Ex Parte Guschin et al

Board of Patent Appeals and Interferences

Sep 13, 2010

11304981 (B.P.A.I. Sep. 13, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
11/304,981 12/15/2005 Dmitry Guschin 8325-0036.20 (S36-US2) 4069
20855 7590 09/13/2010
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO, CA 94303
EXAMINER
DUNSTON, JENNIFER ANN
ART UNIT PAPER NUMBER
1636
MAIL DATE DELIVERY MODE
09/13/2010 PAPER
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
____________________

Ex parte DMITRY GUSCHIN
and
MICHAEL C. HOLMES,
Appellants1
____________________

Appeal 2010-001995
Application 11/304,981
Technology Center 1600
____________________

Before CAROL A. SPIEGEL, FRANCISCO C. PRATS, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

SPIEGEL, Administrative Patent Judge.

DECISION ON APPEAL2

1 The real party in interest is SANGAMO BIOSCIENCES, INC. (Brief on
Appeal under 37 C.F.R. § 41.37 filed 17 March 2009 ("App. Br.") at 2).
This decision also cites the Examiner's Answer mailed 6 July 2009 ("Ans."),
the Reply Brief filed 31 August 2009 ("Reply Br."), and the Specification of
Application 11/304,981 ("the 981 Application") ("Spec.").
2 The two-month period for filing an appeal or commencing a civil action, as
recited in 37 C.F.R. § 1.304, or for filing a request for rehearing, as recited
in 37 C.F.R. § 41.52, begins to run from the "MAIL DATE" (paper delivery
mode) or the "NOTIFICATION DATE" (electronic delivery mode) shown
on the PTOL-90A cover letter attached to this decision.
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Application 11/304,981

2
Appellants appeal under 35 U.S.C. § 134(a) from an Examiner's final
rejection of all pending claims, claims 1, 3-10, and 15-19 (App. Br. 2; Ans.
2; Reply Br. 2). We have jurisdiction under 35 U.S.C. § 134. We AFFIRM.
I. Statement of the Case
The subject matter on appeal is directed to targeted alteration of
genomic sequences. A target sequence at a selected genomic site is
selectively altered by generating four cut DNA ends at two cleavage sites
and rejoining the ends in the presence or absence of a donor sequence (Spec.
3, ¶ 4). The cleavage sites may be present on the same chromosome or on
different chromosomes. For example, a double-stranded break ("DSB") may
be introduced at or near each end of a target sequence with a pair of fusion
proteins, which have endonuclease activity when dimerized, and the cleaved
ends may be allowed to rejoin by non-homologous end joining ("NHEJ")
(resulting in a deletion) or by homologous recombination ("HR") (resulting
in an insertion). (Id. at 3, ¶ 4, to 4, ¶ 2.) The size of the deletion is
determined by the distance between the first and second cleavage sites (id. at
4, ¶ 5; 64, ¶ 1). HR requires a "donor" sequence to template repair of the
target sequence (i.e., the sequence with the DSB) and often results in part or
all of the donor sequence being incorporated into the target site (id. at ¶
bridging 16-17; ¶ bridging 37-38; 39, ¶ 2).
The disclosed methods involve the use of fusion proteins, each of
which comprises a zinc finger DNA binding domain operably linked to a
"half" cleavage domain. The zinc finger DNA binding domains of each pair
of fusion proteins straddle a cleavage site separated by a number of base
pairs compatible with dimerizing their respective "half" cleavage domains.
Cleavage half-domains can be obtained from various restriction
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endonucleases and/or homing endonucleases. For example, a first zinc
finger DNA binding domain binds a sequence on one chromosome in one
direction, while a second zinc finger DNA binding domain binds the
corresponding sequence on the other chromosome in the other direction at a
defined location on genomic DNA. Each "half" of a cleavage domain is a
monomeric restriction endonuclease, e.g., Fok I (a Type II restriction
enzyme), which requires dimerization for enzymatic activity. The first and
second zinc finger DNA binding domains clamp onto one end of the
chromosome at or near one end of the target sequence, the two Fok I
monomers dimerize, activating enzymatic cleavage, and a DSB is introduced
between the two zinc finger DNA binding domains. Third and fourth fusion
proteins are used to introduce a DSB between the two zinc finger DNA
binding domains at the other end of the chromosome at or near the other end
of the target sequence. (Id. at 2, ¶ 6, to 7, ¶ 3; 9, ¶ 3, to 10, ¶ 3; 24, ¶ 4, to
36, ¶ 2; 64, ¶ 1.)
According to the 981 Specification, "[d]ouble-stranded DNA includes
that present in chromosomes, episomes, organellar genomes (e.g.,
mitochondria, chloroplasts), artificial chromosomes and any other type of
nucleic acid present in a cell such as, for example, amplified sequences,
double minute chromosomes and the genomes of endogenous or infecting
bacteria and viruses " (id. at 9, ¶ 3.)
Claims 1, 15, and 19 are illustrative and read (App. Br. 14-16,
emphasis added):
1. A method for deleting sequences in a region of interest in
double-stranded DNA of genomic cellular chromatin in a cell,
the method comprising:
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expressing first, second, third and fourth fusion proteins
in the cell, each of the fusion proteins comprising:
(i) a zinc finger DNA-binding domain that
binds to a target site in the DNA, and
(ii) a cleavage half-domain;
further wherein:
(a) the first and second fusion proteins bind to first and
second target sites respectively, wherein a first cleavage site lies
between the first and second target sites, and
(b) the third and fourth fusion proteins bind to third and
fourth target sites respectively, wherein a second cleavage site lies
between the third and fourth target sites;
such that the first and second fusion proteins cleave the DNA
at the first cleavage site, the third and fourth fusion proteins cleave
the DNA at the second cleavage site, and DNA ends are rejoined
such that sequences between the first and second cleavage sites are
deleted.

