Ex Parte Rozwadowski et alDownload PDFPatent Trial and Appeal BoardDec 9, 201610467639 (P.T.A.B. Dec. 9, 2016) Copy Citation 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. 10/467,639 12/05/2003 Kevin L Rozwadowski 4810-66535-01 6896 24197 7590 12/13/2016 KLARQUIST SPARKMAN, LLP 121 SW SALMON STREET SUITE 1600 PORTLAND, OR 97204 EXAMINER POPA, ILEANA ART UNIT PAPER NUMBER 1633 NOTIFICATION DATE DELIVERY MODE 12/13/2016 ELECTRONIC 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. Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the following e-mail address(es): tanya.harding@klarquist.com docketing @klarquist.com PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte KEVIN L ROZWADOWSKI and DEREK J LYDIATE1 Appeal 2015-006247 Application 10/467,639 Technology Center 1600 Before DONALD E. ADAMS, JOHN G. NEW, and DAVID COTTA, Administrative Patent Judges. NEW, Administrative Patent Judge. DECISION ON APPEAL appellants state the real party-in-interest is Her Majesty, the Queen in Right of Canada, as Represented by the Minister of Agriculture and Agri- Food. App. Br. 2. Appeal 2015-006247 Application 10/467,639 SUMMARY Appellants file this appeal under 35 U.S.C. § 134(a) from the Examiner’s Final Rejection of claims 1, 2, 4—8, 15, 59, 60, 62, and 63. Specifically, claims 1, 2, 4—8, 15, 60, 62 and 63 stand rejected as unpatentable under U.S.C. § 103(a) as being obvious over Baszczynski et al. (US 6,300,545 Bl, October 9, 2001) (“Baszczynski”), Yadav (US 6,077,992, June 20, 2000) (“Yadav”), and Hodges et al. (US 6,110,736, August 29, 2000) (“Hodges”).2 Claims 1, 2, 4—8, 15, 59, 60, 62, and 63 stand rejected as unpatentable under U.S.C. § 103(a) as being obvious over Baszczynski, Yadav, Hodges, and Mason et al. (US 6,392,121, May 21, 2002) (“Mason”). We have jurisdiction under 35 U.S.C. § 6(b). We AFFIRM-IN-PART. NATURE OF THE CFAIMED INVENTION Appellants’ invention is directed to recombinant nucleic acid technology, particularly constructs and methods for targeted gene modification by nucleic acid recombination and/or repair using various nucleic acid replication systems. Spec. 1. 2 An additional reference, E. Valencia-Morales and D. Romero, Recombination Enhancement by Replication (RER) in Rhizobium etli, 154(3) Genetics 971—983 (2000), was included in the Examiner’s rejection. However, the Examiner acknowledges, and agrees with, Appellants’ position that the teachings of the reference are not applicable to the instant invention because they only teach intramolecular recombination. Ans. 3. The reference, being thus withdrawn, plays no part in our analysis. 2 Appeal 2015-006247 Application 10/467,639 REPRESENTATIVE CLAIM Claim 1 is representative of the claims on appeal and recites: 1. A eukaryotic host cell comprising a gene targeting cassette comprised of recombinant nucleic acid sequences integrated into a genome of the host cell, or a progenitor of the host cell, wherein the gene targeting cassette comprises: a) a replication initiator sequence recognized in the host cell by a replication factor to mediate DNA replication in the host cell initiated at the replication initiator sequence, wherein the replication initiator sequence is a DNA sequence encoding a recognition and nicking site where DNA replication is initiated by the replication factor to initiate rolling circle replication; and b) a reproducible sequence homologous to a target sequence in the genome of the host cell operably linked to the replication initiator sequence so that DNA replication initiated at the replication initiator sequence replicates the reproducible sequence, to release a copy of the reproducible sequence, wherein the target sequence and the gene targeting cassette are at different loci in the genome; and, wherein DNA replication initiated at the replication initiator sequence results in the regeneration of the gene targeting cassette for subsequent rounds of DNA replication to produce multiple copies of the reproducible sequence, wherein the gene targeting cassette remains integrated into the genome during replication; and wherein at least a portion of one of the copies of the reproducible sequence mediates a heritable genetic change in the target sequence in the genome of the host cell by recombination with the target sequence. App. Br. 17. 3 Appeal 2015-006247 Application 10/467,639 ISSUES AND ANALYSES We agree with, and adopt, the Examiner’s findings and conclusion that the appealed claims are obvious the combined cited prior art references. We address the arguments raised on appeal by Appellants below. Issue 1 Appellants argue the Examiner erred in finding the combined cited prior art references teach or suggest all of the limitations of the claims. App. Br. 6. Findings of Fact F. 1: Baszczynski teaches that it was well known in the art that T-DNA inserted into a plant cell can be integrated into the plant genome. Baszczynski col. 1,11. 