails to cite any evidence that these frustrations pertained to transitioning the system to
eukaryotic cells. BO4, at 26:18-20. In fact, numerous independent research groups moved the
system into eukaryotic cells very rapidly using only well-known, conventional molecular biology
10
techniques, and Broad fails to rebut this uncontroverted objective evidence. UC Motion 4, at
11
19:15-20:23; Fact 62; Ex. 1022, 158-162; Ex. 1024, 150-154; Ex. 1534, 75-76; Ex.
12
13
At page 7, line 23 to page 8, line 4 and at page 27, lines 13-17 of BO4, it is argued that
14
Broads scientists used special techniques or non-routine techniques when applying the Type
15
II CRISPR-Cas system to eukaryotic cells, including the use of two NLSs, the use of an
16
expression vector, and the use of a tracrRNA that was four nucleotides longer. The response is
17
that the techniques used by Broad are irrelevant to the benefit inquiry. Moreover, Drs. Breaker
18
and Simons admitted that the inclusion of NLSs and the use of expression vectors are not
19
special techniques, but rather these are conventional techniques that were well understood and
20
widely used by persons of ordinary skill in the art when applying a prokaryotic system to
21
22
149:16, 150:8-13, 152:5-153:21, 168:4-169:11; Ex. 1556, at 238:8-16; Ex. 1638, at 179:24-
23
180:2; see also Ex. 1022, 135-150; Ex. 1024, 128-142. Broad fails to present any
-8-
evidence demonstrating that it used a tracrRNA that included an extra four nucleotides. BO4
at 8:3-5; Ex. 2009, 11.107; Ex. 1055, at 821 (Fig. 2B showing a chimeric RNA that has a
tracrRNA component that is identical to the one used in Jinek 2012); Ex. 1639, at 124:24-125:8.
Dr. Simons admitted that he is unaware of anything in Cong 2013 that shows the extra four
nucleotides or that even indicates extra nucleotides were added to the tracrRNA portion of the
chimeric DNA-targeting RNA. Ex. 1639, at 124:8-12, 126:22-127:7. Thus, this argument is
entitled to no weight as it is merely a bald assertion by Broad and Dr. Simons with no
evidentiary support in the underlying Cong 2013 publication or any other reference. See 37
C.F.R. 41.158(a) (Expert testimony that does not disclose the underlying facts or data on
10
which the opinion is based is entitled to little or no weight.); Meitznzer v. Mindick, 549 F.2d
11
775, 782 (C.C.P.A. 1977). Broads argument also lacks merit because UCs Provisionals
12
disclose each of the features to which Broad points (the use of NLSs, expression vectors, and
13
tracrRNA ranging from truncated versions to naturally occurring full length). See e.g. UC
14
Motion 4, at 15:5-17:6; Facts 36-47; Ex. 1003, 0012, 0033, 00115, 00121, Fig. 9; Ex. 1022,
15
16
At page 27, line 19 to page 28, line 1 of BO4, it is argued that the work of Dr. Kims
17
research group, published in Cho 2013 (Ex. 1059), was non-routine because those researchers
18
added 2-15 times more plasmids and RNA to the eukaryotic cells than was recommended by the
19
manufacturer of their nucleofection equipment. The response is that this assertion lacks
20
evidentiary support. Cho 2013 specifically states that the nucleofection procedure was done
21
according to the manufacturers protocol. Ex. 1059, at Supp. Info. p. 3. Broad relies only on
22
Dr. Simonss opinions and provides no evidence of a manufacturers protocol from which Dr.
23
Kims group allegedly deviated. See BO4, at 27:21-28:1 Ex. 2009, 11.132; Ex. 1059, at 6.
-9-
Dr. Simonss Declaration does not include any citation to a manufacturers protocol and he
admitted that he was unaware of the date or version of the alleged protocol on which he was
relying, and he further admitted that the protocol likely changed over time. Ex. 2009, 11.132-
11.133; Ex. 1639, at 59:2-7, 59:11-21. Dr. Simons also admitted that these studies in fact show
the successful use of the Chimera A construct in eukaryotic cells. Ex. 1639, at 130:2-13.
6
7
8
9
10
containing both DNA-targeting RNA and Cas9 protein (lanes 6, 8, and 9 from the left) possess
11
additional bands that could be cleavage product. Ex. 1005, at Fig. 38B; BO4, at 29:8-15.
12
Instead, Broad focuses on the fact that the bands do not appear to be the proper size based on the
13
marker lane (lane 1), and concludes that any cleavage product is the wrong size so the
14
experiment must have failed. BO4, at 29:8-15. Yet, Dr. Simons admitted that the marker lane
15
upon which Broad relies could simply have been mislabeled. Ex. 1638, at 150:19-152:19.
16
Further, Broad ignores the fact that the cleavage product band in the lanes for the Type II
17
CRISPR-Cas samples is within the same size range as the cleavage product bands in the lanes for
18
the ZFN positive control sample. Ex. 1005, at Fig. 38B (compare the lower band in lanes 6, 8,
19
and 9 to the lower bands in lane 10). From this, one of ordinary skill in the art would conclude
20
that the Type II CRISPR-Cas samples had successfully cleaved the target DNA.
21
Figure 36E of UCs Third Provisional clearly shows a cleavage product band in the lane
22
containing a DNA-targeting RNA and a Cas9 protein (lane 5) that is within the size range of the
23
cleavage product bands in the ZFN positive control sample (lane 7) and of the proper size
24
according to the marker lane (lane 1). Ex. 1003, at Fig. 36E. This cleavage band is not present
- 10 -
in any of the negative control lanes. One of ordinary skill in the art would again conclude from
Figure 36E that the Type II CRISPR-Cas sample had successfully cleaved the target DNA. Id.
Furthermore, data presented in Figures 36E and 38B of UCs Third Provisional were also
presented in Jinek 2013. See Ex. 1057, at Figs. 1E and 3B. As Dr. Breaker admitted, Jinek 2013
was peer-reviewed and recommended for publication by persons of ordinary skill in the art, and
he is not aware of any publication criticizing its data. Ex. 1638, at 131:4-6, 131:24-132:6,
153:22-154:6. Further, Drs. Breaker and Simons both admitted that Jinek 2013 has been widely
cited in other publications by persons of ordinary skill in the art. Ex. 1638, at 153:13-16; 154:7-
172:24; Ex. 1639, at 101:18--121:1; see also Exs. 1604-1610, 1621-1627. This provides
10
objective evidence that one of ordinary skill in the art would have viewed and indeed did view
11
the same results presented in Figures 36E and 38B of UCs Third Provisional as successful.
12
13
At page 2, lines 4-6 and page 12, lines 6-8 of BO4, it is argued that Count 1 is directed to
14
a target DNA molecule in a eukaryotic cell, which is not a natural target of the Type II CRISPR-
15
Cas system. The response is that Count 1 does not require cleavage of a eukaryotic target or a
16
non-natural target.