15. A method for targeted replacement of a genomic
sequence, the method comprising:
(a) expressing first, second, third and fourth fusion
proteins in the cell, each of the fusion proteins comprising:
(i) a zinc finger DNA-binding domain that
binds to a target site in the DNA, and
(ii) a cleavage half-domain;
wherein the first and second fusion proteins bind to first
and second target sites respectively, wherein a first
cleavage site lies between the first and second target
sites, and
the third and fourth fusion proteins bind to third and
fourth target sites respectively, wherein a second cleavage
site lies between the third and fourth target sites;
such that the first and second fusion proteins cleave the
DNA at the first cleavage site, and the third and fourth
fusion proteins cleave the DNA at the second cleavage
site; and
(b) contacting the cell with a donor polynucleotide,
wherein the donor polynucleotide comprises:
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(i) sequences homologous to genomic
sequences flanking the first and second cleavage
sites; and
(ii) sequences homologous but non-identical to
genomic sequences between the first and second
cleavage sites:
whereby genomic sequences between the first and second cleavage
sites are replaced by the homologous but non-identical sequences of
the donor polynucleotide.

19. A method for targeted replacement of a genomic
sequence, the method comprising:
(a) expressing first, second, third and fourth fusion
proteins in the cell, each of the fusion proteins comprising:
(i) a zinc finger DNA-binding domain that
binds to a target site in the DNA, and
(ii) a cleavage half-domain;
wherein the first and second fusion proteins bind to first
and second target sites respectively, wherein a first
cleavage site lies between the first and second target
sites, and
the third and fourth fusion proteins bind to third and
fourth target sites respectively, wherein a second cleavage
site lies between the third and fourth target sites;
such that the first and second fusion proteins cleave the
DNA at the first cleavage site, and the third and fourth
fusion proteins cleave the DNA at the second cleavage
site; and
(b) contacting the cell with a donor polynucleotide,
wherein the donor polynucleotide comprises:
(i) sequences homologous to genomic
sequences flanking the first and second cleavage
sites; and
(ii) sequences that are non-homologous to
genomic sequences between the first and second
cleavage sites:
whereby genomic sequences between the first and second cleavage
sites are replaced by the non-homologous sequences of the donor
polynucleotide.

