Ex Parte 7309605 et al

Board of Patent Appeals and Interferences

Mar 14, 2012

95000490 (B.P.A.I. Mar. 14, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
____________

PRECISION BIOSCIENCES, INC.
Requester & Respondent

v.

INSTITUT PASTEUR &
UNIVERSITÉ PIERRE ET MARIE CURIE
Patent Owner & Appellant
____________

Appeal 2011-012285
Reexamination 95/000,490
Patent 7,309,605 B2
Technology Center 3900
____________

Before SALLY G. LANE, RICHARD M. LEBOVITZ, and JEFFREY B.
ROBERTSON, Administrative Patent Judges.

LEBOVITZ, Administrative Patent Judge.

DECISION ON APPEAL
Appeal 2011-012285
Reexamination 95/000,490
Patent 7,309,605 B1

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This is a decision on appeal by the Patent Owner from the Patent
Examiner’s rejections of claims in an inter partes reexamination of U.S.
Patent No. 7,309,605 B1. The Board’s jurisdiction for this appeal is under
35 U.S.C. §§ 6(b), 134, and 315. We affirm all rejections.

STATEMENT OF THE CASE
The patent in dispute in this appeal is U.S. Patent No. 7,309,605 B1
(hereinafter, “the ‘605 patent”), which issued December 18, 2007. The
named inventors are Bernard Dujon, Andre Choulika, Arnaud Perrin, and
Jean-Francois Nicolas.
The claims in the ‘605 patent are directed to a method for introducing
a double-stranded break into chromosomal DNA of a viable cell of an
organism. The method is accomplished using a Group I intron encoded
endonuclease (“GIIEE” or “GIIE endonuclease”). GIIE endonucleases are
endonucleases which are encoded in the intron of certain genes and are
responsible for intron mobility (‘605 patent, col. 2, ll. 6-60). More
background on this process is described in the Decision on the
reexamination of the related U.S. Patent No. 7,214,536 B2 (Reexamination
95/000,427, Appeal 2011-010572).
A request for inter partes reexamination under 35 U.S.C. §§ 311-318
and 37 C.F.R. §§ 1.902-1.997 for the ‘605 patent was filed on July 31, 2009
by a Third-Party Requester (Request for Inter Partes Reexamination
Transmittal Form). The Third-Party Requester is Precision BioSciences,
Inc., who is the Respondent in this appeal (Respondent Br. iii, dated
February 22, 2011). The Patent Owners and Appellants in this appeal are
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the Institut Pasteur and Université Pierre et Marie Curie (Appellant App. Br.
1, dated January 19, 2011). The patent has been licensed to Cellectis SA, of
Paris, France (id.).
There are three additional pending reexamination proceedings
involving the same parties and related patents:
1. Reexamination Control No. 95/000,443 for U.S. Patent No.
6,833,252 B1;
2. Reexamination Control No. 95/000,427 for U.S. Patent No.
7,214,536 B2; and
3. Reexamination Control No. 95/000,491 for U.S. Patent No.
6,610,545 B2.
All three reexaminations are on appeal before the Board.
In addition, there is a pending litigation in a district court asserting
U.S. Pat. Nos. 7,309,605 B1 and 6,610,545 B2 (Cellectis SA v. Precision
Biosciences, Inc., No. 5:08-cv-119 (E.D.N.C.)) (Respondent Br. R-
1;Appellant App. Br., Appendix D.)
Claims 1-18 are pending and stand finally rejected by the Examiner.
There are 32 rejections, each of which is appealed by the Patent Owner
(Appellant App. Br., Appendix E).
Grounds 1 and 2 are anticipation rejections under 35 U.S.C. § 102(b)
over Bell-Pedersen1 and Quirk,2 respectively.

1 Deborah Bell-Pedersen et al., Intron mobility in phage T4 is dependent
upon a distinctive class of endonucleases and independent of DNA
sequences encoding the intron core: mechanistic and evolutionary
implications, 18 Nucleic Acid Research 3763 (1990).
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Grounds 5 and 6 are obviousness rejections 35 U.S.C. § 103(a) over
Schiestl3 in combination with “admissions” or Frey.4
The remaining rejections are for obviousness involving either Bell-
Pedersen or Quirk, and additionally cited secondary publications. In these
rejections, the Examiner made a rejection over Bell-Pedersen and a
secondary publication, and then the exact same rejection using Quirk instead
of Bell-Pedersen. For example Ground 7 rejects claim 2 over Bell-Pedersen
and Seraphin,5 and Ground 8 rejects claim 2 over Quirk and Seraphin. Bell-
Pedersen and Quirk were relied upon by the Examiner for same teachings.
Consequently, we limit our discussion largely to Bell-Pederson, as the
rejections based on Quirk are cumulative.
A number of different declarations by experts were cited as evidence
by both parties in this proceeding and in the related three reexamination
proceedings. For consistency and ease of reference, we have renumbered
the declarations as Exhibits 1001 to 1015, and attached them to
Reexamination 95/000,427 of U.S. Patent No. 7,214,536 B2, which is
Appeal 2011-010572.

2 Susan M. Quirk et al., Intron Mobility in the T-Even Phages: High
Frequency Inheritance of Group I Introns Promoted by Intron Open
Reading Frames, 56 Cell 455 (1989).
3 Robert H. Schiestl & Thomas D. Petes, Integration of DNA Fragments by
Illegitimate Recombination in Saccharomyces Cerevisiae, 88 Proceedings
Nat’l Acad. Sci. 7585 (1991).
4 B. Frey et al., Specific Cleavage of the Yeast and Bacterial Genomes at a
Single Site Using the Rare Cutter Endonuclease I-SceI (Meganuclease I-
SceI), 343 Fresenius’ J. Analytical Chemistry 122-123 (1992).
5 Bertrand Seraphin et al., The Yeast Mitochondrial Intron a15α Associated
Endonuclease Activity and In Vivo Mobility, 113 Genetics 1-8 (1992).
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Claims 1 and 18 are representative and read as follows (underlining
and brackets indicate amendments relative to the issued patent claim):
1. A method for inducing at least one site directed double-stranded
break in the chromosomal DNA of an organism comprising: (a)
providing an isolated, viable cell of said organism containing at least one
Group I intron encoded endonuclease recognition site at a location in the
chromosomal DNA of the cell, (b) providing said Group I intron
encoded endonuclease to said cell by genetically modifying the cell with
a nucleic acid comprising said Group I intron encoded endonuclease or
by introducing said Group I intron encoded endonuclease protein into the
cell such that the Group I intron encoded endonuclease cleaves said
Group I intron encoded endonuclease site at the location in the
chromosomal DNA of the cell.
18. The method of any one of claims 1 to 17, wherein the isolated, viable
cell of the organism is a eukaryotic cell.
Claim 1
Claim 1 is directed to a method of inducing a double-stranded break in
the chromosome of an organism. The method comprises two recited steps:
a) providing a viable cell with a chromosome containing a GIIE
endonuclease recognition site; and
b) providing a GIIE endonuclease to the cell “such that the Group I
intron encoded endonuclease cleaves said Group I intron encoded
endonuclease site at the location in the chromosomal DNA of the cell.”
The claims in this appeal are similar to those in the related
reexamination of U.S. Patent No. 7,214,536 B2 (Reexamination Control No.
95/000,427; Appeal 2011-010572). However, the latter claims were limited
to plant and animal cells, where the claims in this appeal are broader and are
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drawn to the cells of an organism, with dependent claims drawn to
eukaryotic cells.

Claim interpretation
Claim 1 was amended during the reexamination proceeding by adding
the term “viable” to the step of providing a viable cell of an organism with a
GIIE endonuclease recognition site to the chromosomal DNA of the cell.
The term “chromosomal” was also added to the claim at the same time.
(Patent Owner Response 5, dated Nov. 17, 2009.) The term “viable” means
that the cell is a living cell (id.).
The claim requires that endonuclease is provided to the viable cell
“such that the endonuclease cleaves the endonuclease site in the target
nucleic acid sequence” which resides in the chromosome of the viable cell.
We interpret the “viable” requirement to mean that the cell is alive, and not
dying, upon introduction of the endonuclease and after cleavage of the
chromosomal DNA by the endonuclease.