17
There is nothing in Count 1 that precludes the cleavage of natural targets of the Type II
18
CRISPR-Cas system (DNA of viruses and plasmids that invade bacteria in nature) within a
19
eukaryotic cell. See, e.g., Ex. 1022, 48; Ex. 1024, 42. One could utilize a Type II CRISPR-
20
Cas system to cleave natural targets of the Type II CRISPR-Cas system within a eukaryotic cell,
21
e.g., by targeting in a eukaryotic cell the same viral or plasmid DNA sequences that the Type II
22
CRISPR-Cas system naturally targets in prokaryotic cells. This plainly falls within the scope of
23
24
At page 1, lines 17-18 of BO4, it is argued that UCs First Provisional fails to describe
- 11 -
adjacent motif (PAM). At page 2, lines 7-13 of BO4, it is argued that it was known in the art by
May of 2012 that PAM sequences played a role in the ability of a CRISPR system in bacteria to
recognize natural DNA targets, and that the lack of discussion in the First Provisional of the
role of PAM would have indicated to persons of ordinary skill in the art that the inventors failed
to describe an invention configured for use against non-natural targets in a eukaryotic cell. At
page 12, lines 8-10 of BO4, it is argued that, [b]ased on the lack of any PAM discussion, skilled
persons would have concluded that the inventors had not begun to provide information needed to
use CRISPR systems with non-natural targets. The response is that Broads arguments about
10
11
12
require a target sequence with a PAM. See, e.g., Ex. 1639, at 21:1-4. Count 1 only requires that
13
target DNA is cleaved or edited or transcription of at least one gene encoded by the target DNA
14
15
both single-stranded and double-stranded target DNA. See, e.g., Ex. 1003, 0042; see also Ex.
16
1639, at 22:22-23:3. The Type II CRISPR-Cas system can target and cleave single-stranded
17
target DNA and such cleavage by Cas9 does not require a PAM sequence. See, e.g., Ex. 1155 at
18
p. 817; see also Ex. 1154 at E2584; Ex. 1639, at 36:20-23, 23:8-24:5, 27:4-9. Thus, Count 1
19
clearly encompasses embodiments that do not require a PAM sequence, so the presence or
20
absence of a PAM discussion in the UC Provisionals has no bearing on the benefit analysis.
21
As Broad admits, the existence of PAM sequences and their role in the Type II CRISPR-
22
Cas system were well-known in the art by May of 2012. See BO4 at 12:11-13; see also Ex. 1639
23
at 37:14-38:14, 39:6-40:20; Ex. 1298 at 736; Ex. 1022, 306-312; Ex. 102, 297-303. As
- 12 -
such, under well-settled law, the existence and role of the PAM sequence did not need to be
specifically recited in the Provisionals. Capon v. Eshhar, 418 F.3d 1349, 1357 (Fed. Cir. 2005).
Further, Broads arguments attempting to confine this knowledge to so-called natural targets
lack merit. Broad fails to provide evidence of any actual differences between natural and
non-natural targets of the Type II CRISPR-Cas system likely because no such differences
exist. See BO4, at 11:22-14:8. The knowledge in the art was not that the PAM sequence plays a
role in cleavage of natural double-stranded targets, as Broad states, but rather that it played a
role in all double-strand cleavage, regardless of whether the target was natural or not. See Ex.
1033, at 9276, 9278-9; Ex. 1022, 225-226, 310; Ex. 1024, 217-218, 301. For example,
10
studies performed by Sapranauskas et al. in 2011 showed that the PAM sequence played the
11
12
it was previously known to play in the cleavage of natural bacteriophage targets. Id.
13
To the extent that Broad is arguing that the target of the method of Count 1 is non-natural
14
merely because the Type II CRISPR-Cas system and/or target DNA is being introduced into a
15
cell in which it does not naturally occur, Sapranauskas is objective evidence that refutes this
16
argument as it demonstrates that PAM played a role even when an S. thermophilus Type II
17
CRISPR locus was introduced into an E. coli host cell, which does not naturally possess a Type
18
II CRISPR locus, and used to cleave a heterologously introduced target DNA. Id.
19
Thus, the knowledge in the art as of May of 2012 regarding the role of the PAM sequence
20
was not limited to natural targets, but rather it was well known that a PAM sequence played the
21
22
23
At page 12, line 18 to page 13, line 4 of BO4, it is argued that the experiment set forth in
Figure 5 of the First Provisional illustrates a failure to appreciate the significance of PAM.
- 13 -
The response is that Broad is clearly mistaken. The DNA-targeting RNA in Figure 5B is from S.
pyogenes, as can be seen by comparing the sequence in Figure 5B to those in Figures 6 and 7.
Ex. 1003, at Figs. 5B, 6, 7. Dr. Simons admitted that the target DNA in Figure 5B included the
appropriate PAM sequence for S. pyogenes. Ex. 1639, at 42:5-43:20; Ex. 1611. If UCs
inventors truly fail[ed] to appreciate the significance of PAM, as Broad argues, they would not
have utilized a target DNA that included the appropriate PAM sequence. Ex. 1003, at Fig. 5B.
At page 13, line 5 to page 14, line 5 of BO4, it is argued that the absence of a label for the
PAM sequence in Figure 1 of the First Provisional shows that UC chose to conceal that
information [about the PAM sequence] for some reason. The response is that this grossly
10
mischaracterizes Figure 1. Broad compares Figure 1A of Jinek 2012 (PAM labeled) and Figure
11
1 of UCs First Provisional (PAM not labeled) and alleges that the figure in the First Provisional
12
had been scrubbed to remove any reference to PAM sequences. BO4, at 13:10-14:5. Broads
13
allegation conveniently ignores the fact that all of the labels from the figure in the First
14
Provisional are different from those in Jinek 2012 and that the two figures were used in different
15
contexts. Compare Ex. 1003, Fig. 1, with Ex. 1155, Fig. 5A. Broad has no evidence to support
16
17
18
19
Dr. Zhang began his efforts to use CRISPR systems in eukaryotic cells before Jinek 2012
20
published. The response is that Broads alleged priority evidence has nothing to do with UCs
21
request for benefit. To the extent it is considered, the Zhang NIH grant application actually
22
supports UCs assertion that Broad was not able to utilize the Type II CRISPR-Cas system in
23
eukaryotic cells until after Jinek 2012 because it shows that Dr. Zhang did not understand the
24
components of the Type II CRISPR cleavage system Dr. Zhang did not understand the
- 14 -
At page 11, lines 3-12 of BO4, it is argued that [t]he industry has recognized the
pioneering nature of Dr. Zhangs work as evidenced by the receipt of one award by Dr. Zhang
for his application of the Type II CRISPR-Cas system to eukaryotic cells. The response is that
this is again irrelevant to the issue of benefit. To the extent it is considered, UC notes that Broad
conveniently omits from its discussion that the award Dr. Zhang shared with Drs. Doudna and
Charpentier was for development of CRISPR-Cas as a genome editing tool for eukaryotes. Ex.
2419, p. 2 (emphasis added). Further, as Dr. Simons admitted, Drs. Doudna and Charpentier
have received numerous other prizes for their contributions to the CRISPR gene-editing field.
10
See Ex. 1639, at 143:22-155:23; see also Exs. 1629-1635. Thus, the eukaryotic work of Drs.