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The Examiner rejected (i) claims 1, 3-5, 8, 10, and 15-19 as
unpatentable under 35 U.S.C. § 103(a) over Carroll3 in view of Dujon;4 (ii)
claims 6 and 7 as unpatentable under 35 U.S.C. § 103(a) over Carroll in
view of Dujon, as applied to claims 1 and 3, and further in view of Zhou;5
and, (iii) claim 9 as unpatentable under 35 U.S.C. § 103(a) over Carroll in
view of Dujon, as applied to claims 1 and 8, and further in view of Jasin6
(Ans. 4-9).
The Examiner found that Carroll teaches methods for targeted DNA
deletions and targeted DNA insertions comprising cleaving DNA target loci
in a host cell/organism with a pair of fusion proteins called zinc finger
nucleases ("ZFNs") (id. at 4). Each ZFN contains a zinc finger DNA
binding domain that binds to a target site and a Fok I cleavage domain (id.).
The Examiner further found that Carroll teaches that the two cut DNA ends
may be rejoined such that sequences between the two cleavage points are
deleted or, in the presence of a donor polynucleotide, such that at least part
of the donor polynucleotide is integrated into the target DNA (id. at 4-5 and
12). The Examiner found that Carroll failed to teach using a second pair of

3 WO 03/087341 A2, Targeted Chromosomal Mutagenesis Using Zinc
Finger Nucleases, published 23 October 2003, by Carroll et al. ("Carroll").
Appellants did not challenge Carroll as prior art.
4 US Patent 5,948,678, Nucleotide Sequence Encoding the Enzyme I-SceI and
the Uses Thereof, issued 7 September 1999, to Dujon et al. ("Dujon").
5 Zhou et al., Impaired Macrophage Function and Enhanced T Cell-
Dependent Immune Response in Mice Lacking CCR5, the Mouse Homologue
of the Major HIV-1 Coreceptor, 160 THE JOURNAL OF IMMUNOLOGY 4018-
4025 (1998) ("Zhou").
6 Jasin et al., Gene targeting at the human CD4 locus by epitope addition, 4
GENES & DEVELOPMENT 157-166 (1990) ("Jasin").
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7
ZFNs that cleaved a target genomic sequence at a second site on the same
chromosome as claimed (id. at 5).
The Examiner found that Dujon teaches that forming two DSBs, i.e.,
four cut DNA ends, with a restriction endonuclease can result in deletion of
chromosomal sequences or stimulation of HR at increased frequencies of at
least 100-fold and 500-fold, respectively, than occur spontaneously (id. at 5-
6, 13, and 16).
The Examiner concluded that it would have been obvious to one of
ordinary skill in the art to modify the methods of Carroll by introducing a
second DSB, i.e., using four ZFNs working as two pairs, as taught by Dujon
to obtain the expected benefit of increased frequencies of deletions and HRs
as taught by Dujon (id. at 6, 13, 18, and 21).
According to the Examiner, the predictability of the two DSB sites is
based on the design of the ZFNs to endogenous chromosomal target sites as
taught by Carroll (id. at 14-18). In other words, "[t]here are no apparent
differences between the zinc finger nucleases taught by Carroll … and those
used in the claimed methods…. [T]he combination of Carroll … and Dujon
… requires only the predictable use of prior art elements in a manner
consistent with their known function" (id. at 15).
Finding that Dujon teaches the introduction of Sce-I restriction sites
into genomic cellular DNA, the Examiner further stated that one of ordinary
skill in the art would have been motivated to use the ZFNs of Carroll in
place of the Sce-I endonuclease of Dujon to eliminate the need to insert Sce-
I sites into the genomic DNA in order for cleavage to occur (id. at 19), i.e.,
Carroll teaches the design of ZFNs to known sequences (id. at 20).
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Appellants argue that using four ZFNs that cleave their targets by
forming two dimers in order to make two cuts, i.e., DSBs, in genomic