ANTICIPATION REJECTIONS
Issue
The Examiner rejected claim 1, and dependent claim 15, as
anticipated by Quirk, and claims 1, 10, 11, 14, and 15 as anticipated by Bell-
Pedersen (Grounds 1 & 2). The issue in each rejection is the same: whether
a GIIEE recognition site was necessarily cleaved in the chromosome of a
viable cell of a bacterial cell in the experiments described in Quirk and Bell-
Pedersen.
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Legal Principles
A “prior art reference may anticipate without disclosing a feature of
the claimed invention if that missing characteristic is necessarily present, or
inherent, in the single anticipating reference.” SmithKline Beecham Corp. v.
Apotex Corp., 403 F.3d 1331, 1343 (Fed. Cir. 2005). Inherency asks
whether a subject matter is “necessarily” present in the prior art reference,
“not merely probably or possibly present, in the prior art.” Trintec Indus.,
Inc. v. Top-U.S.A., Corp., 295 F.3d 1292, 1295 (Fed. Cir. 2002). It is the
Examiner’s burden to provide “reason to believe that . . . the claimed subject
matter may, in fact, be an inherent characteristic of the prior art.” In re
Schreiber, 128 F.3d 1473, 1478 (Fed. Cir. 1997). Once the Examiner has
satisfied this duty, the burden shifts to Appellant to provide evidence to the
contrary. In re Spada, 911 F.2d 705, 708 (Fed. Cir. 1990) (once “the PTO
shows sound basis for believing that the products of the applicant and the
prior art are the same, the applicant has the burden of showing that they are
not.”).

Quirk publication
Quirk described experiments in E. coli designed to study intron
mobility of the td and sunY introns, introns contained in the td and sunY
genes which are present in a class of phage know as T-even bacteriophages
(Quirk 455) [FF1].6 The “td” refers to the thymidylate synthase gene and
“sunY” refers to a gene having an unknown function at the time the

6 When a finding of fact (“FF”) first appears in the decision, it is defined by
a number within brackets. Thereafter, reference to the finding is made by
the specific fact number.
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publication was published (Quirk 455) [FF2]. Both the td and sunY genes
include introns containing open reading frames (ORFs), which is a sequence
of nucleotides encoding a protein (Quirk 455) [FF3]. The ORF of the td
intron encodes I-TevI, and the ORF of the sunY intron encodes I-TevII
(Bell-Pedersen Decl. of May 19, 2010, ¶ 17, Exhibit 1001) [FF4], both
which are Group I intron encoded endonucleases.
Quirk demonstrated that the td and sunY introns could be transferred
to the corresponding intronless gene, and that such transfer (“mobility”)
required the intact ORF encoding the GIIE endonuclease (Quirk 461) [FF5].
The intronless gene contains a site-specific recognition site for the GIIE
endonuclease (Quirk 458-459, 463, col. 2 (section titled “Phages,” T2L and
T2H); Bell-Pedersen Decl. of May 19, 2010, ¶ 27, Exhibit 1001) [FF6]. The
GIIE endonuclease recognition site is a sequence of DNA nucleotides that
must be present in the DNA (i.e., gene) for it to be cleaved by the
endonuclease (Quirk 458-459, 463, col. 2 (section titled “Phages,” T2L and
T2H); Bell-Pedersen Decl. of May 19, 2010, ¶ 27, Exhibit 1001) [FF7].
During the course of the experiments, Quirk observed that E. coli
containing the td and sunY genes with an intact ORF (i.e., the intact and
functional endonuclease) grew poorly because the ORF product was
apparently toxic to the cells (Quirk 456, col. 2) [FF8].
To address the toxicity issue, td fragments were cloned into a plasmid
and placed under control of the “more tightly regulated phage λ pL
promoter.” (Quirk 457, col. 1) [FF9]. The pL promoter (also referred to as a
“temperature inducible” or “temperature sensitive” promoter) is controlled
by the temperature repressor cI857 “which allows low level expression at
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permissive temperatures (30°-32°C) and high level expression at elevated
temperature (37°-42°C).” (Quirk 457, col. 1) [FF10] The expression of the
GIIE endonuclease from this plasmid “did not result in significantly poor
growth” of the E. coli cells, suggesting that lower expression levels of the
endonuclease were less or not toxic to the cells (Bell-Pedersen Decl. of May
19, 2010, ¶ 23, Exhibit 1001; Quirk 457, col. 2) [FF11].
To determine whether the intron had inserted into the site-specific
recognition site, Quirk harvested phage from the E. coli after temperature
induction (Quirk 458-59) [FF12]. The harvested phage were examined to
determine whether the intron had inserted into the phage in the vicinity of
the site-specific GIIEE recognition site (Quirk 458-59); at the “cognate
site[]”). Quirk found that the intron had inserted into the cognate site (Quirk
458-59) [FF13].

Bell-Pedersen publication
The experiments described in the Bell-Pedersen publication were
performed in the same laboratory as those in the Quirk publication, the
laboratory directed by Dr. Marlene Belfort, who is one of the experts for the
Patent Owner. Drs. Deborah Bell-Pedersen, Susan Quirk, and Marlene
Belfort were listed as coauthors of both Quirk and Bell-Pedersen. Quirk was
published in 1989. Bell-Pedersen was published in 1990. Dr. Bell-Pedersen
is also an expert for the Requester.
Bell-Pedersen performed experiments in E. coli using similar systems
to those described in the Quirk publication. The experiments described in
Bell-Pedersen showed that an intron comprising a marker gene was
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mobilized from a plasmid and inserted into a recipient phage containing a
recognition site for a GIIE endonuclease (Bell-Pedersen 3763) [FF14], the
site in the DNA where the endonuclease cleaves (FF7, Quirk 458-459, 463,
col. 2 (section titled “Phages,” T2L and T2H); Bell-Pedersen Decl. of May
19, 2010, ¶ 27, Exhibit 1001). As explained below, the recognition site is
provided by a phage; the endonuclease is provided by a plasmid. Once the
site is recognized by the endonuclease, the endonuclease cleaves the DNA
into two pieces, creating a site where the intron can be inserted into it
[FF15]. As stated above, the issue is whether the cleavage occurred when
the recognition site was present in the bacterial chromosome.
In one set of experiments performed by Bell-Pedersen, the following
DNA constructs were made (Bell-Pedersen 3764-3765; Bell-Pedersen Decl.
of May 19, 2010, ¶¶ 32-44, Exhibit 1001).
• a plasmid containing a GIIEE intron into which a gene conferring
antibiotic resistance (a marker gene) had been inserted. The antibiotic-
resistance gene was inserted into the intron as a marker to determine when
the intron had been inserted into the phage (Bell-Pedersen 3764-3765; Bell-
Pedersen Decl. of May 19, 2010, ¶¶ 32-44, Exhibit 1001) [FF16];
• a plasmid containing a GIIE endonuclease under control of a
temperature inducible promoter (pL) (Bell-Pedersen 3764-3765; Bell-
Pedersen Decl. of May 19, 2010, ¶¶ 32-44, Exhibit 1001) [FF17]; and
• a phage containing the recognition site for the GIIE endonuclease.
The phage genes are under control of the same temperature inducible
promoter present in the plasmid containing the GIIE endonuclease (Bell-
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Pedersen 3764-3765; Bell-Pedersen Decl. of May 19, 2010, ¶¶ 32-44,
Exhibit 1001) [FF18].
The plasmids and phage were introduced into E. coli hosts. When the
GIIE endonuclease was expressed by the plasmid and manufactured in the
host E. coli cell, the endonuclease would cleave the recognition site in the
phage and then the antibiotic resistance gene would integrate into the phage
Bell-Pedersen 3764-3765; Bell-Pedersen Decl. of May 19, 2010, ¶¶ 32-44,
Exhibit 1001) [FF19]. As one of the steps in the experiment, the phage first
integrates into the E. coli chromosome Bell-Pedersen 3764-3765; Bell-
Pedersen Decl. of May 19, 2010, ¶¶ 32-44, Exhibit 1001) [FF20].
After integration into the bacterial chromosomal DNA, the
temperature of the cells is raised – turning on the temperature inducible
promoter. As a result, the endonuclease is manufactured from the plasmid
and the phage is excised from the bacterial chromosome, beginning the cycle
of phage reproduction. (Bell-Pedersen 3765 [FF21].)
As with Quirk, to determine whether the intron had inserted into the
site-specific recognition site, phage are harvested from the E. coli after
temperature induction and the harvested phage were examined to determine
the presence of the inserted intron comprising the marker gene (Bell-
Pedersen 3765, col. 1; Bell-Pedersen Decl. of May 19, 2010, ¶ 54, Exhibit
1001) [FF22].