11
Doudna and Charpentier has been as celebrated, if not more celebrated, as that of Dr. Zhang. Id.
12
At page 16, line 7 to page 17, line 2 of BO4, it is argued that the rejection of the
13
14
within Count 1. The response is that the Patent Offices rejection of an unrelated application
15
regarding different claimed subject matter is not relevant to the benefit issue. Broad suggests
16
that the Patent Office rejected Sontheimer for lack of written description and enablement based
17
on the absence in that application of a single working example, and analogizes that to UCs
18
Provisionals by stating that the Doudna P1 and P2 applications are entirely prophetic. BO4, at
19
20
the UC Provisionals include working examples of the Type II CRISPR-Cas system cleaving a
21
target DNA outside of the prokaryotic cellular environment. See, e.g., Ex. 1003, 00248-
22
00252; Facts 10-21; UC Motion 4, at 4:11-9:16. Those examples teach one of ordinary skill in
23
the art the components that are necessary and sufficient to operate the system in a heterologous
- 15 -
At page 18, line 5 to page 20, line 16 of BO4, it is argued that Jinek 2012 is not sufficient
to demonstrate that the inventors were in possession of the invention of Count 1. The response is
that it is each of UCs Provisionals that must describe and enable an embodiment within the
scope of Count 1, not Jinek 2012. As a further response, Broad mischaracterizes UCs
discussion of Jinek 2012 by alleging that UC 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. BO4, at 18:5-8. In reality, UCs Motion states
that Jinek 2012 contains much of the same information as the First Provisional, albeit with
10
much less detail about how one would apply the system in other organisms. UC Motion 4, at
11
18:14-16 (emphasis added). As explained in UC Motion 4, even this far less detailed disclosure
12
was sufficient to allow multiple research groups to rapidly move the system into eukaryotic cells.
13
14
V.
15
CONCLUSION
The absence in BO4 of any rebuttal to UCs positive benefit case is telling. Broads
16
arguments all rely upon an improper legal standard for written description and enablement and
17
mischaracterizations of both the record evidence and the knowledge in the art. Because each of
18
UCs Provisionals describes and enables an embodiment within the scope of Count 1, UC
19
20
Respectfully submitted,
By /Todd R. Walters/
Todd R. Walters, Esq. (Reg. No. 34,040)
BUCHANAN INGERSOLL & ROONEY PC
1737 King Street, Suite 500
Alexandria, Virginia 22314
todd.walters@bipc.com
Counsel for UC and Vienna
Date: September 28, 2016
DESCRIPTION
NO.
1001
U.S. Patent Application No. 13/842,859, filed on March 15, 2013, to Jennifer
Doudna et al. (the 859 Application)
1002
1003
1004
1005
1007
U.S. Patent No. 8,697,359, issued on April 15, 2014, to Feng Zhang (the 359
Patent)
1011
U.S. Patent No. 8,932,814, issued on January 13, 2015, to Le Cong and Feng
Zhang (the 814 Patent)
1012
U.S. Patent No. 8,795,965, issued on August 5, 2014, to Feng Zhang (the
965 Patent)
1013
U.S. Patent No. 8,871,445, issued on October 28, 2014, to Le Cong and Feng
Zhang (the 445 Patent)
1-1
DESCRIPTION
NO.
1020
1022
1023
1024
1025
1029
Dai et al., The Transcription Factors GATA4 and dHAND Physically Interact
to Synergistically Activate Cardiac Gene Expression Through a p300dependent Mechanism, 277(27) J. BIOL. CHEM. 24390-24398 (2002) (Dai)
1030
1031
1032
1033
1-2
DESCRIPTION
NO.
1035
1039
1040
1055
1056
1057
1058
1059
Cho et al., Targeted Genome Engineering in Human Cells With the Cas9
RNA-Guided Endonuclease, 31(3) NAT. BIOTECHNOL. 230-232 (2013) with
Supplemental Information (Cho)
1060
1-3
DESCRIPTION
NO.
1125
1126
1152
1153
1154
1155
1156
1161
1-4
DESCRIPTION
NO.
1194
1199
1200
Behr et al., Efficient gene transfer into mammalian primary endocrine cells
with lipopolyamine-coated DNA, 86 PNAS 6982-6986 (1989)
1203
1208
1211
Brouns et al., Small CRISPR RNAs guide antiviral defense in prokaryotes, 321
SCIENCE 960-964 (2008)
1213
1214
465-471 (1999)
1-5
DESCRIPTION
NO.
1217
Chiu et al., Engineered GFP as a vital reporter in plants, 6(3) CURR. BIO.
325-330 (1996)
1218
1225
Davis and Cui, Zinc Finger Nucleases for Genome Editing, 30(13) GEN. ENG.
& BIOTECH NEWS 1-3 (2010)
1227
1229
Dykxhoorn et al., Killing the Messenger: Short RNAs That Silence Gene
Expression, 4 NATURE REV. 457-467 (2003)
1235
1236
1237
1-6
DESCRIPTION
NO.
1248
Gorman et al., High efficiency gene transfer into mammalian cells, B307
PHIL. TRANS. R. SEC. LAND. 343-346 (1984)
1254
1255
1260
1261
1262
1270
Ishino et al., Nucleotide sequence of the iap gene, responsible for alkaline
phosphates isozyme conversion in Escherichia coli, and identification of the
gene product, 169(12) J. BACTERIOL. 5429-5433 (1987)
1-7
DESCRIPTION
NO.
1271
Jansen et al., Identification of genes that are associated with DNA repeats in
prokaryotes, 43(6) MOLECULAR MICROBIOLOGY 1565-1575 (2002)
1283
1285
Lombardo et al., Gene editing in human stem cells using zinc finger nucleases
and integrase-defective lentiviral vector delivery, 25(11) NAT. BIOTECHNOL.
1298-1306 (2007)
1288
1293
1298
Mojica et al., Short motif sequences determine the targets of the prokaryotic
CRISPR defence system, 155(3) MICROBIOLOGY 733-740 (2009) with
Supplementary Data
1299
1-8
DESCRIPTION
NO.
1301
1302
1304
Mussolino et al., A novel TALE nuclease scaffold enables high genome editing
activity in combination with low toxicity, 39(21) NUCL. ACIDS RES. 9283-9293
(2011)
1307
1310
1319
1322
1-9
DESCRIPTION
NO.
1327
1329
1332
1334
1335
1336
1337
1-10
DESCRIPTION
NO.
1353
1370
1371
Gratz et al., Genome Engineering of Drosophila with the CRISPR RNAGuided Cas9 Nuclease, 194 GENETICS 1029-1035 (2013) (Gratz)
1372
1502
1508
Kim et al., Highly efficient RNA-guided genome editing in human cells via
delivery of purified Cas9 ribonucleoproteins, 24 GENOME RES. 1012-1019
(2014) with Supplemental Information
1509
1-11
DESCRIPTION
NO.
1510
Sung et al., Highly efficient gene knockout in mice and zebrafish with RNAguided endonucleases, 24 GENOME RES. 125-131 (2014) with Supplemental
Material
1512
1513
Office Action dated January 13, 2014 in U.S. Patent Application No.