cellular chromatin/DNA such that the cleaved DNA ends are rejoined or
such that targeted replacement occurs between the cleavage sites is not
predictable in light of Carroll's use of two ZFNs and of Dujon's use of one
restriction enzyme (App. Br. 6-9; Reply Br. 3-4). According to Appellants,
"the Examiner has provided no evidence for the assertion that substitution of
four engineered zinc finger nucleases is a predictable substitution for a
single naturally occurring enzyme" (App. Br. 9, original emphasis).
Appellants contend that Carroll appears to reference Dujon in specifically
teaching that one cut is sufficient and two cuts unnecessary (App. Br. 10-11;
Reply Br. 5). Based on this contention, Appellants argue that a skilled
artisan would have had no motivation to look to Dujon for motivation to
make two cuts when one cut was more than sufficient to facilitate HR and/or
promote NHEJ (App. Br. 11; Reply Br. 5).
According to Appellants, "the issue is not whether, once cleavage has
occurred, one or two cuts 'act in the same predictable manner.' Rather, the
issue is how those cuts are made … Carroll teaches nothing about how 4
ZFNs (2 pairs) would be able to dimerize with the appropriate partner and
cut in the same region" (Reply Br. 6). In essence, Appellants argue that
Carroll and Dujon use different mechanisms to cleave DNA (App. Br. 9-10).
Appellants further argue that Dujon's inserted sites are not genomic
cellular chromatin and are not binding sites for ZFNs (App. Br. 7; Reply Br.
6-7). To wit, Dujon is describing a nuclease that acts on circular DNA
targets and, therefore, does not establish any predictability vis-à-vis DNA
targets packaged in chromatin (App. Br. 8-9).
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Appellants still further argue that since both Zhou and Janis fail to
teach or suggest using four ZFNs to make two cuts in genomic cellular
chromatin as claimed, the rejections of claims 6, 7, and 9 cannot be
sustained (App. Br. 11-12; Reply Br. 7-8).
Since Appellants have not separately argued the patentability of any
of the dependent claims, we decide this appeal on the basis of the
independent claims, claims 1, 15, and 19. 37 C.F.R. § 41.37(c)(1)(vii).
Consequently, a discussion of Zhou and Jasin is not necessary to our
decision.
At issue, is whether the evidence of record teaches or suggests that
one of ordinary skill in the art would have been motivated to use four ZFNs
to introduce two DSBs in double-stranded/genomic DNA so as to selectively
alter a target sequence in the double-stranded/genomic DNA, by deletion or
by HR, as claimed with a reasonable expectation of success in so doing.
II. Findings of Fact
The following findings of fact ("FF") are supported by a
preponderance of the evidence of record.
A. Dujon
[1] Dujon discloses using I-SceI for in vivo site directed genetic
recombination (Dujon col. 3, ll. 65-67).
[2] Example 5 of Dujon describes transfecting the G-MtkPL provirus into
NH3T3 mammalian cells to insert I-Sce I sites into the genome of a
host cell (id. at col. 30, ll. 59-col. 31, l. 47).
[3] According to Dujon, the results of Example 5 demonstrated that I-Sce
I induces chromosomal recombination in mammalian cells, suggested
that I-Sce I is able to cut in vivo a chromosome at a predetermined
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target, and showed that the presence of two I-Sce I sites in a proviral
target and the expression of the I-Sce I lead to an increase in the
deletion of a tk gene at a frequency at least 100 fold greater than that
occurring spontaneously (id. at col. 33, ll. 28-58; col. 34, ll. 16-20;
Figs. 27A-27C).
[4] According to Dujon, the results of Example 5 indicate an intra-
chromosomal recombination between the two LTRs (id. at col. 33, ll.
49-52; Fig. 27A-27C).