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Expert testimony
Dr. Bell-Pedersen
The Requester filed three written declarations by Deborah Bell-
Pedersen, Ph.D. Dr. Bell-Pedersen performed her doctoral research in the
laboratory of Dr. Marlene Belfort and is a co-author of the Quirk and Bell-
Pedersen publications. In her declarations, Dr. Pedersen testified that
cleavage of the DNA and insertion of the marker gene into the phage took
place when the phage was resident in the E. coli bacterial chromosome.
According to Dr. Bell-Pedersen:
• Although the temperature inducible pL promoter is induced as 42°C,
the GIIE endonuclease would be expressed at a low level from the pL
promoter when the E. coli cells were grown at 30°C (the “leaky” promoter;
Bell-Pedersen Decl. of Dec. 16, 2009, ¶ 38, Exhibit 1002 [FF23]).
• During this growth period at 30°C, Dr. Bell-Pedersen stated that the
endonuclease would be manufactured, and would cleave the endonuclease
recognition site carried by the E. coli chromosome (Bell-Pedersen Decl. of
Dec. 16, 2009, ¶¶ 39 & 40, Exhibit 1002) [FF24].
Dr. Bell-Pedersen supported her conclusion with evidence from her
own publications and research, as well as from the scientific literature. We
summarize some of the additional points discussed in the Bell-Pedersen
declarations:
• Quirk taught that the pL promoter is controlled by the temperature
repressor cI857 “which allows low level expression at permissive
temperatures (30°-32°C) and high level expression at elevated temperatures
(37°-43°C)” (Quirk 457; Bell-Pedersen Decl. of Dec. 16, 2009, ¶ 14. Exhibit
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1002) [FF25]. Thus, Dr. Bell-Pedersen concluded, some GIIE endonuclease
would be produced at all times during the experiment (Bell-Pedersen Decl.
of May 19, 2010, ¶¶ 29, 42, & 47, Exhibit 1001) [FF26].
• Her own experiments demonstrating that cleavage of double-
stranded DNA required only a GIIE endonuclease, making cleavage of the
chromosome “inevitable at some level” when the endonuclease is expressed
in the cell (Bell-Pedersen Decl. of Aug. 4, 2010, ¶ 13, Exhibit 1003) [FF27].
• Her thesis in which she “suggested that the toxicity associated with
expression of I-TevI could be due to endonuclease cleavage sites on the E.
coli chromosome.” (Bell-Pedersen Decl. of Aug. 4, 2010, ¶ 23, Exhibit
1003) [FF28]. This suggestion was based on evidence from the Quirk
publication, as well as additional experiments involving the SOS response
(Bell-Pedersen Decl. of May 19, 2010, ¶¶ 73-78, Exhibit 1001) [FF29].
• Scientific literature showing that E. coli “possesses a set of
recombination and repair proteins that repair DSBs that are known to
substitute for λ’s proteins.” (Bell-Pedersen Decl. of Aug. 4, 2010, ¶ 20,
Exhibit 1003) [FF30]. Based on this evidence, Dr. Bell Pedersen concluded
that the E. coli would have been able to repair DSBs made by the GIIE
endonuclease (Bell-Pedersen Decl. of Aug. 4, 2010, ¶ 20, Exhibit 1003)
[FF31].
• Testimony that she believed at the time of the publication that
insertion of the intron (with the antibiotic resistance gene) into the phage
occurred while the phage was integrated into the E. coli chromosome (Bell-
Pedersen Decl. of Dec. 16, 2009, ¶¶ 44-46), Exhibit 1002 [FF32].)
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• Testimony that the intent of the Bell-Pedersen experiments was “to
determine whether 1-TevI could function in trans to promote insertion of a
gene of interest (foreign DNA sequences) into an I-TevI recognition site in
a λ prophage integrated into the E. coli chromosome, independent of the
DNA sequences that encode the intron.” (Bell-Pedersen Decl. of May 19,
2010, ¶ 45. Exhibit 1001) [FF33].)
• Testimony that:
The expression of I-TevI from the low copy number pKC30
plasmid in the RRI(λc+) lysogen or the N99(λcI857) lysogen at
30°C did not result in significantly poor growth of the E. coli
lysogens. This suggested that the lower levels of 1-TevI were
not toxic (or less toxic) because they resulted in cleavage of the
naturally-occurring I-TevI recognition sites at rates sufficiently
low to allow for successful repair by the natural E. coli repair
mechanisms.
(Bell-Pedersen Decl. of May 19, 2010, ¶ 23, Exhibit 1001 [FF34].)

Dr. Victoria Derbyshire
Victoria Derbyshire, Ph.D., provided expert testimony on behalf on
the Requester. Dr. Derbyshire was a post-doctoral research affiliate in the
laboratory directed by Dr. Marlene Belfort from 1992 to 1998. Dr.
Derbyshire testified that “one of ordinary skill in the art at the relevant time
would have understood the Bell-Pedersen et al. (1990) reference to show
cleavage of the I-TevI recognition site of the E. coli lysogen occurring
primarily, if not exclusively, before excision of the prophage DNA from the
E. coli chromosome.” (Derbyshire Decl. of Dec. 17, 2009, ¶ 27, Exhibit
1004 [FF35].) Dr. Derbyshire also testified that the majority of cleavage and
integration of the antibiotic resistance gene occurred before excision of the
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phage from the bacterial chromosome (Derbyshire Decl. of Dec. 17, 2009, ¶
28, Exhibit 1004) [FF36]. Derbyshire based her opinion primarily on the
knowledge that the pL promoter used to control the endonuclease expression
was “leaky” and would have been expressed at 30°C, during the growth
phase, prior to intentional induction of the promoter (Derbyshire Decl. of
Dec. 17, 2009, ¶ 31, Exhibit 1004 [FF37]).
Additional points include:
• “even if repression of pL at 30°C were ‘leaky,’ it does not follow that
induction of the λ prophage will result.” (Derbyshire Decl. of May 19, 2010,
¶ 14. Exhibit 1005 [FF38].)
• Citing six pre-filing date publications, Dr. Derbyshire stated one of
ordinary skill in the art in 1992 would have in the art would have known that
E. coli had the capacity to repair double-strand breaks in and DNA and
conduct homologous recombination without phage proteins (Derbyshire
Decl. of August 4, 2010, ¶¶ 19-21, Exhibit 1006 [FF39].)

