Ex Parte Liljedahl et alDownload PDFPatent Trial and Appeal BoardMar 2, 201610395816 (P.T.A.B. Mar. 2, 2016) Copy Citation UNITED STA TES p A TENT AND TRADEMARK OFFICE APPLICATION NO. FILING DATE 10/395,816 0312012003 20855 7590 03/02/2016 PASTERNAK PATENT LAW 1900 EMBARCADERO ROAD SUITE 211 PALO ALTO, CA 94303 FIRST NAMED INVENTOR Monika Liljedahl 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 ATTORNEY DOCKET NO. CONFIRMATION NO. 8325-3003 3642 EXAMINER WILSON, MICHAEL C ART UNIT PAPER NUMBER 1632 MAILDATE DELIVERY MODE 03/02/2016 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 PATENT TRIAL AND APPEAL BOARD Ex parte MONIKA LILJEDAHL, SIMON ERIC ASPLAND, and DAVID J. SEGAL 1 Appeal2013-005228 Application 10/395,816 Technology Center 1600 Before TONI R. SCHEINER, ERIC B. GRIMES, and LORA M. GREEN, Administrative Patent Judges. GRIMES, Administrative Patent Judge. DECISION ON APPEAL 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 claims for obviousness. We have jurisdiction under 35 U.S.C. § 6(b ). We affirm. STATEMENT OF THE CASE This application was the subject of a previous appeal (2010-001206, decided Sept. 21, 2010), in which the Examiner's rejection was affirmed. 1 Appellants identify the Real Party in Interest as Sangamo BioSciences, Inc. (Appeal Br. 2.) Appeal2013-005228 Application 10/395,816 After prosecution was resumed, Appellants amended the claims and introduced new evidence, the Examiner rejected the amended claims, and this appeal followed. The opinion in Appeal 2010-001206 summarizes the background of the invention. Claims 1, 7-9, 11, 12, 15, 37, and 38 are on appeal. Claim 1 is illustrative and reads as follows: 1. A method of generating an isolated genetically modified plant or mammalian cell, comprising: providing an isolated cell containing an endogenous chromosomal target DNA sequence in which it is desired to have homologous recombination occur; providing at least one expression vector that expresses first and second zinc finger endonucleases, each zinc finger endonuclease comprising a Fok! cleavage 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 wherein the first and second zinc finger endonuclease form a dimer which 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. 2 Appeal2013-005228 Application 10/395,816 DISCUSSION Issue The Examiner has rejected all of the claims on appeal under 35 U.S.C. § 103(a) as obvious based on Bibikova, 2 Pabo, 3 and Choulika. 4 (Ans. 2.) The Examiner finds that Bibikova discloses a method of modifying a target DNA sequence in a cell using a zinc finger endonuclease (ZFE) meeting the limitations of claim 1, and discloses that two endonucleases must dimerize to cut the target DNA. (Id.) The Examiner also finds that Bibikova's target DNA is a model of an inactivated chromosomal target (id.) and that Bibikova discloses how to target endogenous chromosomal DNA (id. at 3). The Examiner finds that Pabo discloses zinc finger domains comprising five or more zinc fingers and discloses that "'designer' zinc finger domains had increased affinity and binding." (Id. at 4.) The Examiner finds that Choulika discloses cleaving a chromosomal target DNA using ZFEs in plant or mammalian cells. (Id. at 6.) The Examiner concludes that it would have been obvious to those of ordinary skill in the art to use a ZFE with 5 zinc fingers to target endogenous chromosomal DNA as described by the combined teachings of Bibikova and Pabo wherein the ZFE is introduced into a plant or mammalian cell using a vector encoding the ZFE as described by Choulika. 2 Bibikova et al., Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases, 21 MOLECULAR AND CELLULAR BIOLOGY 289--297 (2001 ). 3 Carl 0. 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 Al, published Aug. 8, 2002. 3 Appeal2013-005228 Application 10/395,816 (Id.) The Examiner also concludes that it would have been obvious "to replace the ZFE protein with a vector encoding the ZFE because Choulika taught the two were interchangeable." (Id.) Appellants contend that the references do not teach or suggest targeting an endogenous gene in a plant or mammalian cell. (Appeal Br. 6- 15, 18-20.) Appellants also contend that the references do not teach or suggest introducing dimerizing ZFEs in polynucleotide form. (Id. at 15-17.) Finally, Appellants contend that the Examiner has improperly dismissed their declaratory evidence. (Id. at 20-23.) The issue presented is whether a preponderance of the evidence of record supports the Examiner's conclusion that the method of claim 1 would have been obvious to a person of ordinary skill in the art based on Bibikova, Pabo, and Choulika. 