30—32. F.2: Baszczynski teaches: The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T- DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. Baszczynski col. 3,11. 6—14. 4 Appeal 2015-006247 Application 10/467,639 F.3: Baszczynski teaches: Depending on the substrates, the recombination event could consist of a single or a double cross-over event. In the simplest case, the plant chromosome and the excised viral replicon each contain a single target site. Site-specific recombination between these two target sites results in the insertion of the viral replicon and any associated DNA of interest into the plant chromosome. Baszczynski col. 4,11. 40-46. F.4: Baszczynski teaches: The site-specific recombinase must be expressed in the plant cell in order for mobilization of the viral DNA from the T- DNA. Accordingly, the recombinase protein must be expressed so that it is present in the cell at an effective concentration sometime between when the T-DNA is transferred to the plant cell and before non-integrated T-DNA is lost from the plant cells. Accordingly, the expression cassette encoding the site- specific recombinase may be supplied on the T-DNA in cis to the viral DNA; in trans on a plant chromosome or extrachromosomal replicon; or may be transferred to the plant near to the time of agro-mediated transformation. Baszczynski col. 11, 3—14 (emphasis added). F.5: Yadav teaches: In one embodiment, the present invention provides a binary transgenic viral expression system for replicating and increasing expression of a target gene comprising a) a heritable proreplicon lacking a functional replication gene for autonomous episomal replication and comprising: i) cis-acting viral elements required for viral replication; 5 Appeal 2015-006247 Application 10/467,639 ii) a target gene comprising at least one suitable regulatory sequence; and iii) flanking sequences that enable the excision of the elements of (i) and (ii); and, b) a heritable chimeric trans-acting replication gene comprising a regulated plant promoter operably-linked to a viral replication protein coding sequence. Yadav cols. 4—5,11. 60—7 (emphasis added). F.6: Yadav teaches: Both components of the system are chromosomally-integrated. One component is a chimeric trans-acting replication gene in which the coding sequence of the geminivirus replication protein(s) is placed under the control of a tissue- or development- specific and/or inducible promoter. The second component is a proreplicon, which is unable to replicate by itself but does so in the presence of viral replication protein(s). Yadav col. 5,11. 15—22. F.7: Yadav teaches: “Regulated expression of a chromosomally integrated chimeric replication gene will result in the release and replication of the replicon from a chromosomally integrated master copy of the proreplicon.” Yadav col. 5,11. 30-33. F.8: Yadav teaches: The proreplicon is incapable of self replication in plant cells because it lacks an essential trans-activating replication gene. The second component of the system, a chimeric trans-acting replication gene, consists of a regulated promoter operably- linked to the coding region for a viral replication protein. Expression of the viral replication protein results in the release and replication of the replicon from the proreplicon. 6 Appeal 2015-006247 Application 10/467,639 Yadav col. 6,11. 4—11. F.9: Yadav teaches: The term “episomal replication” and “replicon replication” refer to replication of DNA or RNA viruses or virus-derived replicons that are not chromosomally-integrated. It requires the presence of viral replication protein(s), is independent of chromosomal replication, and results in the production of multiple copies of virus or replicons per host genome copy. Yadav col. 10,11. 30-36. F.10: Yadav teaches: The term “proreplicon” refers to a replication-defective replicon that is integrated into a bacterial plasmid or host plant chromosome. It is comprised of cis-acting viral sequences required for replication, and flanking sequences that enable the release of the replicon from it. In addition, the proreplicon may contain a target gene. Yadav col. 10,11. 43—48. F. 11: Yadav teaches: These regulatory sequences can be for constitutive or regulated expression. Proreplicon lacks a gene encoding a replication protein essential for replication. Therefore, it is unable to undergo autonomous episomal replication but can undergo episomal replication in the presence of the replication protein provide in trans. Thus, its replication requires both release from the integration and the presence of the essential replication gene in trans. Yadav col. 10,11. 