Dr. Marlene Belfort
During the course of the reexamination proceeding, the Patent Owner
provided series of declarations by Dr. Marlene Belfort, a scientist with more
than twenty years of experience in the field of molecular biology as it relates
to endonucleases. Dr. Belfort was the senior author of both the Quirk and
Bell-Pedersen publications. She was the director of the laboratory in which
the research reported in Quirk and Bell-Pedersen was performed. Dr. Bell-
Pedersen was working under Dr. Belfort’s supervision as a Ph.D. graduate
student. Dr. Derbyshire was a postdoctoral affiliate in her lab. We have
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considered all the testimony in the Belfort declarations. Below we
summarize some of the main points made by Dr. Belfort.
• Bell-Pedersen did not show cleavage of chromosomal DNA by a
GIIE endonuclease or evidence that a GIIE endonuclease could cleave
chromosomal DNA (Belfort Decl. of Nov. 17, 2009, ¶¶ 10 & 16, Exhibit
1007) [FF40]).
• The pL temperature inducible promoter controlled both endonuclease
production and phage excision from the chromosome. Consequently, Dr.
Belfort declared that when there was a low activity of the pL promoter at
30°C, the phage would have been excised from the chromosome where it
could serve as a target for endonuclease cleavage and intron insertion
(Belfort Decl. of Nov. 17, 2009, ¶ 33, Exhibit 1007 [FF41]).
• Belfort acknowledged that “[s]ome baseline integration of the
[antibiotic resistance gene or marker gene] may have occurred prior to
induction, but this was neither the intent of the experiment nor the bulk of
what was measured in the experiment” (Belfort Decl. of Nov. 17, 2009, ¶ 37,
Exhibit 1007). However, Dr. Belfort clarified that it was “theoretically
possible” that the gene integrated into the chromosomal DNA, but “this is
not what Bell-Pedersen and Quirk show, nor is it what a skilled artisan
would conclude from reading these references.” (Belfort Decl. of June 30,
2010, ¶ 30, Exhibit 1008) [FF42]).
• “Since the time of the research, experiments and publications, none
of the authors of either the Bell-Pedersen reference or the Quirk reference
have discussed with me their belief that the research and experiments
described in the two references demonstrated the introduction of a site-
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specific double-strand break at a group I intron encoded endonuclease
recognition site in chromosomal DNA.” (Belfort Decl. of March 8, 2010, ¶
21, Exhibit 1009) [FF43].)
• “Nowhere in Bell-Pedersen or Quirk is it shown or suggested that
cleavage of the I-TevI recognition site occurred while the recognition ' site
was present in chromosomal DNA. In fact, the intent of the experiments
described in Bell-Pedersen and Quirk was for I-TevI to cleave its recognition
site in bacteriophage that was replicating independently of the
chromosome.” (Belfort Decl. of March 8, 2010, ¶ 23, Exhibit 1009) [FF44].
• Dr. Derbyshire’s conclusion that intron insertion would not have
occurred in excised phage because they were replicating too rapidly is
flawed since in other experiments, using phage that were not capable of
integrating into the E. coli chromosome, intron insertion rates were similar
(Belfort Decl. of March 8, 2010, ¶¶ 29 & 30, Exhibit 1009) [FF45].
• “the only explanation for the productive cleavages that are reported
in Bell-Pedersen and Quirk is that repair and recombination are taking place
in developing phage (i.e., phage that are in the lytic phase and expressing
phage proteins) . . . Since phage proteins that are required for recombination
are not present in lysogens [when phage is inserted into the chromosomal
DNA], and since E. coli cells are not able to repair the double-strand break
without the assistance of phage proteins, one must conclude that a
productive cleavage occurred after induction (i.e., after expression of phage
proteins and excision). If cleavage had occurred prior to induction, the cell
would have died, and there would have been no progeny phage to report
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intron acquisition.” (Belfort Decl. of June 30, 2010, ¶ 26, Exhibit 1008)
[FF46].
• “As the person who designed these experiments [reported in the
Bell-Pedersen publication], and directed laboratory members to conduct the
experiments, I can say with certainty that it was not our intent to determine
whether I-TevI could function on a chromosome.” (Belfort Decl. of June 30,
2010, ¶ 36, Exhibit 1008) [FF47].

Rejection
The method of claim 1 comprises two recited steps: 1) providing a
GIIE endonuclease recognition site to the chromosome of a cell; and 2)
providing a GIIE endonuclease to a cell such that the endonuclease cleaves
the chromosome at the recognition site.
The Examiner found (Right of Appeal Notice (“RAN”) 12-14) that
each of Quirk and Bell-Pedersen described: 1) providing a GIIE
endonuclease recognition site to the chromosome of an E. coli cell, where
the site is present in a phage DNA which integrates into the E. coli
chromosome. The Examiner also found (RAN 12-14) that both publications
described 2) providing a GIIE endonuclease to the cell such that it cleaved
the chromosomal DNA. As both steps were found to be described in Quirk
and Bell-Pedersen, the Examiner concluded that claim 1 was anticipated by
the publications (RAN 12-14).
The Patent Owner contends that the Examiner erred in finding that
Bell-Pedersen produced a double-stranded break in a chromosome. The
Patent Owner contends that the Examiner did not establish that cleavage of
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19
the DNA took place when the phage was integrated into the bacterial
chromosome (Appellant App. Br. 15).
The Patent Owner reasoned, based on expert testimony, that when the
temperature inducible promoter (pL) was expressed, excision of the phage
from the bacterial chromosome and the manufacture of the endonuclease
would happen simultaneously. Consequently, the endonuclease would be
made as the phage is excised from the bacterial chromosome, and in this
excised form, would be available for the endonuclease to cleave it.
Cleavage and insertion of the antibiotic resistance, according to the Patent
Owner, would thus have occurred when the phage is no longer part of the E.
coli chromosomal DNA.
The Examiner and Requester challenged the Patent Owner’s
interpretation of the Quirk and Bell-Pedersen publications. The cells are
shifted to a high temperature to activate the promoter which, in turns, directs
manufacture of the endonuclease (FF9-FF10, Quirk 457, col. 1). In
agreement with the Examiner, the Requester contends that a low level of
endonuclease was made during the growth phase of the E. coli (due to
“leakiness” of the promoter), prior to the shift to a higher temperature.
During this time, the Requester argued that cleavage would have occurred
while the GIIEE recognition site was still resident in the E. coli
chromosome. (Requester Brief 7.)
The Examiner also stated “the prior art only needs to teach that at
least some integration of the kanamycin resistance gene occurred in the
bacterial chromosome in the experiments described in Bell-Pedersen. It
need not teach that every single incident of integration occurred while the
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20
recognition site was integrated in the chromosome of the lysogen.” (RAN
56.)
In addressing this issue it is important to recognize that neither the
Patent Owner nor Requester disputed the fact that Bell-Pedersen did not
disclose whether cleavage of the DNA and insertion of the marker gene into
the intron occurred while the phage was still residing in the bacterial
chromosome or after the phage had been excised from the chromosome. It
was silent on where this event took place. Thus, the position of the
Examiner, the Patent Owner, and Requester is based on opinion and
evidence of what the skilled worker would have understood, based on their
knowledge, the level of ordinary skill in the art, and their reading of the
Quirk and Bell-Pedersen publications.