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 Fokl and a DNA-binding domain made up of three Cys2His2 zinc fingers." (Bibikova 289, right col.) 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. at 289, abstract.) 4 Appeal2013-005228 Application 10/395,816 3. Bibikova states that "[ s ]pecific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain." (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, right col.; reference citation omitted.) 5. Bibikova discloses that"[ c ]leaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin." (Id. at 289, abstract.) 6. Bibikova states that its results show[] that these chimeras are capable of locating their target sequences in chromatin, cleaving \~1ith good efficiency, and thereby stimulating homologous recombination. Although the substrates we used were engineered plasmid DNAs and the cellular milieu was that of the Xenopus oocyte, our findings should be applicable to mammalian somatic cells and to many other cells and organisms. (Id. at 295, left col.) 7. Bibikova states that "[ t ]he method of delivery [of the chimeric nucleases and DNA] would depend on the organism, cell type, and other experimental conditions." (Id. at 296, left col.) 8. Bibikova states that "[ s ]ince each set of three fingers binds nine consecutive base pairs, two chimeric nucleases effectively demand an 18-bp target if each zinc finger domain has perfect specificity. Any given sequence 5 Appeal2013-005228 Application 10/395,816 of this length is predicted to be unique in a DNA as complex as the human genome." (Id.) 9. Bibikova states that [ s ]everal additional issues remain to be addressed to confirm the utility of chimeric nucleases as tools for gene targeting. Among these are demonstrating discrimination against related sequences; proving the efficacy of zinc fingers designed to bind arbitrarily chosen sequences; and testing the cleavage of genuine chromosomal targets. The question of discrimination among potential binding sites is a particularly critical one. (Id. at 296, right col.) 10. Bibikova states that its results "show[] that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because 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 289, abstract.) 11. 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 313, abstract.) 12. 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.) 13. 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.) 6 Appeal2013-005228 Application 10/395,816 14. Choulika discloses a method of repairing a specific sequence of interest in chromosomal DNA of a cell comprising (a) inducing in the cell a double stranded break at a site of interest, and (b) introducing into the cell targeting DNA, wherein the targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which repairs the specific sequence of interest upon recombination between the targeting DNA and the chromosomal DNA. (Choulika 3 ii 26.) 15. Choulika states that "[ d]ouble stranded breaks at a site of interest can also be achieved by the chimeric restriction endonucleases ... described herein." (Id. at 2 ii 21.) 16. Choulika states that "[ fJor application of the method to the manipulation of any chromosomal DNA locus, chimeric restriction endonucleases generated by the juxtaposition of specific DNA binding sequences (in some cases generated by the linking of specific zinc finger binding domains) and DNA cleavage domains can be used to elicit cleavage." (Id. at 3 ii 25.) 17. Choulika states that a "model chromosomal loc[ us] was generated in which a site for the meganuclease I-Seel was introduced within the target region for recombination, and double stranded DNA cleavage via introduction of a vector encoding the restriction endonuclease was induced." (Id.) 18. Choulika states that "DNA binding sequences include zinc finger binding domains and meganuclease recognition sites. . . . In [one] embodiment, the chimeric restriction endonuclease is produced by joining a 7 Appeal2013-005228 Application 10/395,816 I-Seel meganuclease recognition site and the FokI cleavage domain." (Id. at 5 ii 42.) 19. Choulika states that "[t]argeting DNA and/or restriction endonucleases introduced into a cell or individual as described above can be inserted in a vector." (Id. at 5 if 44.) "A vector comprising targeting DNA and/or nucleic acid encoding a restriction endonuclease can also be introduced into a cell." (Id. at 6 if 49.) 