[5] Example 4 of Dujon describes introducing duplicated I-Sce I
recognition sites separated by 5.8 or 7.2 kbp on a chromosome in each
LTR of proviral structures in NH3T3 fibroblasts and PCC7-S
multipotent mouse cell lines by retrovirus integration (id. at col. 25, ll.
47-67; col. 26, ll. 21-65; col. 29, ll. 44-50), followed by cotransfecting
with an expression vector for I-Sec I and a donor plasmid pVRneo (id.
at col. 27, ll. 38-45).
[6] According to Dujon, "expression of I-Sce I induces HR between pVR
neo and the proviral site and that site directed HR is ten times more
frequent than random integration of pVR neo near a cellular promoter,
and at least 500 times more frequent than spontaneous HR" (id. at col.
27, ll. 62-67).
[7] Dujon concludes that "in presence of I-Sce I, the donor vector
recombines very efficiently with sequences within the two LTRs to
produce a functional neogene. This suggests that I-Sce I induced very
efficiently double strand breaks in both I-Sce I sites." (Id. at col. 29, ll.
50-54).
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[8] Dujon enumerates a number of variations of its described gene
replacement protocol, including site specific gene insertions and
specific deletions of a chromosomal fragment at predetermined
locations defined by previous integration of two or more I-Sce I sites
(id. at 34:57-38:39).
B. Carroll
[9] By way of background, Carroll notes that "[i]t has been demonstrated
in model experiments that introduction of a double-strand break
(DSB) in host DNA greatly enhances the frequency of localized
recombination. However, those tests required insertion of a
recognition site for a specific endonuclease before cleavage could be
induced." (Carroll 1:20-23.)
[10] Carroll further notes that "[t]argeted genetic recombination or
mutation of a cell or organism is now possible because complete
genomic sequences have been determined for a number of organisms,
and more sequences are being obtained each day" (id. at 1:34-2:1).
[11] Carroll discloses methods of altering genome sequences using HR and
NHEJ recombination processes for targeted DNA deletions and
insertions (id. at 5:1-11; 8:16-24; 10:4-13; 14:11-31).
[12] According to Carroll, "[a]ny segment of endogenous nucleic acid in a
cell or organism can be modified by the method of the invention as
long as the sequence of the target region, or portion of the target
region, is known, or if isolated DNA homologous to the target region
is available" (id. at 2:10-13).
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[13] Specifically, Carroll's method involves use of chimeric zinc finger
nucleases ("ZFNs") and, in some embodiments, donor polynucleotides
(donor DNA) (id. at 5:11-14).
[14] According to Carroll,
[a] zinc finger nuclease … is a chimeric
protein molecule capable of directing targeted
genetic recombination or targeted mutation in a
host cell by causing a double stranded break
(DSB) at the target locus. A ZFN … includes a
DNA-binding domain and a DNA-cleavage
domain, wherein the DNA binding domain is
comprised of at least one zinc finger and is
operably linked to a DNA-cleavage domain. The
zinc finger DNA-binding domain is at the N-
terminus of the chimeric protein molecule and the
DNA-cleavage domain is located at the C-terminus
of said molecule. [Id. at 10:30-11:2.]
* * * * * *
In order to target genetic recombination or
mutation according to a preferred embodiment …,
two 9 bp zinc finger DNA recognition sequences
must be identified in the host DNA. These
recognition sites will be in an inverted orientation
with respect to one another and separated by about
6 bp of DNA. ZFNs are then generated by
designing and producing zinc finger combinations
that bind DNA specifically at the target locus, and
then linking the zinc fingers to a cleavage domain
of a Type II restriction enzyme. [Id. at 12:18-24.]