Analysis
Quirk and Bell-Pedersen are silent on where cleavage of the
recognition site takes place. The recognition site is introduced into the E.
coli cell on a phage (FF6, Quirk 458-459, 463, col. 2 (section titled
“Phages,” T2L and T2H); Bell-Pedersen Decl. of May 19, 2010, ¶ 27,
Exhibit 1001; FF7; FF14, Bell-Pedersen 3763; & FF18, Bell-Pedersen 3764-
3765; Bell-Pedersen Decl. of May 19, 2010, ¶¶ 32-44, Exhibit 1001). The
phage integrates into the bacterial chromosome as part of its replication
cycle, and then, when the temperature inducible promoter is activated by an
increase in temperature, the phage is excised from the chromosome (FF21,
Bell-Pedersen 3765 & FF22, Bell-Pedersen 3765, col. 1; Bell-Pedersen Decl.
of May 19, 2010, ¶ 54, Exhibit 1001). The GIIE endonuclease is controlled
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21
by the same promoter, and thus is produced as well, when the temperature is
shifted upwards (FF10, Quirk 457, col. 1; FF16-FF18, Bell-Pedersen 3764-
3765; Bell-Pedersen Decl. of May 19, 2010, ¶¶ 32-44, Exhibit 1001).
The Requester and Examiner contend cleavage at the recognition site
takes place when the phage is still in the chromosome. The Patent Owner
contends cleavage takes places after excision of the phage.
Expert written testimony was a key component of the arguments made
by both the Patent Owner and the Requester. Expert testimony was provided
that specific endonuclease cleavage at the recognition site occurred a) while
the phage was resident in the chromosome, or b) alternatively after the phage
had been excised from chromosome. After considering the totality of the
evidence before us, including the expert testimony and the disclosures of the
Quirk and Bell-Pedersen publications, we conclude that the Examiner did
not provide sufficient evidence to establish that cleavage “necessarily” took
place while the phage was resident in the bacterial chromosome. SmithKline
Beecham Corp., 403 F.3d at 1343; Trintec Indus., 295 F.3d at 1295.
The Requester provided evidence that the endonuclease cleaved the
bacterial chromosome when the endonuclease was highly expressed during
the growth cycle. Drs. Bell-Pedersen’s and Derbyshire’s theory that
cleavage occurred at the recognition site while it resided in the E. coli
chromosome was factually supported by evidence, e.g., of the leaky
inducible promoter (FF23-25; Quirk 457, Bell-Pedersen Decl. of Dec. 16,
2009, ¶¶ 14, 38-40, Exhibit 1002 & FF37, Derbyshire Decl. of Dec. 17,
2009, ¶ 31, Exhibit 1004), that expression of endonuclease and excision
(“induction”) of prophage would not necessarily both occur under leaky
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22
expression conditions (FF38, Derbyshire Decl. of May 19, 2010, ¶ 14.
Exhibit 1005), and the existence of productive repair mechanisms provided
by E. coli (FF39, Derbyshire Decl. of August 4, 2010, ¶¶ 19-21, Exhibit
1006).
As disclosed by Quirk, E. coli containing the td and sunY genes with
an intact ORF (i.e., a functional endonuclease) grew poorly and were
apparently toxic to the cells (FF8, Quirk 456, col. 2). Dr. Bell-Pedersen
explained, and provided supporting evidence, that the toxicity was a result of
endonuclease cleavage of the bacterial chromosome (FF28, Bell-Pedersen
Decl. of Aug. 4, 2010, ¶ 23, Exhibit 1003 & FF29, Bell-Pedersen Decl. of
May 19, 2010, ¶¶ 73-78, Exhibit 1001). However, such cells were not viable
as required by the claim because the endonuclease was toxic to the cells.
Dr. Bell-Pedersen did not provide adequate evidence that the cleavage
occurred in the cleavage site provided by the phage, rather than cleavage
sites present elsewhere in the bacterial chromosome. In fact, Dr. Bell-
Pedersen referred to toxicity being due to cleavage sites on the chromosome
(FF28, Bell-Pedersen Decl. of Aug. 4, 2010, ¶ 23, Exhibit 1003), but did not
specifically refer to the GIIEE cleavage sites provided by the phage when
integrated into the E. coli chromosome.
On the other hand, Dr. Belfort provided countervailing evidence that
integration occurred only after the phage had been excised from the
chromosome. First, Dr. Belfort testified that Bell-Pedersen did not expressly
disclose chromosomal cleavage (FF40, Belfort Decl. of Nov. 17, 2009, ¶¶ 10
& 16, Exhibit 1007). Dr. Belfort acknowledged that the promoter was leaky,
but explained that since such promoter controlled both phage excision and
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23
endonuclease product, the phage would be available as a target (FFs 41-42,
Belfort Decl. of Nov. 17, 2009, ¶ 33, Exhibit 1007 & Belfort Decl. of June
30, 2010, ¶ 30, Exhibit 1008). Dr. Belfort also provided evidence that
cleavage could occur in the phage as they replicated (FF45, Belfort Decl. of
March 8, 2010, ¶¶ 29 & 30, Exhibit 1009), contrary to Dr. Derbyshire’s
assertions. Finally, Dr. Belfort gave scientific reasons as to why she
believed that cleavage occurred in the excised phage (FF46, Belfort Decl. of
June 30, 2010, ¶ 26, Exhibit 1008).
While the competing theories are each supported by factual scientific
evidence, the preponderance of the evidence does not establish that the Bell-
Pedersen publication disclosed or would have suggested to one of ordinary
skill in the art that cleavage of the GIIE endonuclease site occurred while the
phage was integrated into the bacterial chromosomal DNA. Bell-Pedersen is
silent on where productive endonuclease cleavage occurred. It was thus the
Examiner’s burden to show that a person of ordinary skill in the art would
have necessarily recognized that it occurred at the GIIEE recognition site
when integrated into the E. coli chromosome. This burden was not met by a
preponderance of the evidence. None of the experts on both sides of this
dispute provided definitive experimental data to explain where cleavage of
the GIIEE recognition site took place. For example, the “leakiness” of the
promoter used to control expression of the GIIE endonuclease was one of
Requester’s main arguments as to why endonuclease cleavage would have
occurred in the bacterial chromosome. However, there was no “hard”
evidence that the GIIE endonuclease was actually produced under “leaky”
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Patent 7,309,605 B1

24
conditions in amounts that would enable it to cleave the chromosome at the
GIIEE recognition site.
When there was such sharp disagreement between the experts, and an
absence of conclusive data, it cannot be said with certainty that one of
ordinary skill in the art would have necessarily known that the chromosomal
cleavage occurred at the GIIEE recognition site provided to the cell,
particularly in view of Belfort’s testimony that it did not.
There was testimony as to the intent of the experiments described in
the Bell-Pedersen publication (FF33, Bell-Pedersen Decl. of May 19, 2010,
¶ 45. Exhibit 1001; FF42-FF45, Belfort Decl. of June 30, 2010, ¶ 30, Exhibit
1008; Belfort Decl. of March 8, 2010, ¶¶ 21, 23, 29, 30, Exhibit 1009).
However, the fact that different co-authors may have had different intents in
carrying out the experiments does not establish whether cleavage of the
GIIEE recognition site took place while the phage was integrated into the
bacterial chromosome.

Parker
Both parties cited the Parker publication7 as supporting their position
as to where endonuclease cleavage occurred. Parker was published in 1996,
after the filing date of the ‘605 patent, but Parker is relied upon to prove a
fact in existence on the filing date of the patent, i.e., whether GIIEE cleavage
occurred in the phage while integrated in the chromosome (and as required
by claim 18) or after the phage had been excised from the chromosome

7 Monica M. Parker et al., Homology Requirements for Double-Strand
Break-Mediated Recombination in a Phage λ-td Intron Model System, 143
Genetics 1057 (July 1996).
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Patent 7,309,605 B1