20. Choulika provides a working example that describes cotransfecting NIH 3 T3 Gap 1 and NIH 3 T3 Gap2 cell lines with two vectors ("Expression Plasmid" and "Repair Matrix"). (Id. at 8 iii! 75, 76; Table 1.) 21. The NIH 3 T3 Gap 1 and NIH 3 T3 Gap2 cells had been modified to include a plasmid (pPytknBWSacZ) integrated into their genomic DNA. (Id. at 8, if 74: "[A]nalysis of the mRNA expressed by the integrated pPytknBWSacZ construct showed no expression for one of the clones and signals for the other two clones. These two cell lines, NIH 3T3 Gap 1 and NIH 3T3 Gap2, were selected to be the targets to the gap repair.") 22. Choulika states that "[t]ransfection ofNIH 3T3 Gapl cells with the mix number 1 (pCMV I-Seel(+), 9 µg; pSlacB, 1 µg) gave a 12 to 28% of B-galactosidase positive clones (out of the three experiments) as the higher rate of gap repair recombination of the pPytknBWSacZ deleted plasmid." (Id. at 8 if 79.) Analysis We agree with the Examiner that the cited references would have made obvious a method meeting the limitations of claim 1. Bibikova discloses "chimeric nucleases for DNA cleavage and initiation of 8 Appeal2013-005228 Application 10/395,816 recombination in living cells" (FF 1 ). Bibikova states that its chimeric nucleases are made up of a Fokl cleavage domain and a zinc finger DNA- binding domain (FF 1 ). Bibikova discloses that specific cleavage required dimerization of the cleavage domain (FF3). Bibikova states that "these chimeras are capable of locating their target sequences in chromatin, cleaving with good efficiency, and thereby stimulating homologous recombination" (FF6). Bibikova states that "[a ]lthough the substrates we used were engineered plasmid DNAs and the cellular milieu was that of the Xenopus oocyte, our findings should be applicable to mammalian somatic cells and to many other cells and organisms" (FF6). Bibikova also states that its results "show[] that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. . . . [T]his approach holds great promise for inducing targeted recombination in a variety of organisms" (FF 10). Bibikova states that zinc finger domains like the one it used bind to 9- bp recognition sites, so two such DNA-binding domains require an 18-bp target, which is expected to be unique in the human genome (FF8). Similarly, Pabo states that zinc finger proteins can be designed to target a desired site in double-stranded DNA (FFl 1) and that they can be modified by adding a DNA cleavage domain (FF13). Choulika discloses a vector (FF 17) comprising a chimeric restriction endonuclease comprising the DNA-binding domain of a I-Seel mega- nuclease and the Fokl cleavage domain (FF18). Choulika suggests that the 9 Appeal2013-005228 Application 10/395,816 DNA-binding domain of its chimeric nuclease can be a zinc finger DNA- binding domain (FF 18). Choulika discloses inducing a double-strand break in the chromosomal DNA of a cell (FF14) using its chimeric nuclease to cleave the DNA at the nuclease's recognition site (FF15), and repairing a specific sequence of interest via recombination of the chromosomal DNA with a targeting DNA that "comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which repairs the specific sequence of interest upon recombination between the targeting DNA and the chromosomal DNA" (FF14). Choulika provides a working example of gap repair recombination of a target site on a chromosomally integrated plasmid by cotransfection of NIH 3T3 cells with an expression plasmid and a repair matrix (FF20-FF22). These teachings would have made obvious the method of claim 1. Specifically, it would have been obvious to modify Choulika's vector to include a chimeric nuclease with a zinc finger DNA-binding domain because Choulika expressly suggests doing so. It would have been obvious to include DNA encoding two of the resulting nucleases on Choulika's vector because Bibikova teaches that chimeric nucleases require dimerization in order to produce specific cleavage, and teaches that combining two chimeric nucleases with different binding specificities will produce a dimer having a target site that is expected to be unique in the human genome. Choulika provides a working example of gap repair using its vectors in NIH 3T3 cells, and states that it provides "a method of repairing a specific sequence of interest in chromosomal DNA of a cell" (FF 14). Bibikova states 10 Appeal2013-005228 Application 10/395,816 that its results in Xenopus oocytes "should be applicable to mammalian somatic cells and to many other cells and organisms" (FF6). These disclosures would have provided a person of ordinary skill in the art with a reasonable expectation of successfully