14/054,414
1514
Claims filed on December 12, 2013 in U.S. Patent Application No. 14/105,017
1515
Office Action dated November 13, 2014 in U.S. Patent Application No.
14/105,017
1516
Claims filed on February 18, 2014 in U.S. Patent Application No. 14/183,429
1517
Office Action dated April 9, 2014 in U.S. Patent Application No. 14/183,429
1518
1519
Final Office Action dated November 18, 2014 in U.S. Patent Application No.
14/256,912
1520
1521
Final Office Action dated November 18, 2014 in U.S. Patent Application No.
14/226,274
1-12
DESCRIPTION
NO.
1522
Preliminary Amendment filed on July 27, 2015 in U.S. Patent Application No.
14/704,551
1523
Office Action dated August 14, 2015 in U.S. Patent Application No.
14/704,551
1524
Claims filed on February 18, 2014 in U.S. Patent Application No. 14/183,471
1525
Office Action dated July 1, 2014 in U.S. Patent Application No. 14/183,471
1526
Claims filed on April 22, 2014 in U.S. Patent Application No. 14/258,458
1527
Office Action dated July 14, 2014 in U.S. Patent Application No. 14/258,458
1528
1531
1532
1533
Corrected Filing Receipt, dated December 11, 2015 in U.S. Patent Application
No. 13/842,859
1534
1-13
DESCRIPTION
NO.
1535
1555
1556
1604
1605
1606
1607
1608
1609
1610
1-14
DESCRIPTION
NO.
1611
1621
1622
1623
1624
1625
1626
1627
1-15
DESCRIPTION
NO.
1629
1630
1631
1632
Press Release, FNIH Awards Lurie Prize to Jennifer Doudna (February 24,
2014),
available at http://www.fnih.org/news/press-releases/lurie-prize-in-thebiomedical-sciences-to-jennifer-doudna
1633
Press Release, The Gruber Foundation, Yale University, 2015 Gruber Genetics
(June 16, 2015) available at http://gruber.yale.edu/genetics/press/2015-grubergenetics-press-release
1-16
DESCRIPTION
NO.
1634
1635
1638
1639
Deposition Transcript of Paul Simons, Ph.D., September 15, 2016, with errata
2007
2009
2010
2125
Chen et al., U.S. Patent Application No. 61/734,256, filed December 18, 2013.
2207
2213
1-17
DESCRIPTION
NO.
2230
Pandika, Rising Stars: Jennifer Doudna, CRISPR Code Killer, OZY (Jan. 7,
2014), http://www.ozy.com/rising-stars/jennifer-doudna-crispr-codekiller/4690.
2274
2290
2411
2412
2419
2420
2422
2423
1-18
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.
Broads response: ADMIT
2.
Methods and Compositions for RNA-Directed Site-Specific DNA Modification, was filed on
October 19, 2012, and lists Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, Krzysztof
Chylinski, and James Harrison Doudna Cate as co-inventors. See Ex. 1004.
Broads response: ADMIT
3.
U.S. Provisional Application No. 61/757,640 (the Third Provisional), titled, Methods
and Compositions for RNA-Directed Site-Specific DNA Modification, was filed on January 28,
2013, and lists Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, Krzysztof Chylinski,
and James Harrison Doudna Cate as co-inventors. See Ex. 1005.
Broads response: ADMIT
4.
The technology behind Count 1 is explained in the Declarations of Drs. Carroll and
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).
Broads response: DENY
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,
2-1
The First Provisional describes and enables methods within the scope of Count 1. See
Ex. 1003; see also Ex. 1022, 341-363, 373-431, Ex. 1024, 332-354, 364-422.
Broads response: DENY
7.
Example 1 of the First Provisional sets forth all elements of Count 1 other than the
eukaryotic cell element. See Ex. 1003, 00248-00252, Figures 3, 5; see also Ex. 1022, 341349, Ex. 1024, 332-340.
Broads response: DENY
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.
Broads response: DENY
9.
Count 1 because one of ordinary skill in the art as of May 25, 2012, would have recognized that
the listed inventors were in possession of the methods of Count 1 and could have performed
those methods using only routine and predictable techniques. See Ex. 1003, 00165, 0024800252; see also Ex. 1022, 350-351, Ex. 1024, 341-342.
Broads response: DENY
10.
target DNA, albeit not in a eukaryotic cell. See Ex. 1003, 00248-00252, Figs. 3, 5; see also
see also Ex. 1022, 350-352, Ex. 1024, 341-343.
2-2
Example 1 of the First Provisional satisfies all aspects of the second element of Count 1,
except for contacting in a eukaryotic cell, because it employs Cas9, which is necessarily a Type
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.
Broads response: DENY
12.
Included in the Type II CRISPR-Cas system of Example 1 of the First Provisional were
Contacting a target DNA with the system of Example 1 of the First Provisional in
eukaryotic cells is taught throughout the First Provisional. See Ex. 1003, 00165; see also Ex.
1003, 00167, 00177, 00178, 00201, 00215, 00216; see also Ex. 1022, 352-353, 376, Ex.
1024, 343-344, 367.
Broads response: DENY
14.
Example 1 of the First Provisional satisfies all aspects of the third, fourth, fifth, and sixth
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.
2-3
Figure 3B of the First Provisional illustrates the structure of the engineered and non-
Example 1 of the First Provisional satisfies all aspects of element 7 of Count 1 because
the DNA-targeting RNA formed a complex with the Cas9 protein during incubation [with each
other] in the cleavage buffer for 15 min[utes] at room temperature. See Ex. 1003, 00249; see
also Ex. 1022, 355, Ex. 1024, 346.
Broads response: DENY
17.
Example 1 of the First Provisional explains how the Cas9 protein was targeted to the
target DNA by adding the assembled DNA-targeting RNA/Cas9 complex to target DNA and
incubated for 1 h[ou]r at 37C. See Ex. 1003, 00249; see also Ex. 1022, 355, Ex. 1024,
346.
Broads response: DENY
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,
Ex. 1024, 346.
Broads response: DENY
19.
Figures 3A and 5A of the First Provisional show that cleavage of the target DNA
2-4
The First Provisional states the tested single-molecule DNA-targeting RNAs (RNA
chimera A) supported efficient target DNA cleavage. See Ex. 1003, 00251, Fig. 3B.
Broads response: ADMIT
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.
Broads response: DENY
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.
See Ex. 1003; see also Ex. 1022, 341-358, 373-407, Ex. 1024, 332-349, 364-398.
Broads response: DENY
23.
Claims 54, 58, 61, 66, and 72-75 of the First Provisional describe and enable all elements
of Count 1, including the eukaryotic limitation. See Ex. 1003, pp. 76-78; see also Ex. 1022,
359-363, 380-381, Ex. 1024, 350-354, 371-372.