26
Figure 2 shows a model system for studying integration of a phage
intron (containing the Kan marker gene) into an intronless gene, a process
which is dependent on a double-stranded chromosomal break produced by
the GIIE endonuclease, I-TevI (Parker 1057). The large outside rectangle
represents an E. coli bacterial cell (Parker 1057-1059). The thin wavy line
in the inner rectangle represents bacterial chromosomal DNA and the heavy
solid line represents plasmid DNA. “Kan” is an antibiotic resistance gene
used as marker gene (Parker 1059). Kan is present in the donor phage
intron (represented by the filled bar abutted by unfilled bars). The unfilled
bar in the bacterial chromosome (thin wavy line) represents the recipient
phage DNA, which serves as a recipient for the donor phage intron
comprising Kan (Parker 1059).
[FF49] I-TevI is a GIIE endonuclease shown as a gene on a plasmid
(bar labeled “I-TevI”) and protein (circular Pac-man-like symbol) (Parker
1059) that is made from the gene.
[FF50] The GIIE endonuclease (I-TevI; Pac-man) cleaves the
recipient phage DNA (shown as Pac-man “attacking” unfilled bar) to make a
double-stranded DNA break (Parker 1059-1060). Kan, carried on a plasmid,
integrates in the vicinity of the break (Parker 1059).
[FF51] The temperature of the bacterial cell is shifted to a higher
temperature, inducing the phage to excise from the chromosome, and the
phage (shown below the “Induction” arrow) are examined for the presence
of the Kan marker gene, evidence that the GIIE endonuclease promoted the
genes integration into the recipient phage DNA.
[FF52] In explaining Figure 2, Parker wrote:
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27
A quantitative in vivo assay was developed to analyze the
role of exon homology in the DSB-mediated [double-stranded
breaks] homing process [homing refers to the integration of the
intron into the GIIEE recognition site of an intronless gene]
(Figure 2). This experimental system utilizes a plasmid-borne
intron donor allele [donor phage intron] . . . in which the I-TevI
coding sequence has been deleted from the td intron and
replaced with a kanamycin resistance (kanR) gene (Bell-
Pedersen et al. 1990). I-TevI is provided . . . [on a plasmid with
the temperature inducible promoter PL]. The induced λ
prophage containing an intronless td allele . . . serves as the
intron recipient.
In the lysogenic host containing the intron donor plasmid
and the I-TevI expression plasmid, lysogeny and repression of
I-TevI are maintained at the permissive temperature . . . [by the
temperature inducible promoter system]. Upon shift of mid-log
phase cells to the nonpermissive temperature (42°C), synthesis
of I-TevI and λ lytic functions are induced, resulting in
endonucleolytic cleavage of λ at the [recipient phage intron].
(Parker 1059-60 (emphases added).)
The Examiner found that Figure 2 of Parker showed cleavage of the
recipient phage DNA while integrated in the chromosomal DNA (wavy line
in Figure 2) by the GIIE endonuclease (I-TevI, represented as Pac-Man-like
shapes). The Examiner stated:
The experiments (figure 2) in Parker represent the experiment
from Bell-Pedersen. Dr. Belfort and her co-authors described
the experiment of Bell-Pedersen as showing cleavage of the
chromosome of the E. coli lysogen, and insertion of the kanR
gene into the prophage integrated into the chromosome (see ¶¶
25-29 of the Dr. Bell-Pedersen declaration filed on 8/6/10).
(RAN 48.)
Dr. Belfort, an author of the Parker publication, acknowledged “that
the cartoon shown in Figure 2 [(FF48, Parker 1059)] shows Pac-Man-like
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28
shapes to represent I-TevI, and that it appears at a passing glance of this
cartoon that these Pac-Man-like shapes are cleaving I-TevI recognition sites
in the λ prophage.” (Belfort Decl. of June 10, 2010, ¶ 74, Exhibit 1008.)
However, Dr. Belfort testified in her written declaration that Parker taught
that the temperature shift resulted in concurrent production of TevI and
phage excision (induction of lytic functions), making “it highly likely that
productive cleavage of I-TevI occurs after induction.” (Id. at ¶ 75.) In other
words, since the endonuclease was produced at the same time the phage was
excised from the bacterial chromosome, Dr. Belfort argued that it was
“highly likely” cleavage took place after the phage was free of the
chromosome, despite how the cleavage was illustrated in the conflicting
figure.
Additionally, Dr. Belfort pointed to Parker’s subsequent statement as
evidence that cleavage did not take place in the chromosome. Parker wrote:
“The induced λ prophage containing an intronless td allele . . . serves as the
intron recipient.” (FF52, Parker 1059-60.) Prophage induction, Dr. Belfort
testified, means that prophage was induced to excise from the chromosome
(Belfort Decl. of Jun. 30, 2010, ¶¶ 75-79, Exhibit 1008). Thus, according to
Dr. Belfort, an induced prophage is one which is excised from the
chromosome. Once induced, the phage would be available for cleavage by
the GIIE endonuclease (Belfort Decl. of Jun. 30, 2010, ¶¶ 75-79, Exhibit
1008).
The Requester took the opposite position, relying on testimony by its
expert, Dr. Deborah Bell-Pedersen. Dr. Bell-Pedersen testified that a
“prophage” refers to the phage when integrated into the bacterial
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29
chromosome (Bell-Pedersen Decl. of May 19, 2010, ¶ 62, Exhibit 1001).
Thus, based on Figure 2 and Packer’s statement about the “prophage”
serving as the intron recipient, Dr. Bell-Pedersen testified that Parker teaches
that cleavage and integration occurred while the prophage was still
integrated in the chromosome (id.).
We have considered both positions advanced by the parties, but
conclude that Parker does not show that Bell-Pedersen’s experiments
necessarily resulted in cleavage of the bacterial chromosome by the GIIE
endonuclease. It is true, as acknowledged by Dr. Belfort, that Figure 2
(FF48, Parker 1059) shows the endonuclease cleaving the recipient phage
DNA while residing in the bacterial chromosome. However, the teaching in
this figure is offset by the text of Parker stating that the “induced λ
prophage” was the recipient for the intron/marker gene DNA (FF52, Parker
1059-60). As explained by Dr. Belfort, and supported by factual evidence,
induction would result in excision of the phage from the chromosome –
indicating that the prophage had been excised from the chromosome when
cleaved by the endonuclease in order to serve as the intron recipient.
Although Dr. Bell-Pedersen testified that term “prophage” indicated the
phage was still integrated in the bacterial chromosome, Dr. Belfort pointed
out that the text referred to an “induced prophage” which meant it had been
excised from the chromosome. In other words, inducing a prophage would
result in phage excision and thus an “induced prophage” would reasonably
be understood to refer to an excised phage free of the bacterial chromosome.
When the Parker publication is read in its entirety, we find that Parker
does not unambiguously teach that the endonuclease cleavage occurred in
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30
the chromosome. Figure 2, said to support this fact conflicts with Parker’s
own explanation of the figure.

Conclusion
In sum, the Examiner did not meet the burden of showing that Quirk
and Bell-Pedersen described cleaving the bacterial chromosome with a GIIE
endonuclease as required by claim 1. Consequently, we reverse the
anticipation rejection under 35 U.S.C. 102 of claim 1, and dependent claims
10, 11, 14, and 15.

OBVIOUSNESS REJECTIONS BASED ON QUIRK AND BELL-
PEDERSEN
Grounds 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 38,
41, 42, 45, 46, 51, 52, 55, 61, 62, 65, and 66 each cite Quirk or Bell-
Pedersen combined with additional secondary prior art publications cited for
specific GIIE endonuclease recognition sites and other narrowing limitations
of the claim 1, the only independent claim on appeal.
We select claim 18, which depends on claim 1, as representative.
Claim 1 is drawn to a method comprising the step of providing a GIIE
endonuclease to a viable cell of an organism “such that the Group I intron
encoded endonuclease cleaves said Group I intron encoded endonuclease
site at the location in the chromosomal DNA of the cell.” Claim 18 further
recites that “the isolated, viable cell of the organism is a eukaryotic cell.”

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Legal Principles
To decide whether a composition, device, or process would have been
obvious in light of the prior art, it must be determined whether, at the time of
invention, “a person of ordinary skill in the art would have had reason to
attempt to make the composition or device, or carry out the claimed process,
and would have had a reasonable expectation of success in doing so.”
PharmaStem Therapeutics, Inc. v. ViaCell, Inc., 491 F.3d 1342, 1360 (Fed.
Cir. 2007).

