Ex Parte Liljedahl et al

Board of Patent Appeals and Interferences

Sep 21, 2010

10395816 (B.P.A.I. Sep. 21, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte MONIKA LILJEDAHL, SIMON ERIC ASPLAND, and
DAVID J. SEGAL
__________

Appeal 2010-001206
Application 10/395,816
Technology Center 1600
__________

Before ERIC GRIMES, TONI R. SCHEINER, and LORA M. GREEN,
Administrative Patent Judges.

GRIMES, Administrative Patent Judge.

DECISION ON APPEAL1
This is an appeal under 35 U.S.C. § 134 involving claims to a method
of generating a genetically modified cell. The Examiner has rejected the

1 The two-month time 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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claims for obviousness. We have jurisdiction under 35 U.S.C. § 6(b). We
affirm.
STATEMENT OF THE CASE
The Specification discloses that “[b]reaking DNA using site specific
endonucleases can increase the rate of homologous recombination in the
region of the breakage” (Spec. 4, ¶ 0014). The Specification discloses a
method of genetically modifying a cell by “providing a zinc finger
endonuclease (ZFE) that includes an endonuclease domain that cuts DNA,
and a zinc finger domain that includes a plurality of zinc fingers that bind to
a specific nucleotide sequence within the endogenous chromosomal target
DNA” (id. at 6, ¶ 0019).
Claims 1-15, 37 and 38 are on appeal. Claim 1 is the only
independent claim on appeal and reads as follows:
1. A method of generating an isolated genetically modified cell,
comprising:
providing an isolated cell containing an endogenous chromosomal
target DNA sequence in which it is desired to have homologous
recombination occur;
providing a zinc finger endonuclease comprising an endonuclease
domain that cuts DNA, and a non-naturally-occurring zinc finger domain
comprising a plurality of zinc fingers that bind to a specific nucleotide
sequence within said endogenous chromosomal target DNA in said cell;
contacting said endogenous chromosomal target DNA sequence with
said zinc finger endonuclease in said cell such that said zinc finger
endonuclease cuts both strands of a nucleotide sequence within said
endogenous chromosomal target DNA sequence in said cell, thereby
enhancing the frequency of homologous recombination in said endogenous
chromosomal target DNA sequence; and
providing a nucleic acid comprising a sequence homologous to at least
a portion of said endogenous chromosomal target DNA such that
homologous recombination occurs between said endogenous chromosomal
target DNA sequence and said nucleic acid.
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Issue
The Examiner has rejected claims 1-3, 5-9, and 37 under 35 U.S.C.
§ 103(a) as being obvious in view of Bibikova2 and Pabo 3 (Answer 3). The
Examiner has also rejected claims 1-15, 37, and 38 under 35 U.S.C. § 103(a)
as being obvious in view of Bibikova, Pabo, and Choulika4 (Answer 5). The
same issue is dispositive with respect to both rejections so we will discuss
them together. In addition, the claims have not been argued separately and
the dependent claims therefore stand or fall with independent claim 1. 37
C.F.R. § 41.37(c)(1)(vii).
The Examiner finds that Bibikova discloses that a zinc finger
endonuclease (ZFE) cleaves a target site on a plasmid in a Xenopus oocyte
and allows homologous recombination to occur (Answer 3). The Examiner
also finds that Bibikova suggests targeting chromosomal DNA with an
engineered ZFE and a linear donor DNA for homologous recombination
(id.). The Examiner finds that Pabo discloses “‘designer’ zinc finger
domains [that] had increased affinity and binding” (id.).
The Examiner concludes that “it would have been obvious to those of
ordinary skill in the art … to genetically modify a cell using a ZFE as
described by Bibikova wherein the zinc finger domain of the ZFE was
‘designer’” (id. at 5). The Examiner reasons that “those of ordinary skill in
the art … would have been motivated to replace the zinc finger domain of