54—60. 7 Appeal 2015-006247 Application 10/467,639 F.12: Yadav teaches: “The terms ‘genetically stable’ and ‘heritable’ refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.” Yadav col. 11,11. 59-62. F.13: Yadav teaches: The invention provides a two-component, viral expression system for the production of transgenic plants. Both components are chromosomally-integrated and, thus, stably maintained by themselves. One component is the proreplicon that is unable to replicate by itself. The second component is a chimeric trans acting replication gene in which the coding sequence of a viral replication is operably-linked to a regulated promoter. Expression of the viral replication protein-under appropriate stimulus will result in the release of replicon from the proreplicon and its episomal replication in a cell autonomous manner. Thus, replicon replication can be targeted to specific plant cells by controlling the expression of replication protein(s) to those cells. Yadav col. 12,11. 10-22. Analysis Appellants allege the Examiner mischaracterizes the teachings of Baszczynski; specifically, Appellants argue, Baszczynski does not teach a genome-integrated cassette, or regeneration of the gene targeting cassette for subsequent rounds of DNA replication of the reproducible sequence, in which the gene targeting cassette remains integrated into the genome during replication. App. Br. 6—7. Appellants also argue that Baszczynski does not teach a “reproducible sequence homologous to a target sequence in the genome” that also “mediates a heritable genetic change in [the] target sequence.” Id. at 7. 8 Appeal 2015-006247 Application 10/467,639 Appellants contend that Baszczynski neither teaches nor suggests a genome-integrated cassette that is regenerated. App. Br. 7. According to Appellants, Baszczynski describes a T-DNA containing “a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon ... An expression cassette for the site specific-recombinase is present on the T-DNA....” Id. (citing Baszczynski, col. 3,11. 2—9). Appellants assert the “cassette” of Baszczynski is the T-DNA, which is not integrated in the host cell genome. Id. Appellants contend that Baszczynski in fact teaches the opposite; the T- DNA is not integrated into the host genome. Id. Appellants point to Baszczynski’s teaching that a site-specific recombinase acts on the T-DNA to excise the viral replicon “before non-integrated T-DNA is lost....” Id. (citing Baszczynski col. 11,11. 3—9). Appellants argue that the T-DNA of Baszczynski is therefore not integrated into the host cell genome and Baszczynski clearly does not teach a “genome integrated cassette.” Appellants thus contend that Baszcynski does not teach that its T-DBA does not “remain[] integrated into the genome during replication,” as required by claim 1. Id. at 7—8. Appellants argue further that Baszczynski also neither teaches nor suggests that there is no regeneration of the cassette upon production of “copies of the reproducible sequence” for mediation of a heritable genetic change. App. Br. 8. Appellants point to Figures 1 and 2 of Baszczynski, which, they assert demonstrate that the viral replicon is excised from the T- DNA vector by the site-specific recombinase. Id. Therefore, Appellants argue even if the T-DNA is defined by the Office as the “gene-targeting 9 Appeal 2015-006247 Application 10/467,639 cassette,” it is subsequently destroyed during the production of any copies of the “reproducible sequence” and not regenerated, as required claim 1. Id. Alternatively, argue Appellants, even if the excised viral replicon is the “gene targeting cassette,” that cassette is not a cassette integrated into the genome and that remains integrated during replication. App Br. 8. Therefore, argue Appellants, the teachings of Baszczynski cannot provide the advantage of enabling numerous opportunities for gene targeting to occur at different times during development and in different generations. Id. Rather, Appellants assert, the viral replicon of Baszczynski is never integrated into the genome, but only the loxP' and loxP sites and the intervening “DNA of interest.” Id. (citing, e.g., Baszczynski Fig. 3, col. 4, 11. 