[15] A preferred ZFN comprises three zinc fingers and a DNA cleavage
domain from Fok I (id. at 11:26-28); and, requires dimerization of two
ZFN DNA-cleavage domains for effective cleavage of double-
stranded DNA (id. at 11:30-32).
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[16] A donor DNA is a DNA sequence with sufficient homology to the
region of the target locus to allow participation in HR at the site of the
DSB (id. at 9:17-21).
III. Discussion
A. Legal principles
"The consistent criterion for determination of obviousness is whether
the prior art would have suggested to one of ordinary skill in the art that this
process should be carried out and would have a reasonable likelihood of
success, viewed in the light of the prior art." In re Dow Chem. Co., 837 F.2d
469, 473 (Fed. Cir. 1988). In determining whether obviousness is
established by combining the teachings of the prior art, "the test is what the
combined teachings of the references would have suggested to those of
ordinary skill in the art." In re Keller, 642 F.2d 413, 425 (CCPA 1981).
Furthermore, the "[obviousness] analysis need not seek out precise teachings
directed to the specific subject matter of the challenged claim, for a court
can take account of the inferences and creative steps that a person of
ordinary skill in the art would employ." KSR Int'l Co. v. Teleflex Inc., 550
U.S. 398, 418 (2007). A person of ordinary skill must be presumed to have
some skill, In re Sovish, 769 F.2d 738, 743 (Fed. Cir. 1985), and ordinary
creativity, KSR, 550 U.S. at 418.
All that is required for obviousness under 35 U.S.C. § 103 is a
reasonable expectation of success. In re O'Farrell, 853 F.2d 894, 904 (Fed.
Cir. 1988).
B. Discussion
Here, we agree with the Examiner that it would have been obvious to
one of ordinary skill in the art to modify the methods of Carroll by
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introducing a second DSB as taught by Dujon to increase the frequency of
localized genomic recombination with a reasonable expectation of success
based on the combined disclosures of Carroll and Dujon.
Both Carroll and Dujon teach methods of targeted alterations of
genome sequences using HR and NHEJ recombination processes to repair
DSBs made at predetermined locations in genomic DNA by endonucleases
(FF 1, 3, 4, 6, 7, 11, 12, 14). Both Carroll and Dujon teach that it is the
recognition site of an endonuclease that determines where the DNA will be
cut by the endonuclease. Dujon inserts exogenous I-Sce I recognition sites
for the endonuclease I-Sce I in a target region; Carroll relies on endogenous
recognition sites already present in a target region. (FF 2, 3, 7, 12-14.)
Thus, both Carroll and Dujon induced DSBs in target regions of genomic
sequences using the same generic mechanism albeit with different
endonucleases. As noted by the Examiner (Ans. 20), Carroll teaches
designing ZFNs to selectively cleave a known genomic sequence at a
specific location (FF 14). As also noted by the Examiner (Ans. 5-6, 13, 16),
Dujon teaches that forming two DSBs with a restriction enzyme can result in
deletion of chromosomal sequences or stimulation of HR at increased
frequencies of at least 100-fold and 500-fold, respectively (FF 3, 6). Thus, it
reasonably appears that a cell or organism would increase its DSB repairs,
i.e., HR and/or NHEJ recombination processes, in response to DSBs.
Indeed, Appellants themselves argue that the issue is not whether what
happens after the DNA cuts have occurred, but rather how the cuts
themselves are made (Reply Br. 6).
It is well known and predictable that an endonuclease will cleave at its
art-recognized restriction site (see e.g., Dujon and Carroll). As noted by the
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Examiner (Ans. 15), there are no apparent differences between the ZFN
endonucleases used by Carroll and the claimed methods. For example, the
981 Specification explains that the zinc finger DNA binding domain of each
pair straddle a cleavage site separated by a number of base pairs compatible
with dimerizing their respective "half" cleavage domains and that a preferred
"half" cleavage domain is a Fok I cleavage domain (Spec. 2, ¶ 6, to 7, ¶ 3; 9,
¶ 3, to 10, ¶ 3; 24, ¶ 4, to 36, ¶ 2; 64, ¶ 1). Similarly, Carroll teaches
designing ZFNs to bind to recognition sites in an inverted orientation with
respect to one another and separated by about 6 bp (FF 14) and the necessity