Rejection
Quirk and Bell-Pedersen were cited by the Examiner as evidence that
chromosomal cleavage by a GIIE endonuclease was known to take place in
an organism. The Examiner found that Quirk and Bell-Pedersen differed
from claim 18 in not describing cleavage of a eukaryotic chromosome as
required by the claim (RAN 43 & 44). However, the Examiner found that
Frey disclosed in vitro cleavage of a eukaryotic chromosome from yeast by a
GIIE endonuclease (id.). The Examiner concluded it would have been
obvious to one of ordinary skill in the art to apply Quirk and Bell-Pedersen
to Frey in order to facilitate site directed insertion of a gene of interest into a
eukaryotic chromosome (id. at 43-45).
Patent Owner contends that the Examiner did not establish the
interchangeability of bacterial and yeast cells, or of phage DNA and
eukaryotic chromosomal DNA, nor a suggestion to use eukaryotic cells,
rather than bacterial cells in the methods of Quirk and Bell-Pedersen
(Appellant App. Br. 35-36). Patent Owner also contends there would have
been no reasonable expectation of success that a eukaryotic chromosome
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32
would be cleaved by a GIIE endonuclease as required by claim 18 (id. at
37).
Analysis
In making an obviousness determination, it is necessary to take the
following factors into consideration: (a) the scope and content of the prior
art; (b) the differences between the prior art and the claimed invention; (c)
the level of skill in the pertinent art; and (d) evidence of secondary
considerations. Graham v. John Deere, 383 U.S. 1, 17-18 (1966).
In this case, there were numerous publications cited by the Examiner
and both parties in this proceeding. Although the rejections were limited to a
specific combinations of prior art, in evaluating the obviousness of the
claimed subject matter, we cannot ignore the other publications in
determining the scope and content of the prior art and level of skill in the art,
factors that must be considered in making an obviousness determination.
(id.).
Patent Owner argues that the Examiner did not establish that the
bacterial cells utilized in Quirk’s and Bell-Pedersen’s experiments were
interchangeable with the yeast cells of Frey (Appellant Appeal Br. 35-36;
Appellant Rebuttal Br. 17-18). However, “interchangeability” is only one
factor to be considered. The pertinent issue in an obviousness
determination is whether the skilled worker would have had a reason to
combine the prior art. KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418
(2007). It is unnecessary that such reason is expressly stated in the prior art.
KSR, 550 U.S. at 418; In re Kahn, 441 F.3d 977, 987-88 (Fed. Cir. 2006).
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The Examiner provided a reason to have expressed a GIIE
endonuclease in a yeast cell (RAN 43 & 45). In addition to this, as pointed
out by the Requester, Eddy (1992)9 (hereinafter, Eddy92) also provided a
reason to have used Quick or Bell-Pedersen’s system in other organisms,
such as yeast (Requester’s Respondent Br. 18-19).
Eddy92 constructed an artificial mobile element from the gene for the
restriction enzyme Eco RI that is capable of site-specific insertions into a
phage genome (Eddy 92 1544 [FF53]). Although Eddy92 did not use a GIIE
endonuclease, Eddy “configured the experimental system to closely mirror
that used to measure the mobility of the mobile T4 td and sunY introns”
described in Bell-Pedersen (Eddy92 1546, col. 2) [FF54]. Based on these
experiments, Eddy92 concluded:
It should be possible to use DSBR-based targeting of
homologous recombination to effect site-directed gene
conversions in a variety of organisms. The extreme specificity
of intron-encoded endonucleases . . . could allow targeting of a
single specific site in a genome, either by the good fortune of
having a usable site already in the genome or by the one-time
transgenic introduction of a "landing site" containing the
endonuclease recognition site and flanking exon homology to
some shuttle vector construct.
(Eddy92 1547, col. 2) [FF55].
Thus, Eddy92 directly suggested using a GIIE endonuclease for
engineering a genome (FF55, Eddy92 1547, col. 2) and referred specifically
Bell-Pedersen’s GIIEE system in the publication (FF54, Eddy92 1546, col.
2). Consequently, there was an express suggestion in the prior art to have

9 Sean R. Eddy & Larry Gold, Artificial Mobile DNA Element Constructed
From the EcoRI Endonuclease Gene, 89 Proc. Nat’l Acad. Sci.1544 (1992).
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34
utilized a GIIE endonucleases “in a variety of organisms.” (FF55, Eddy92
1547, col. 2.)
The Patent Owner states that Eddy92 “did not involve eukaryotic
cells, did not involve chromosomal DNA (eukaryotic or otherwise), and did
not involve any GIIEEs.” (Appellant Rebuttal Brief 18.) Patent Owner also
stated that Eddy92 noted “limitations and unpredictability” of its restriction
enzyme system (id. at 19.)
We agree that Eddy92 did not utilize a GIIE system, but that does not
nullify Eddy92’s express suggestion to have used a GIIE for site-directed
gene conversion in a variety of organisms (FF55, Eddy92 1547, col. 2).
Claim 1 simply involves cleaving a cleaving a chromosome of an
“organism,” and is not limited to a particular type. Furthermore, even where
unpredictability is shown for Eddy92’s restriction enzyme system, such
unpredictability is mooted in light of Frey and Dujon (1990) as discussed
below.

Expectation of success
Once a reason has been established to combine the prior art, it is the
Examiner’s burden to show that that there would have been a reasonable
expectation of success that a GIIE endonuclease could cut chromosomal
DNA in a viable cell as required by the claims. PharmaStem Therapeutics,
Inc. 491 F.3d at 1360.
As found by the Examiner, and undisputed by the Patent Owner, Frey
teaches that purified, intact chromosomes from yeast were digested by I-
SceI, a GIIE endonuclease (Frey 122-123; RAN 43) [FF56]. In addition, the
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35
Examiner cited Dujon (1990) (hereinafter, “Dujon90”) as providing “direct
evidence that at least one group I encoded endonuclease was known in the
art at the time of the invention to introduce a double-stranded break in the
chromosomal DNA of a viable yeast cell and was known to provide the
endonuclease to the cell through double stranded break repair and
homologous recombination.” (RAN 51, ll. 3-7.) According to Dujon:
I-Sce I can be expressed in the yeast nucleus from artificial
constructs and the protein is able to cleave efficiently both its
natural site within mitochondria and an artificially placed site
within the nucleus. In the latter case, double strand break repair
and homologous recombination follow the formation of the cut
in a manner which parallels that obtained with the HO
endonuclease.
(Dujon90, Abstract [FF57].) This disclosure meets the cleavage step recited
in claims 1 and 18 because yeast is an “organism” as recited in claim 1 and a
“eukaryotic” cell as in claim 18.
Patent Owner contends that Frey “involved experiments in vitro (not
in a viable cell), and, thus, did not teach or suggest the invention.”
(Appellant Rebuttal Br. 18.) However, Frey explicitly taught cleavage of
eukaryotic chromosomal DNA by a GIIEE (FF56, Frey 122-123), as recited
in claims 1 and 18. Patent Owner did not explain nor provide expert
testimony as to why the in vitro results could not be extrapolated to the in
vivo environment when the enzyme (GIIEE) and substrate (chromosome)
would be the same. Moreover, the Examiner found that in vivo cleavage of
chromosomal DNA by a GIIEE was known at the time of the invention as
taught by Dujon90 (RAN 51, ll. 3-7; FF57, Dujon90 Abstract.)
Patent Owner’s argument about Quirk and Bell-Pedersen’s failure to
teach or suggestion chromosomal cleavage (Appellant’s Appeal Br. 30-31)
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36
is noted, but not persuasive in view of the express teaching in the prior art of
the ability of a GIIEE to cleave yeast chromosomal DNA (FFs 56-57).
Summary
Based on the evidence as a whole, we conclude the skilled worker
would have had a reason to have made the claimed subject matter and would
have had a reasonable expectation of success in doing so. Consequently, we
affirm the rejection of claim 18, and claim 1 upon which it depends
(Grounds 65 and 66). Claim 13 was not separately argued and thus falls
with claims 1 and 18. Appellant did not separately address grounds 7, 8, 11,
12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 41, 42, 45, and 46. We thus
we affirm the rejections of claims 2-9, 12, and 13 which correspond to
grounds 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 41, 42, 45,
and 46.
On pages 32-34 of the Appeal Brief, Appellant separately addressed
grounds 38, 51, 52, 55, 61, and 62 rejecting claim 1, 9-11, and 14-17,
traversing the rejections on the grounds they do not provide the teachings
missing from Bell-Pedersen or Quirk. As we did not find these arguments
persuasive for the latter publications, we affirm the rejections of claims 1, 9-
11, and 14-17.