Broads response: DENY
24.
illustrated in Figure 1B, provides the framework of Count 1s second, third, fourth, and fifth
elements of relying upon a DNA-targeting RNA comprising i) a targeter-RNA or guide
sequence that hybridizes with the target sequence, and ii) an activator-RNA, or a trans-activating
CRISPR RNA (tracrRNA), that hybridizes with the targeter-RNA to form a double-stranded
2-5
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.
Broads response: DENY
26.
Claim 72 of the First Provisional satisfies the second element of Count 1 because it
specifies that the DNA-modifying polypeptide used in the method of Claim 54 comprises an
amino acid sequence having at least about 75% amino acid sequence identity to amino acids 7166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted in Figure 2, or to the
corresponding domains in any of the amino acid sequences depicted in Figure 12. See Ex.
1003, p. 78; see also Ex. 1022, 362, Ex. 1024, 353.
Broads response: DENY
27.
Because Cas9 proteins are necessarily part of a Type II CRISPR system and because the
Cas9 protein may be mutated up to 25% from a naturally-occurring Cas9, the second and sixth
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.
Broads response: DENY
28.
cleavage. See Ex. 1003, p. 78; see also Ex. 1022, 363, Ex. 1024, 354.
Broads response: DENY
29.
Claims 54 and 73-75 of the First Provisional, especially in view of Figure 1, satisfy
2-6
From at least Claims 54, 58, 61, 66, and 72-75 of the First Provisional, one of ordinary
skill in the art would have recognized that the inventors were in possession of the methods of
Count 1 and would have been able to perform those methods from the enabling disclosures
throughout the First Provisional. Ex. 1003, pp. 76-78; see also Appendix 3; see also Ex. 1022,
380, Ex. 1024, 371.
Broads response: DENY
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.
Broads response: DENY
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.
1024, 347.
Broads response: ADMIT
33.
The First Provisional explains that the target DNA used in the disclosed methods may
include a cell from any organism and specifically identifies, among others, a cell of a singlecell eukaryotic organism. See Ex. 1003, 0165; see also Ex. 1022, 356, Ex. 1024, 347.
Broads response: DENY
34.
demonstrating that the inventors were in possession of an engineered and non-naturally occurring
2-7
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.
Broads response: DENY
36.
Guidance provided in Example 1 and Paragraph 00165 of the First Provisional, Claims
54, 58, 61, 66, and 72-75, and numerous passages in the Specification demonstrate that one of
ordinary skill in the art as of May 25, 2012, would have readily performed the methods of Count
1 without undue experimentation. See Ex. 1003, 00248-00252; see also Ex. 1022, 373407, Ex. 1024, 364-398.
Broads response: DENY
37.
The First Provisional teaches that the Cas9 protein and/or the DNA-targeting RNAs can
be provided in the form of a nucleic acid containing a nucleotide sequence encoding those
components, and further explains that the nucleotide sequences can be contained on an
expression vector, such as a viral vector, as would be commonly done when performing the
method in a eukaryotic cell. See Ex. 1003, 00120-00123; see also Ex. 1022, 387, Ex. 1024,
378.
Broads response: DENY
38.
Suitable expression vectors are specifically identified in the First Provisional and it was
2-8
The First Provisional explains that [i]n some embodiments, a nucleotide sequence
The First Provisional provides numerous examples of promoters that were known by May
25, 2012, to be functional in eukaryotic cells. See Ex. 1003, 00127; see also Exs. 1229, 1237,
1310, 1332, 1337; see also Ex. 1003, 00127; see also Ex. 1022, 390-392, Ex. 1024, 381383.
Broads response: DENY
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 sitedirected modifying polypeptide, and explains that there are numerous well-known methods that
can be used to accomplish this introduction. See Ex. 1003, 00121, 00129; see also Ex. 1022,
393, Ex. 1024, 384.
Broads response: DENY
2-9
Methods for introducing a nucleic acid into a eukaryotic cell were known in the art for
over 30 years before the First Provisional was filed. See Exs. 1040, 1200, 1248, 1260, 1307; see
also Ex. 1022, 393-394, Ex. 1024, 384-385.
Broads response: DENY
43.
The First Provisional explains the use of elements to target the Cas9 protein to a desired
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.
Broads response: DENY
45.
The First Provisional discloses that the Cas9 protein can include a conjugate to facilitate
traversing a cell or organelle membrane (a PTD). See Ex. 1003, 00115; see also Ex. 1022,
395, Ex. 1024, 386.
Broads response: ADMIT
46.
Some of the PTDs discussed in the First Provisional were well known NLSs, including
the sequence RKKRRQRRR. See Ex. 1003, 00115, 00179; see also Ex. 1022, 395, Ex.
1024, 386.
Broads response: DENY
47.
The First Provisional also provides examples of expression vectors that contain an NLS,
such as the vector pSVK3. See Ex. 1003, 00124; see also Ex. 1022, 395, Ex. 1024, 386.
Broads response: DENY
48.
For decades prior to the filing of the First Provisional, researchers had used nuclear
2-10
The First Provisional explains that standard recombinant nucleic acid manipulation
techniques, such as codon optimization, can be used to practice the methods in eukaryotic cells.
See Ex. 1003, 0033; see also Exs. 1029, 1030, 1035, 1213, 1217, 1235, 1236, 1293, 1302,
1319, 1327, 1329, 1335, 1336, 1502; see also Ex. 1003, 0033; see also Ex. 1022, 396-404,
Ex. 1024, 387-395.
Broads response: DENY
50.
For many years prior to the filing of the First Provisional, researchers had used codon
The First Provisional would have allowed persons of ordinary skill in the art to carry out
the methods of Count 1 without undue experimentation. See Ex. 1003; see also Ex. 1022,
405-407, 431, Ex. 1024, 396-398, 422.
Broads response: DENY
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.
Broads response: DENY
53.
Numerous passages throughout the First Provisional set forth each and every element of
Count 1 and the application explains how to perform the methods. See Ex. 1003; see also Ex.
2-11
Because the First Provisional provides detailed descriptions and the reagents and
techniques necessary to perform the methods were well-known, minimal experimentation was
needed to perform the methods of Count 1. See Ex. 1003; see also Ex. 1022, 406, Ex. 1024,
397.
Broads response: DENY
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.
Broads response: DENY
56.
Example 1 in the First Provisional is an actual, working example of all but the eukaryotic
element of Count 1, and one of ordinary skill in the art could readily and predictably have
transitioned Example 1 to eukaryotic cells based upon the guidance provided in the application.
See Ex. 1003; see also Ex. 1022, 406, Ex. 1024, 397.
Broads response: DENY
57.
The rapid success of numerous other research groups in applying the system of Example
1 of the First Provisional to eukaryotic cells immediately after the invention was published is
empirical evidence that the First Provisional describes and enables embodiments within the
scope of Count 1. See Exs. 1003, 1055, 1056, 1058, 1059, 1508-1510; see also Ex. 1022,
408-430, Ex. 1024, 399-421.
Broads response: DENY
2-12
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.
Broads response: DENY
59.
Jinek 2012 explained the basic components required for the CRISPR system to operate,
demonstrated its effectiveness outside of a cell, and discussed its usefulness within a eukaryotic
cell. See Ex. 1155; see also Ex. 1022, 413, Ex. 1024, 404.