to dimerize two Fok I DNA cleavage domains for effective cleavage of
double-stranded DNA (FF 15). Thus, both Appellants' and Carroll's ZFN
pairs dimerize Fok I cleavage domains to induce a DSB at an Fok I cleavage
site straddled by zinc finger DNA binding domains in an inverted orientation
with respect to each other. The only apparent difference between the
methods of Carroll and the claimed methods is the number of DSBs
introduced into the genomic DNA, one versus two, respectively.
However, Dujon teaches that forming two DSBs with a restriction
enzyme can result in deletion of chromosomal sequences or stimulation of
HR at increased frequencies of at least 100-fold and 500-fold, respectively
(FF 3, 6). Forming two DSBs requires the use of two pairs of ZFNs.
Therefore, the weight of the evidence of record favors the conclusion that
one of ordinary skill in the art would have been motivated to modify the
methods of Carroll by using two pairs of ZFNs with a reasonable
expectation of success that in so doing the frequency of repair, i.e., of HR
and NHEJ processes, would increase. Appellants' arguments do not
persuade us otherwise.
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Appellants misunderstand the Examiner's position when they argue
that the Examiner has provided no evidence showing that four engineered
ZFNs is a predictable substitute for a single naturally occurring enzyme
(App. Br. 9). What is predictable is that an endonuclease will cleave at its
restriction site, whether that endonuclease is I-Sce I or a ZFN. What is
predictable is that if one pair of ZFNs is needed to induce one DSB, two
pairs of ZFNS are needed to induce two DSBs. What is predictable is that
two DSBs results in an increased recombination repair response by a cell as
taught and/or suggested by Dujon.
The argument that Carroll specifically teaches that one cut is
sufficient and two cuts unnecessary is similarly unpersuasive (App. Br. 10-
11; Reply Br. 5). To the extent Carroll only exemplifies methods
comprising inducing a single DSB, we note that a prior art reference must be
considered in its entirety and is not limited to preferred embodiments or
specific working examples. In re Lamberti, 545 F.2d 747, 750 (CCPA
1976). Furthermore, in determining whether obviousness is established by
combining the teachings of the prior art, "the test is what the combined
teachings of the references would have suggested to those of ordinary skill
in the art," Keller, 642 F.2d at 425, and here this argument ignores the
combined teachings of Carroll and Dujon. Similarly, Appellants' arguments
attacking Dujon individually, e.g., Dujon's inserted sites are not binding sites
for ZFNs, ignores the combined teachings of Carroll and Dujon and are
likewise unpersuasive.
Therefore, the weight of the evidence of record supports the
Examiner's conclusion of obviousness.
C. Conclusion
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We will sustain the rejection of claims 1, 15, and 19, as well as the
claims dependent thereon, under § 103(a) over the combined teachings of
Carroll and Dujon, alone or further in view of Zhou or Jasin.
The evidence of record supports a conclusion that one of ordinary
skill in the art would have been motivated to use four ZFNs to introduce two
DSBs in double-stranded/genomic DNA so as to selectively alter a target
sequence in the double-stranded/genomic DNA, by deletion or by HR, as
claimed with a reasonable expectation of success in so doing based on the
combined teachings of Carroll and Dujon.
IV. Order
Upon consideration of the record, and for the reasons given, it is
ORDERED that the decision of the Examiner to reject claims 1, 3-5,
8, 10, and 15-19 as unpatentable under 35 U.S.C. § 103(a) over Carroll in
view of Dujon is AFFIRMED;
FURTHER ORDERED that the decision of the Examiner to reject
claims 6 and 7 as unpatentable under 35 U.S.C. § 103(a) over Carroll in
view of Dujon, as applied to claims 1 and 3, and further in view of Zhou is
AFFIRMED;
FURTHER ORDERED that the decision of the Examiner to reject
claim 9 as unpatentable under 35 U.S.C. § 103(a) over Carroll in view of
Dujon, as applied to claims 1 and 8, and further in view Janis is
AFFIRMED; and,
FURTHER ORDERED that no time period for taking any subsequent
action in connection with this appeal may be extended under 37 C.F.R. §
1.136(a)(1)(vi).
AFFIRMED
Appeal 2010-001995
Application 11/304,981

18

cdc

ROBINS & PASTERNAK
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