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37
OBVIOUSNESS REJECTIONS BASED ON SCHIESTL
Claim 1 stands rejected under 35 U.S.C. § 103(a) as obvious over
Schiestl10 combined with admissions in the written description of the ‘605
patent or with Frey (grounds 5 & 6). The issue in these rejections is whether
it would have been obvious to one of ordinary skill in the art to have utilized
a GIIE endonuclease in the method described by Schiestl.

Findings of Fact
Schiestl contains the following pertinent disclosure:
[FF58] Saccharomyces cerevisiae (“yeast”) cells were transformed
with BamHI DNA fragments in the presence of BamHI enzyme (Schiestl
7585 & 7586, col. 2).
[FF59] The fragments were inserted into the yeast genome at BamHI
sites (Schiestl 7587, col. 2).
[FF60]
The simplest explanation of the restriction enzyme mediated
event is that the BamHI enzyme enters the cells, perhaps in
association with the transforming DNA. The enzyme cleaves
the chromosomal DNA at BamHI sites, the cohesive ends of the
URA3 fragment pair with those of the chromosomal DNA, and
the resulting nicks are sealed by DNA ligase . . . .
(Schiestl 7587, col. 2.)
[FF61]
A number of studies indicate that restriction enzymes can enter
mammalian cells that had been made permeable by

10 Robert H. Schiestl & Thomas D. Petes, Integration of DNA Fragments by
Illegitimate Recombination in Saccharomyces Cerevisiae, 88 Proceedings
Nat’l Acad. Sci. 7585 (1991).
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38
electroporation (29). These enzymes reduced the viability of
the cells and induced chromosomal aberrations. W. Morgan
(personal communication) has observed deletions and
rearrangements of restriction fragments contained within
plasmids in mammalian cells as a result of the enzyme
treatment.
(Schiestl 7588-7589).

[FF62]
Potential uses of Restricion enzyme-mediated events include:
insertional mutagenesis, generation of chromosome aberrations,
stimulation os mitotic recombination at aspecific sites, and
improvements in the efficiency of gene targeting in organisims
with high levels of non homologous integration. In addition,
since there is no reason to assme that th eiptake of proteins
during transformation is limited to restriction enzzymes, one
might be able to observe the effecvts of other proteins
(topoisomerases, helicases, etc.) on recombination in vivo.
(Schiestl 7589, col. 1.)

Morgan publication11
[FF63] Morgan describes the introduction of bacterial restriction
endonucleases into mammalian hamster cells (Morgan 191).
[FF64] The endonucleases caused DNA double-strand breakage and
chromosome aberrations (Morgan 191).
[FF65]

11 William F. Morgan et al., The Use of Restriction Endonucleases to Mimic
the Cytogenetic Damage Induced by Ionizing Radiations, in 136 Ionizing
Radiation Damage to DNA: Molecular Aspects 191-199 (S.S. Wallace &
R.B. Painter, eds., Wiley-Liss, Inc. 1990).
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39
There was an increase in the number of breaks observed with
all three enzymes at 2 h and 3 h after electroporation (Fig 1).
We believe this is due to nonspecific DNA degradation in a
population of enzyme treated cells. We have proposed that
DNA cleavage is so severe that these cells cannot recover and
are dying cells (10).
(Morgan 193.)
[FF66]
Consistent with their ability to induce aberrant chromosomes,
both ionizing radiations and restriction enzymes significantly
enhance cell killing.
(Morgan 194.)

Rejection
In determining that the claims were obvious, the Examiner made the
following findings:
• Schiestl described cleavage of chromosomal DNA with a restriction
enzyme, BamHI, and integration of a fragment into the cleaved site (RAN
14-15; FFs 60-61, Schiestl 7585 & 7586, col. 2; 7587, col. 2; 7588-89; &
7589, col. 1).
• Schiestl taught that its methods can be used to improve gene
targeting (RAN 15; FF62, Schiestl 7589, col. 1).
• “Schiestl et al. teaches that restriction enzymes can be introduced
into mammalian [animal] cells by electroporation, which suggests site-
directed introduction of a gene of interest into an animal cell using a
restriction enzyme according to the methods described by Schiestl et al.”
(RAN 15; Schiestl 7589, col. 1).
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40
• Schiestl did not describe using a GIIE endonuclease or a GIIE
endonuclease recognition site in its methods (RAN 15).
• However, “patent owner admits that two site-specific endonucleases,
I-SceI and I-SceII, were known in the prior art as restriction enzymes at the
time of filing of the application which became the ‘605 patent (see column
2). Patent owner further admits that I-SceI was known to generate a double
stranded break in DNA and to induce repair events (see column 2, lines 34-
53).” (RAN 15.) The Examiner made a similar finding based on Frey’s
teachings (id. at 16).
Based on these findings, the Examiner determined it would have been
obvious for one of ordinary skill in the art
to have used the I-SceI endonuclease disclosed in the prior art
[or in Frey] as an alternative restriction enzyme in the method
disclosed by Schiestl et al for introducing a double stranded
break into a target nucleic acid in a [plant or animal] cell. The
substitution of one known restriction enzyme (I-SceI) for
another (BamHI), to achieve the predictable result of generating
a double stranded break in the DNA of a target cell; in order to
facilitate site directed insertion of a gene of interest, would have
been obvious, absent evidence to the contrary.
(RAN 15.)
Patent Owner contends that the Examiner did not establish
obviousness of the claimed invention. Patent Owner argued (App. Br. 41-
42):
• The Examiner did not establish that I-SceI is equivalent to BamHI.
• “Schiestl teaches that the introduction of bacterial restriction
enzyme[s] into a cell will reduce the cell’s viability, induce chromosomal
aberrations, and cause DNA deletions and rearrangements.” (App. Br. 42).
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Analysis
The key issue in this rejection is whether it would been obvious to one
of ordinary skill in the art to have replaced Schiestl’s BamHI with I-SceI in
order to cleave chromosomal DNA of an animal cell in a gene targeting
technique. The Examiner found that I-SceI, a GIIE endonuclease, was
equivalent to the restriction enzyme BamHI described in Schiestl. Based on
this finding, the Examiner concluded that it would have been obvious to one
of ordinary in the art to have replaced BamHI with a known GIIE
endonuclease.
We do not agree. Schiestl, citing a communication from Morgan,
expressly stated restriction enzymes “reduced the viability of cells and
induced chromosomal aberrations” in mammalian cells (FF61, Schiestl
7588-7589). The Morgan publication, by the same Morgan mentioned in
Schiestl, was subsequently cited by the Patent Owner. The Morgan
publication consistently reported that restriction enzymes caused DNA
cleavage “so severe that these cells cannot recover and are dying cells.”
(FF65, Morgan 193.) Based on this disclosure, we agree with the Patent
Owner that the skilled worker would have been led away from the claimed
invention. The disclosure that restriction enzymes induced cell killing (FFs
64-66) is a teaching away from the claimed invention.
A reference may be said to teach away when a person of ordinary
skill, upon reading the reference, would be discouraged from
following the path set out in the reference, or would be led in a
direction divergent from the path that was taken by the applicant. .
. . [I]n general, a reference will teach away if it suggests that the
line of development flowing from the reference’s disclosure is
unlikely to be productive of the result sought by the applicant.
In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994).
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In view of this teaching away, we reverse the Examiner’s decision to
reject the claim 1 over Schiestl combined with the admissions in the written
description of the ‘605 patent or with Frey.

TIME PERIOD FOR RESPONSE
Requests for extensions of time in this inter partes reexamination
proceeding are governed by 37 C.F.R. § 1.956. See also 37 C.F.R. § 41.79.

AFFIRMED

KMF

For Patent Owner:
Kenneth J. Meyers, Esq.
Finnegan, Henderson, Farabow, Garrett & Dunner LLP
901 New York Avenue, NW
Washington, DC 20001-4413

For Third Party Requester
Michael J. Twomey, Esq.
Wilmer Cutler Pickering Hale and Dorr LLP
60 State Street
Boston, MA 02109