using the vector made obvious by the cited references to target a site in an endogenous mammalian gene, and therefore would have provided a reason to introduce a targeting DNA into a mammalian cell having the sequence to be repaired. Appellants argue that "the combination of references fails to teach or suggest cleavage of an endogenous mammalian or plant gene." (Appeal Br. 6.) Appellants argue that "Bibikova's teachings relate solely to extra- chromosomal targets in frog (Xenopus) oocytes" and Bibikova "states that homologous recombination in Xenopus oocytes occurs via single-stranded annealing of exogenous DNA targets." (Id. at 7 .) Similarly, Appellants argue that Bibikova "teaches that the cleaved (nonendogenous) substrate also acts as the exogenous sequence for recombination with itself." (Id. at 18.) Appellants also argue that the other references do not make up for Bibikova's deficiency, because "Pabo includes only one sentence relating to ZFEs" (id. at 10) and "Choulika uses the homing endonuclease Seel to cleave a single Seel target site that is either episomal or artificially integrated into the genome" (id. at 12). These arguments are unpersuasive. Bibikova states that its injected circular DNAs are assembled into apparently normal chromatin, in which form they are inert to recombination but can be induced to recombine if cleaved (FF4). Bibikova states that, therefore, its circular DNAs serve as effective models for inactive chromosomal targets (FF4). Bibikova also 11 Appeal2013-005228 Application 10/395,816 states that its chimeric nucleases "are capable of locating their target sequences in chromatin, cleaving with good efficiency, and thereby stimulating homologous recombination" (FF6), and that its findings "should be applicable to mammalian somatic cells and to many other cells and organisms" (FF6). Choulika states that a "model chromosomal loc[us] was generated in which a site for the meganuclease I-Seel was introduced within the target region for recombination" (FF 17, emphasis added). Choulika thus characterizes its own work as modelling recombination with a chromosomal target. In addition, Choulika repeatedly characterizes its disclosure as allowing targeted recombination with "chromosomal DNA" (FF 14, FF 16), and its Example 3 is carried out in mouse (NIH 3T3) cells (FF20). Appellants' argument that the cited references would not have suggested targeting an endogenous mammalian gene is therefore not persuasive. Appellants cite the 2nd Umov Declaration5 (Appeal Br. 8-9) as evidence that "Xenopus oocytes containing extrachromosomal, injected substrates are not models for endogenous mammalian or plant genomes" (id. at 9). Appellants also cite the 2nd Umov Declaration as evidence that "Bibikova does not in any teach or suggest ZFE cleavage of endogenous mammalian or plant genes." (Id. at 10.) We have considered the cited declaratory evidence but are not persuaded that it supports Appellants' position. Appellants cite i-fi-1 5-7 of the declaration as showing that Bibikova's Xenopus system is not a model 5 Declaration under 37 C.F.R. § 1.132 of Fyodor Umov, dated Dec. 7, 2011. 12 Appeal2013-005228 Application 10/395,816 for mammalian or plant genomes. (Appeal Br. 8-9.) Dr. Umov states that Xenopus oocytes differ from mammalian or plant cells in various ways and that they have "completely different nucleoplasm dynamics." (2nd Umov Deel. i-f 5.) Dr. Umov states that "Bibikova's 'chromatinized' substrates are not normal, endogenous chromatin." (Id.) Dr. Umov does not, however, explain what characteristics are implied by the term "nucleoplasm dynamic," nor does Dr. Umov explain why the cited differences between Xenopus oocytes and mammalian cells support his conclusion that Xenopus oocytes are inadequate models for the interaction of DNA binding proteins and their templates. Bibikova acknowledges that its injected DNA is not endogenous chromatin, but concludes that it "serves as an effective model for an inactive chromosomal target" that undergoes homologous recombination if cleaved (FF4). Bibikova also states that, contrary to Dr. Umov's opinion, its findings inXenopus oocyte experiments "should be applicable to mammalian somatic cells and to many other cells and organisms" (FF6). Choulika supports this conclusion because it teaches that causing a double-stranded break in the chromosomal DNA, of a mammalian cell, with a chimeric nuclease allows homologous recombination of the cleaved site with a repair DNA introduced into the cell (FF14, FF15, FF20-FF22). Bibikova also states that homologous