Broads response: DENY
60.
Within just seven months after Jinek 2012 published, the Cong (Ex. 1055), Mali (Ex.
1056), Cho (Ex. 1059), and Hwang (Ex. 1058) groups had successfully performed experiments,
prepared and submitted manuscripts for review, and published papers on their results showing
that the system published in Jinek 2012 (disclosed in the First Provisional) worked in eukaryotic
cells. See, e.g., Ex. 1055, p. 819, middle col.; Ex. 1056, p. 1, Ex. 1059, p. 230, left col, and Ex.
1058, pp. 1-2; see Exs. 1002, 1011-1013, 1057, 1371, 1372; see also Ex. 1022, 414-423, Ex.
1024, 405-414.
Broads response: DENY
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.
Broads response: DENY
2-13
codon-optimization, and nuclear localization signals) employed by Cong, Mali, Cho, and Hwang
to successfully move the system of Jinek 2012 into eukaryotic cells are disclosed in the First
Provisional. See Exs. 1003, 1055, 1056, 1058, 1059; see also Ex. 1022, 423, Ex. 1024, 414.
Broads response: DENY
63.
Less than two years after Jinek 2012 published, at least three more groups Kim (Ex.
1508), Cho (Ex. 1509), and Sung (Ex. 1510) successfully transitioned the methods of the First
Provisional to eukaryotic cells. See Exs. 1508-1510, 1531, 1532; see also Ex. 1022, 424-430,
Ex. 1024, 415-421.
Broads response: DENY
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.
Broads response: DENY
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.
Broads response: DENY
66.
The Second Provisional includes all relevant content from the First Provisional to show at
least one constructive reduction to practice of Count 1. See Ex. 1004; see also Ex. 1022, 434436, Ex. 1024, 425-427.
Broads response: DENY
2-14
Claims 60, 65, 66, 69-72, and 98-100 of the Second Provisional disclose embodiments
within the scope of Count 1. See Ex. 1004, pp. 111, 112, 115-117; see also Appendices 6-8; see
also Ex. 1022, 435, Ex. 1024, 426.
Broads response: DENY
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.
Broads response: DENY
69.
That in vitro working Example 1 of the First Provisional could have readily been
transitioned to a eukaryotic cell is evidenced by Cho, Mali, Cong, and Hwang and their reliance
on Jinek 2012. See Exs. 1003, 1004, 1055, 1056, 1058, 1059; see also Ex. 1022, 437, Ex.
1024, 428.
Broads response: DENY
70.
The information in the Second Provisional that is not in the First Provisional provides
more guidance to one of ordinary skill in the art for carrying out the methods of Count 1. See Ex.
1004, 0016-0018, 0053-0061, 0084-0089, 00178, 00255-00256, 00289-00359, Figs. 13-35;
see also Ex. 1022, 437-439, Ex. 1024, 428-430.
Broads response: DENY
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.
Broads response: DENY
2-15
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.
Broads response: DENY
73.
Claims 64, 69, 70, 73-76, and 102-104 of the Third Provisional disclose embodiments
within the scope of Count 1. See Ex. 1005, pp. 152, 153, 155-160; see also Ex. 1022, 443, Ex.
1024, 434.
Broads response: DENY
74.
All of the pertinent disclosure found in the First Provisional and the Second Provisional
to describe and enable an embodiment within the scope of Count 1 is carried through and
included in the Third Provisional. Compare Ex. 1003 with Ex. 1004 and Ex. 1005; see also Ex.
1022, 445-448, Ex. 1024, 436-439.
Broads response: DENY
75.
Example 1 of the First Provisional could have readily been transitioned 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.
Broads response: DENY
76.
The rapid successes at performing the methods of Example 1 in eukaryotic cells by Mali,
Cho, Cong, Hwang, Kim, Cho, and Sung further evidence that one of ordinary skill in the art was
readily and predictably able to transition those methods to eukaryotic cells. See Exs. 1055, 1056,
1058, 1059, 1508-1510; see also Ex. 1022, 445, Ex. 1024, 436.
Broads response: DENY
77.
Additional information included in the Third Provisional not included in the First
2-16
The Third Provisional includes working Examples 2 and 3. See Ex. 1005, 00408-
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.
Broads response: DENY
80.
U.S. Patent Application Serial No. 13/842,859 currently shares four inventors from each
of the Provisional Applications, i.e., Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier,
and Krzysztof Chylinski. See Ex. 1001, pp. 346-347; see also Exs. 1003-1005, 1533.
Broads response: ADMIT
2-17
Neither Doudna P1 nor P2 includes an experiment that falls within the scope of Count 1.
Ex. 1003; Ex. 1004; Ex. 2009 11.22. Response: Insufficient information, therefore unable
to admit or deny.
82.
P1 lacks any discussion of PAM sequences. Ex. 1003; Ex. 2009 11.43. Response:
Denied.
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. Response:
Admitted.
84.
Persons of ordinary skill (POSITA) in 2012 would have expected the role of PAM to
be addressed when adapting a CRISPR-Cas9 system for non-natural target DNA. Ex. 2009
11.43. Response: Denied.
85.
The state of the art in May 2012 relating to CRISPR-Cas9 systems was nascent and
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-
Cas9 systems to function in eukaryotic cells. See Ex. 2412; Paper 53; Ex. 2009 11.102.
Response: Denied.
89.
By 2011, publications had identified certain components that potentially played role in
CRISPR-Cas9 system in prokaryotes. Ex. 1032; Ex. 1153; Ex. 1227; Ex. 2009 11.98-11.99.
2-18
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
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.
Response: Denied.
92.
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
B), with shorter tracrRNA components than the wild-type tracrRNA. Ex. 1003 at 2, 0006.
Response: Denied.
94.
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
RNA, but instead suggested to those of ordinary skill the use of Chimera A with its relatively
short tracrRNA component as compared to wild-type tracrRNA. Ex. 1155; Ex. 2009 11.66.
Response: Denied.
96.
Dr. Zhangs group at the Broad compared a modified version of Chimera A of Jinek 2012
to the CRISPR-Cas9 system previously developed in their laboratory. Ex. 1155 at 20;
Response: Denied.
2-19
Dr. Zhangs group at the Broad adapted CRISPR-Cas9 with Chimera A-type RNA to
function in eukaryotic cells, using non-routine techniques, adding two NLSs to target the Cas9
protein to the nucleus, and a vector to express Cas9 and crRNA, with four nucleotides not
present on Chimera A. Ex. 1055 at 2. Response: Denied.
98.
Eight of the 14 Chimera A-type samples tested by the Broad scientists did not work in a
eukaryotic cell. Ex. 1055 at 20; Ex. 2009 11.130. Response: Denied.
99.
Dr. Churchs group at Harvard published the Mali 2013 article in January 2013 using
longer tracrRNA. Ex. 1056 at 824; Ex. 2009 11.109. Response: Insufficient information,
therefore unable to admit or deny.
100.
The Junior Party includes both the Broad and Harvard, which is affiliated with the Broad,
and some Broad scientists have advisors at both institutions. Ex. 2423; Ex. 2009 11.90.