2 Marina Bibikova et al., Stimulation of Homologous Recombination through
Targeted Cleavage by Chimeric Nucleases, 21 MOLECULAR AND CELLULAR
BIOLOGY 289-297 (2001)
3 Carl O. Pabo et al., Design and Selection of Novel Cys2His2 Zinc Finger
Proteins, 70 ANNU. REV. BIOCHEM. 313-340 (2001)
4 Choulika et al., US 2002/0107214 A1, Aug. 8, 2002
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Bibikova with the ‘designer’ zinc finger domain of Pabo to increase affinity
and binding of the zinc fingers to the target sequence” (id.).
Appellants contend that the cited references do not suggest contacting
an endogenous chromosomal target DNA sequence within a cell with a non-
naturally occurring ZFE to cut both strands of the target DNA sequence,
with a reasonable expectation of success, because Bibikova does not show
cleavage of endogenous target DNA (Appeal Br. 6), the zinc finger domain
of Bibikova is not “non naturally occurring” (id.), and Pabo does not
specifically disclose or suggest non-naturally occurring zinc finger domains
in conjunction with an endonuclease (id. at 8).
The issue presented is: Does the evidence of record support the
Examiner’s conclusion that it would have been obvious to contact an
endogenous chromosomal target DNA sequence within a cell with a non-
naturally occurring ZFE, with a reasonable expectation that the ZFE would
cut the target DNA sequence?
Findings of Fact
1. Bibikova discloses “chimeric nucleases for DNA cleavage and
initiation of recombination in living cells. These enzymes are hybrids
between the nonspecific cleavage domain of the type IIs restriction
endonuclease FokI and a DNA-binding domain made up of three Cys2His2
zinc fingers” (Bibikova, 289).
2. Bibikova discloses that “DNA substrates and the nucleases were
injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and
subsequent homologous recombination were observed” (id., abstract).
3. Bibikova discloses that “[c]leaved DNA molecules were activated
for homologous recombination; in optimum conditions, essentially 100% of
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the substrate recombined, even though the DNA was assembled into
chromatin” (id.).
4. Bibikova discloses that the “[i]njected circular DNAs are
assembled into apparently normal chromatin and are inert for recombination,
but they can be induced to interact with a homologous partner, if they are
cleaved.… A circular DNA thus serves as an effective model for an inactive
chromosomal target.” (Id. at 290.)
5. Bibikova discloses that “[b]ecause the recognition specificity of
zinc fingers can be altered experimentally, this approach holds great promise
for inducing targeted recombination in a variety of organisms” (id. at
abstract).
6. Pabo discloses that “Cys2His2 zinc finger proteins offer a stable
and versatile framework for the design of proteins that recognize desired
target sites on double-stranded DNA” (Pabo, abstract).
7. Pabo discloses that “selection strategies have been developed that
allow these proteins to be targeted to almost any desired site on double-
stranded DNA” (id.).
8. Pabo discloses that “[t]hese new proteins can then be modified by
adding other domains—for activation or repression of transcription, for
DNA cleavage, or for other activities” (id.).
Principles of Law
“Obviousness does not require absolute predictability of success…
For obviousness under § 103, all that is required is a reasonable expectation
of success.” In re O’Farrell, 853 F.2d 894, 903-04 (Fed. Cir. 1988).
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Analysis
Claim 1 is directed to a method for genetically modifying a cell using
a zinc finger endonuclease comprising a non-naturally-occurring zinc finger
domain that binds to and cuts a target sequence in the chromosomal DNA,
enhancing the frequency of homologous recombination between the target
sequence and a homologous nucleic acid.
Bibikova discloses the use of zinc finger endonucleases (ZFEs) to
target and cleave plasmid DNA with the result that homologous
recombination was promoted. Bibikova also discloses that the circular
plasmid DNA is an effective model for a chromosomal target DNA, and
suggests that its approach could be used to induce targeted recombination
based on altering the recognition specificity of zinc fingers. Pabo discloses
that zinc finger proteins can be targeted to almost any desired site in double-
stranded DNA and can be modified by addition of a DNA cleavage domain.
In view of these disclosures, it would have been obvious to one of ordinary
skill in the art to construct a zinc finger endonuclease comprising a non-
naturally-occurring zinc finger domain, as suggested by Pabo, that binds to
an endogenous chromosomal target DNA and to use the ZFE to promote
homologous recombination within a cell, as suggested by Bibikova.
Appellants argue that the cited references do not provide a reasonable
expectation of success for cleaving an endogenous chromosomal target DNA
sequence within a cell with a non-naturally occurring ZFE because Bibikova
does not show cleavage of endogenous target DNA (Appeal Br. 6-7).
This argument is not persuasive. Although Bibikova discloses the
cleavage of plasmid, not chromosomal, DNA with a ZFE, Bibikova
discloses that the plasmid “DNAs are assembled into apparently normal
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chromatin.… A circular DNA thus serves as an effective model for an
inactive chromosomal target.” (FF 4.) Bibikova’s disclosure that its plasmid
DNA was “an effective model” of a chromosomal target would have
provided a skilled worker with a reasonable expectation that a ZFE would
cleave a target sequence in endogenous chromosomal DNA. In accord with
In re O’Farrell, obviousness does not require an absolute probability of
success but only a reasonable expectation of success.
Appellants also argue that the cited references do not provide a
reasonable expectation of success because the zinc finger domain of
Bibikova is not “non naturally occurring” and Pabo does not specifically
disclose or suggest non-naturally occurring zinc finger domains in
conjunction with an endonuclease (Appeal Br. 8). Along the same line,
Appellants argue that “neither Bibikova nor Pabo teach or suggest cleavage
using zinc finger nucleases comprising a non-naturally occurring zinc finger
domain” (Reply Br. 4).
These arguments are not persuasive. Although neither of the cited
references expressly discloses a ZFE having a non-naturally occurring zinc
finger domain, they nonetheless would have made obvious such a ZFE
because Pabo discloses that zinc finger proteins can be targeted to almost
any desired site (FF 7) and Bibikova states that altering the recognition
specificity of zinc fingers was a promising approach for inducing targeted
recombination (FF 5). 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).
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We also do not agree that the lack of an express disclosure of a ZFE
combined with a non-naturally occurring zinc finger domain would have
cast doubt on the expectation of success in combining the references. Given
the demonstrated success in Bibikova of a ZFE cleaving an effective model
of chromosomal DNA and Pabo’s disclosure of designing zinc finger
proteins to target a particular DNA sequence, one of skill in the art would
have had a reasonable expectation of successfully constructing a ZFE with a
non-naturally occurring zinc finger domain that targets and cleaves
chromosomal DNA.
Appellants also assert that certain references cited in Pabo are
evidence of unpredictability in the design of zinc finger transcription factors
that target chromosomal DNA (Appeal Br. 9-10; Reply Br. 6-8). However,
Appellants have not persuasively explained why these references, which
concern zinc finger transcription factors, should be given greater evidentiary
weight than Bibikova, which specifically discloses that a zinc finger
endonuclease efficiently binds to and cleaves an effective model of
chromosomoal DNA in a cell. Appellants therefore have not established that
the cited references provide a basis for doubting the reasonable expectation
of success based on Bibikova and Pabo with respect to the invention of
claim 1.
Conclusion of Law
The evidence of record supports the Examiner’s conclusion that it
would have been obvious to contact an endogenous chromosomal target
DNA sequence within a cell with a non-naturally occurring ZFE, with a
reasonable expectation that the ZFE would cut the target DNA sequence.
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SUMMARY
We affirm the rejection of claims 1-15, 37 and 38 under 35 U.S.C.
§ 103(a).
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED

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