recombination "in Xenopus laevis oocytes ... proceeds by the same single-strand annealing mechanism that is the principal pathway available to exogenous DNAs in cultured mammalian cells." (Bibikova 290, bridging para.) Dr. Umov questions the applicability of this mechanism to the claimed method, stating that "this 13 Appeal2013-005228 Application 10/395,816 pathway is irrelevant to the homologous recombination process involving endogenous genes that is claimed." (2nd Umov Deel. i-f 5.) Dr. Umov also states that "every scientist working in this field knew (and knows) that extra chromosomal substrates injected into Xenopus oocytes do not undergo homologous recombination in the same way as endogenous targets in mammalian and plant cells." (Id. at i-f 6.) Similarly, Dr. Umov states that Bibikova's statement that "[s]everal additional issues remain to be addressed" (see FF9) "clearly indicates that Bibikova' s Xenopus system is not relevant to endogenous mammalian or plant genes." (2nd Umov Deel. ,-r 8.) We are not persuaded that the cited evidence demonstrates lack of a reasonable expectation of success based on the prior art. The claimed method involves recombination between a nucleic acid provided to the cell of interest and an endogenous chromosomal target. Dr. Umov does not provide any technical explanation of why the pathway described by Bibikova would be irrelevant to the recombination contemplated in the claimed method. In addition, Choulika demonstrates that a method similar to that of Bibikova, in which a chimeric nuclease cleaves a chromosomally integrated target site and induces homologous recombination with an exogenously introduced DNA, can be carried out in mammalian cells (FF20-FF22). The evidence of record therefore does not support Dr. Umov's conclusion that those skilled in the art would have considered Bibikova's experiments to be irrelevant to recombination in mammalian cells. We are also not persuaded that Bibikova's reference to "issues [that] remain to be addressed" shows the irrelevance of its results to mammalian 14 Appeal2013-005228 Application 10/395,816 cells. Bibikova states that "[ s ]everal additional issues remain to be addressed to confirm the utility of chimeric nucleases as tools for gene targeting" (FF9, emphasis added). The acknowledgement that additional work remains to be done to confirm a technique's utility does not mean that previous work is irrelevant to that utility. In addition, the quoted statement must be considered in combination with Bibikova's statement that its "findings should be applicable to mammalian somatic cells and to many other cells and organisms" (FF6) and Choulika's disclosure that its similar method is applicable to chromosomal targets in mammalian cells, among others. A preponderance of the evidence therefore supports a conclusion that those skilled in the art would have considered Bibikova to be relevant to the claimed method. Dr. Umov also states that "Bibikova teaches explicitly that the ZFEs must be delivered in protein form via microinjection" rather than via a nucleic acid encoding the ZFE, as claimed. (2nct Umov Deel. i-f 7 .) Appellants argue that the declaration shows that Bibikova does not suggest an expression vector encoding a ZFE. (Appeal Br. 17 .) This argument is also unsupported by the evidence, because Bibikova expressly states that "[t ]he method of delivery [of the chimeric nucleases and DNA] would depend on the organism, cell type, and other experimental conditions" (FF7). Bibikova therefore does not limit the delivery of its chimeric nuclease to microinjection of the nuclease itself. In addition, Choulika expressly suggests delivering a chimeric nuclease to a cell via a vector encoding the nuclease (FF 1 7, FF 19). Thus, the cited references 15 Appeal2013-005228 Application 10/395,816 would have made obvious a vector encoding a chimeric nuclease, as recited in claim 1 . Appellants argue that "Choulika clearly teaches away from cleaving an endogenous mammalian or plant gene, as claimed, in that it clearly states that cleavage of an endogenous gene may lead to death of the cells." (Appeal Br. 13.) Appellants cite Dr. Umov's statement that "[t]he entire purpose of Choulika is to insert a single restriction endonuclease target site that is non-endogenous so the resulting cleavage by the single restriction endonuclease is not toxic to the cell." (2nd Umov Deel. i-f 12.) This argument is also unpersuasive. In support of their position, Appellants cite the following statement in Choulika: A restriction endonuclease used in the