Response: Insufficient information, therefore unable to admit or deny.
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. Response: Insufficient information, therefore
unable to admit or deny.
102.
Dr. Hwangs group thanked Dr. Church for sharing unpublished results. Ex. 1058 at 6.
Response: Admitted.
103.
Senior Party does not assert actual reduction to practice until October 29, 2012. Paper 58.
Response: Denied.
104.
The Senior Partys in vitro work published in Jinek 2012. UC Motion 4 at 18:14-19:5
Response: Admitted.
105.
The Jinek system with Chimera A did not work in eukaryotic cells using routine
2-20
Dr. Doudnas laboratory was surprised when they learned that Dr. Churchs laboratory at
After Dr. Churchs laboratory shared unpublished results, Dr. Doudnas laboratory
published Jinek 2013 paper with attempt to adapt CRISPR-Cas9 system to function in eukaryotic
cells. Ex. 2009 11.141. Response: Denied.
108.
Senior Partys inventors filed P3 containing the same eukaryotic test results as in Jinek
2013 using a chimeric RNA with longer tracrRNA components than Chimera A. Ex. 1005 at
Figures 36-38; Ex. 1055 at Figures 3-5. Response: Denied.
109.
stated that Its not trivial to make CRISPR/Cas systems work in eukaryotic cells, and that
[o]ne thing is to have them in silico and have a sequence and realize theyre smaller, and
another thing is to do the [eukaryotic] experiments and make it work. Ex. 2213. Response:
Insufficient information, therefore unable to admit or deny.
110.
Drs. Zhang, Horvath, Doudna, Charpentier and Barrangou jointly received the 2016
Gairdner Award for their work with CRISPR-Cas systems. Ex. 2419. Response: Denied.
111.
The Gairdner Foundation noted Dr. Zhangs work in the development of the microbial
CRISPR-Cas system as a genome editing tools for function in eukaryotic cells. Ex. 2419 at 2.
Response: Admitted.
112.
Count 1 recites a target DNA molecule in a eukaryotic cell that is not a natural target of
Figure 5 of Doudna P1 shows results using six Cas9 orthologs from different species of
bacteria, of which three did not show any cleavage. Ex. 1003 at 95; Ex. 2009 11.49. Response:
Denied.
2-21
Persons of ordinary skill would have understood that the Doudna P1 inventors did not use
the correct PAM for all six orthologs in Figure 5 of Doudna P1. Ex. 2009 11.49. Response:
Denied.
115.
Example 1 of Doudna P1 and P2 describes only in vitro testing and not testing in
eukaryotic cells. Ex. 1003 at 248-253; Carroll Tr. 156:20-157:8; Ex. 2009 11.5. Response:
Admitted.
116.
Dr. Church characterized the move of CRISPR-Cas from bacteria to eukaryotic cells as a
Example 1 and Paragraph 00165 of P1 and P2 do not provide any evidence that the
CRISPR-Cas9 system of Example 1 actually works in eukaryotic cells. Ex. 2009 11.22.
Response: Denied.
118.
None of the portions of P1 or P2 cited by Senior Party provide evidence that the
CRISPR-Cas9 actually worked in eukaryotic cells, or identify a structural aspect of a CRISPRCas9 system that would make it adapted for eukaryotic cells. Ex. 2009 11.25. Response:
Denied.
119.
Persons of ordinary skill in the art in 2012 would not predict the operability of a
CRISPR-Cas system in a eukaryotic cell based on in vitro experiments. Ex.2009 11.6, 11.92.
Response: Denied.
120.
Skilled persons in 2012 would have required positive eukaryotic tests results conclude
that the Doudna inventors provided sufficient information to show possession of an invention of
an embodiment of a CRISPR-Cas system functioning in eukaryotic cells. Ex.2009 11.6, 11.92.
Response: Denied.
121.
Senior Party did not make an embodiment of Count 1 until more than a year after the
2-22
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. Response:
Denied.
123.
Sontheimer application stated that its CRISPR system could be used in eukaryotes and
taught techniques for eukaryotic experiments. Ex. 1161 at 0054-0060; Ex. 2009 11.33.
Response: Admitted.
124.
The Sontheimer application did not include actual eukaryotic experiments. Ex. 1161.
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. Response: Admitted.
126.
The lack of tests in eukaryotic cells in Doudna P2 four months after Jinek 2012 would
Dr. Carroll admitted that all of the information in P1 and P2 either appears in Jinek 2012
or was known. Carroll Tr. at 255:4-258:17; 305:6-338:10; Ex. 2009 11.86-87. Response:
Denied.
128.
Dr. Carroll observed in a September 2012 article that [a]ll the experiments described in
Jinek 2012 were performed in vitro with purified components and expressed doubt about
whether the CRISPR-Cas system would function in eukaryotic cells. Ex. 1152 at 1660.
Response: Denied.
129.
Dr. Carroll claims to have knowledge of one of ordinary skill in the art. Ex. 1024 at 19.
2-23
Dr. Carroll did not dispute that his 2012 article does not state any expectation of success
for CRISPR-Cas9 systems in eukaryotic cells. Carroll Tr. at 117:5-118:2. Response: Denied.
131.
In 2012, Dr. Carroll stated in reference to Jinek 2012 that [o]nly attempts to apply the
system in eukaryotes will address concerns he had raised. Ex. 1152 at 1660. Response:
Admitted.
132.
In a July 2014 article, Dr. Doudna confirmed that after publication of Jinek 2012, she did
not know if the CRISPR-Cas9 system would work in eukaryotic cells. Ex. 2207. Response:
Denied.
133.
An article from July 28, 2012 stated that [t]he next steps according to Dr. Charpentier
and Dr. Doudna were to test the single-RNA construct with Cas9 to find out whether it works in
eukaryotic organisms. Ex. 2290. Response: Admitted.
134.
A POSITA in May 2012 would have recognized that an in vitro environment is very
different from the environment of eukaryotic cells. Ex. 2009 11.114. Response: Denied.
135.
Differences between eukaryotic and prokaryotic cells could have unpredictable effects on
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. Response: Denied.
136.
It was known in May 2012 that it may not be possible to deliver components of CRISPR
systems to eukaryotic cells so as to be properly transcribed and translated. Ex. 2009 11.31.
Response: Denied.
137.
It was known in May 2012 that proteins or nucleic acids present in eukaryotic cells could
2-24
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
prevent CRISPR-Cas9 from functioning in eukaryotic cells. Ex. 2009 11.31. Response:
Denied.
140.
It was known in May 2012 that CRISPR-Cas9 evolved in prokaryotes. Ex. 2009 11.88.
antithetical to its natural function in prokaryotic cells. Ex. 2009 10.5. Response: Denied.
142.
A POSITA in May 2012 understood that the human genome is at least a thousand-fold
larger than that of bacteria. Ex. 2009 10.7. Response: Insufficient information, therefore
unable to admit or deny.
143.
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
system may be toxic to eukaryotic cells. Ex. 2009 11.31. Response: Denied.
145.