present invention recognizes a target DNA sequence (e.g., a restriction endonuclease site) which would not lead to death of the cells upon cleavage of the DNA sequence by the restriction endonuclease. i~\~ meganuclease enzyme, \~1hich recognizes a very large DNA sequence, is an example of a restriction endonuclease which can be used in the present invention. An example of a meganuclease enzyme is I-Seel, which recognizes an 18-bp site (DNA sequence) that does not appear to be represented in murine or human DNA. ( Choulika 3 i-f 24.) This statement does not support Appellants' position that "cleavage of an endogenous gene may lead to death of the cells." (Appeal Br. 13.) Rather, the above statement means that the restriction endonuclease target site used in Choulika's method must be one that is unique in the target genome (as the I-Seel site would be in the human or murine genome, after it is introduced), because cleavage of the genome at additional sites could lead to cell death. This reading of the above statement is consistent with 16 Appeal2013-005228 Application 10/395,816 Choulika's description of the inserted I-Seel recognition site as a "model chromosomal loc[ us]" (FF 17) and its references to repairing a specific sequence in chromosomal DNA (FF14) or manipulating a chromosomal DNA locus (FF16). Choulika also makes clear that an inserted meganuclease site is not the only way to target a unique site in a genome. "[C]himeric restriction endonucleases capable of recognizing specific DNA sequences unique to a disease allele can be generated through juxtaposition of zinc finger DNA binding domains and restriction endonuclease cleavage domains." (Choulika 5 i-f 41.) Similarly, Bibikova states that two chimeric enzymes, each with three zinc fingers, demand an 18-bp target sequence that should be unique in the human genome (FF8). Thus, whether Choulika is considered by itself or together with the other cited references, it does not teach away from cleaving an endogenous mammalian gene, as Appellants argue. Appellants also argue that "the claims clearly require cleavage of an endogenous mammalian or plant gene by dimerizing ZFEs expressed within the cells from at least one expression vector. There is no combination of the references that teaches such methods." (Appeal Br. 15.) This argument is unpersuasive. For the reasons previously discussed, the cited references would have made obvious the claimed method, even though none of them identically disclose it. See In re Young, 927 F.2d 588, 591 (Fed. Cir. 1991) ("The test for obviousness is what the combined teachings of the references would have suggested to one of ordinary skill in the art."). 17 Appeal2013-005228 Application 10/395,816 Appellants argue that the Examiner improperly dismissed the declaratory evidence submitted to show lack of predictability. (Appeal Br. 20-23.) Appellants' arguments, however, focus on the Examiner's recitation of fact-finding from the previous Board decision in this application. Appellants do not address the Examiner's response to the declaratory evidence set out on pages 12-18 of the Office Action mailed Feb. 21, 2012, or on pages 11-17 of the Answer. Because the Examiner directly addressed Appellants' declaratory evidence, we do not find Appellants' argument persuasive. Finally, Appellants present additional arguments regarding the dependent claims (Appeal Br. 23-24). In each case, however, Appellants argue only that Bibikova does not teach cleaving an endogenous plant or mammalian gene using a ZFE expressed from a polynucleotide, and also does not teach the limitations of the dependent claims. (See id.) Appellants, however, provide no reasoned basis for concluding that the limitations of the dependent claims would not have been obvious based on the combined teachings of the references. See Young, 927 F.2d at 591 ("The test for obviousness is what the combined teachings of the references would have suggested to one of ordinary skill in the art.") The arguments regarding the dependent claims are therefore unpersuasive. Conclusion of Law A preponderance of the evidence of record supports the Examiner's conclusion that the method of claim 1 would have been obvious to a person of ordinary skill in the art based on Bibikova, Pabo, and Choulika. 18 Appeal2013-005228 Application 10/395,816 SUMMARY We affinn the rejection of claims 1, 7-9, 11, 12, 15, 37, and 38 under 35 U.S.C. § 103(a) based on Bibikova, Pabo, and Choulika. 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 19 Copy with citationCopy as parenthetical citation