Toxic effects include the effect of genome editing in locations other than the target DNA,
which are called off-target effects. Ex. 2009 11.31. Response: Denied.
146.
Dr. Carroll testified that off-target effects are an important concern for genome editing
and that off-target effects could, in fact, be lethal. Carroll Tr. at 227:3-6, 229:2-12. Response:
Denied.
147.
Dr. Greider testified that POSITA would have had no expectation regarding off-target
effects from CRISPR-Cas9 in eukaryotes without doing experiments. Greider Tr. at 405:14-22.
2-25
It was known in May 2012 that it may not be possible to localize the components of the
CRISPR-Cas9 system to target DNA in the environment of a eukaryotic cell. Ex. 2009 11.31.
Response: Denied.
149.
P1 and P2 do not teach using an NLS in eukaryotic cells. Ex. 2009 11.31; Ex. 1003.
Response: Denied.
150.
Dr. Greider testified DNA target of interest is in nucleus. Greider Tr. at 285:20-286:1.
Response: Denied.
151.
Dr. Greider provided no opinion that the Senior Party inventors possessed an
embodiment of Count 1 for delivering a functioning CRISPR-Cas9 system, with the requisite
RNA, into a mitochondria or chloroplast. Greider Tr. (7/20/16) 422:4-18. Response: Denied.
152.
A POSITA reading P1 and P2 at the times they were filed would have known of prior
difficulties adapting a prokaryotic system to eukaryotic cells. Ex. 2010 1.45-1.54. Response:
Insufficient information, therefore unable to admit or deny.
153.
A POSITA in May 2012 would have also been aware of the history of research relating to
targetrons, gene editing tools based on group II self-splicing ribozymes. Ex. 2010 1.54
Response: Admitted.
154.
to adapt them to function in eukaryotic cells. Ex. 2010 1.45-1.48 Response: Denied.
155.
Targetrons include protein and RNA components, like CRISPR systems. Ex. 2010 1.45.
Response: Admitted.
156.
Group II self-splicing ribozymes were initially described in 1986, and targetrons were
developed that could act on bacterial genes by 2003. Ex. 2010 1.46. Response: Admitted.
2-26
After nine more years of research, researchers had not been able to adapt targetrons to
A POSITA in May 2012 reading the P1 and P2 applications would have understood that
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. Response: Denied.
159.
History of targetrons taught that ability of prokaryotic system to edit genes in other
environments did not show adaptation to function in eukaryotic cells. Ex. 2010 1.45-1.54.
Response: Denied.
160.
A POSITA in May 2012 would have known that the level of Mg2+ ions could be a
problem for CRISPR-Cas9 as with targetrons, since both systems evolved to function in
prokaryotic cells. Ex. 2010 1.51-1.53. Response: Denied.
161.
Given the history of prior failed attempts to transfer prokaryotic systems to eukaryotes,
POSITA would have needed experimental results in eukaryotic cells to show possession of a
CRISPR-Cas9 invention for eukaryotic cells. Ex. 2009 11.35. Response: Denied.
162.
Dr. Zhang achieved success with a CRISPR-Cas9 system in eukaryotic cells prior to the
publication of Jinek 2012. Ex. 1055 at 2; Ex. 2411; Ex. 2009 11.97. Response: Denied.
163.
Dr. Zhang showed that his CRISPR-Cas9 system with longer tracrRNA worked better
than modified Chimera A-type system for eukaryotic cells. Ex. 1055 at 20; Ex. 2009 11.130.
Response: Denied.
164.
Senior Party has not shown that the research groups responsible for the Cong 2013, Mali
2013, Cho 2013 and Hwang 2013 articles were inspired by Jinek 2012 or that those groups were
independent with respect to CRISPR research. Ex. 2009 11.6. Response: Denied.
165.
The research groups responsible for the Cong 2013, Mali 2013, Cho 2013 and Hwang
2-27
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. Response: Insufficient
information, therefore unable to admit or deny.
167.
P1 and P2 provide no guidance for practicing Count 1 in eukaryotes. Ex. 2009 11.114.
Response: Denied.
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. Response: Denied.
169.
Dr. Church acknowledged that his graduate student Dr. Cong worked with Dr. Zhang on
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. Ex. 2422 at 11, 20-24. Response: Admitted.
172.
Cho 2013 added more plasmids and RNA to eukaryotic cells than recommended by
manufacturer of their nucleofection equipment, which was not routine. Ex. 2009 11.132.
Response: Denied.
173.
The testing in Cho 2013 confirms that a POSITA would have needed extensive
experimentation to make unmodified Chimera A function in eukaryotic cells. Ex. 2009 11.133.
Response: Denied.
2-28
In later papers from Cho laboratory, researchers abandoned Chimera A and used a longer
tracr. Ex. 1509; Ex. 1508; Carroll Tr. at 279:20-291:7; Ex. 2009 11.134. Response: Denied.
175.
The journal SCIENCE has large reach in scientific community, with 570,400 readers
each week and over five million monthly visits to Science website. Ex. 2420; Ex. 2009 11.113.
Response: Insufficient information, therefore unable to admit or deny.
176.
Only Dr. Chos group using non-routine techniques showed that a CRISPR-Cas9 system
with unmodified chimera A could function in eukaryotic cells. Ex. 2009 11.113. Response:
Denied.
177.
The Doudna P3 application was filed January 29, 2013. Ex. 1005. Response: Denied.
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. Response: Denied.
179.
Two experiments in P3 purport to show cleavage in living eukaryotic cells. Ex. 1005.
Response: Admitted that at least two experiments in P3 show cleavage in living eukaryotic
cells.
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. Response:
Admitted.
181.
In P3, Figure 38B, the gel should show bands between 160 and 200 bp if the CRISPR-
Cas9 successfully cleaved the target DNA. Ex. 2009 11.140. Response: Admitted.
182.
The samples in P3 containing Cas9 and sgRNA do not show any diagnostic bands
between 160 and 200 bp. Ex. 1005 at 36E; Ex. 2009 11.140. Response: Denied.
183.
A POSITA would have concluded from Figure 38B of P3 that there was no successful
2-29
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
purify their PCR product as evidenced by the additional bands visible in lanes 2 and 4 and that
the test results were inconclusive at best. Ex. 2009 11.151-11.159. Response: Denied.
186.
Figure 36B reports results of an experiment that according to P3, revealed abundant
Cas9 expression and nuclear localization. Ex. 1005 at 416. Response: Admitted.
187.
To a POSITA, the results in Fig. 36B of P3 would not have indicated that the Senior
Partys inventors obtained nuclear localization. Ex. 2009 11.154. Response: Denied.
188.
The failure in Fig. 36B of P3 to localize the Cas9 protein in the nucleus could account for
the poor results shown in Figures 36E and 38B. Ex. 2009 11.162. Response: Denied.
2-30
/Todd R. Walters/
Todd R. Walters, Esq.
Registration No. 34,040
Buchanan Ingersoll & Rooney PC
1737 King Street, Suite 500
Alexandria, Virginia 22314
Telephone (703) 836-6620
Facsimile (703) 836-2021
todd.walters@bipc.com
Counsel for UC and Vienna