47-64). Appellants argue further that Baszczynski neither teaches nor suggests a “reproducible sequence homologous to a target sequence in the genome” that also “mediates a heritable genetic change in the target sequence” as recited in the claims. App. Br. 8. Appellants contend this necessitates that the DNA sequence of the reproducible sequence be homologous to the target sequence, and also be at least one base different than the DNA sequence of the target so as to be able to mediate a genetic change. Id. at 8—9. According to Appellants, Baszczynski does not teach this limitation because the only sequences in the viral replicon taught by Baszczynski that are homologous to a target sequence are the loxP' and loxP sites. Id. at 9. Finally, Appellants admit Baszczynski teaches that an intervening sequence could potentially be inserted between two recombination sites (citing Baszczynski Fig. 2), such a sequence cannot qualify as the “reproducible sequence” of Appellants’ claims because it is not homologous 10 Appeal 2015-006247 Application 10/467,639 to any target sequence. App. Br. 9. Appellants assert that mere proximity to the recombination sites (lox P' and loxP sites), which themselves are homologous to recombination sites in the genome, cannot quality the intervening sequences as part of a “reproducible sequence.” Id. Correspondingly, Appellants explain, an insertion into the genome between two recombination sites would not qualify as a heritable genetic change in a target sequence because the genetic change is occurring outside the region of homology (in proximity to the recombination sites, but not within them). Id. Appellants admit Yadav teaches a chromosomally-integrated cassette (the proreplicon), however, Appellants argue, this proreplicon must be excised from the genome for replication. App. Br. 10. Consequently, Appellants argue, replication initiated at the replication initiator sequence does not result in the regeneration of the cassette and the cassette does not remain integrated into the genome during replication, as required in the claims. Id. Appellants argue further that Hodges and Valencia-Morales do not cure the alleged deficiencies of Baszczynski and Yadav. App. Br.10. The Examiner responds that above, Baszczynski teaches a gene targeting cassette integrated into the genome of a eukaryotic host cell. Ans. 11. The Examiner finds Baszczynski’s gene targeting cassette is a geminiviral replicon comprising an origin of replication (i.e., a replication initiator sequence) operably linked to a reproducible sequence homologous to a target sequence in the host cell genome. Id. The Examiner finds that at least a portion of the reproducible sequence mediates a heritable change in the target sequence in the genome of the host cell by recombination with the target sequence. Id. The Examiner finds Baszczynski teaches the gene 11 Appeal 2015-006247 Application 10/467,639 targeting cassette is flanked by recombination sites recognized by a site- specific recombinase and that controlled expression of the site-specific recombinase results in the excision of the replicon from the genome and its amplification to high copy numbers within the host cell nucleus. Id. The Examiner finds the high copy replicon numbers increase replicon integration efficiency into the target chromosome. Id. Therefore, the Examiner finds, the advantage of providing high copy numbers of replicons to increase the efficiency of their integration into a target locus in the genome was known in the prior art. Id. The Examiner finds that although the gene targeting cassette (i.e., the geminiviral replicon) of Baszczynski is excised by the site-specific recombinase, and does not remain integrated into the genome of the host plant cell during replication, the advantages of using geminiviral replicons that remain integrated into the host plant cell genome during replication is taught by Yadav. Ans. 12. Specifically, the Examiner finds Yadav teaches use a geminiviral replicon (or “proreplicon”) that remains integrated during replication and provides the advantage of yielding heritable traits, thus overcoming the need for infection in each generation. Id. The Examiner finds Yadav teaches the geminiviral proreplicon is amplified to high copy numbers by rolling circle replication, in which the proreplicon remains integrated into the genome as a master copy from which replicon copies are released and amplified. Id. The Examiner finds that, since the master proreplicon is heritable, rolling circle replication can be triggered in the same cell or its progeny. The Examiner therefore concludes that it was known in the art at the time of filing, that using a stably integrated replicon (and thus obviating the need for transfection in each generation and enabling 12 Appeal 2015-006247 Application 10/467,639 gene targeting at desired time in the same cell or its progeny) was advantageous. Id. The Examiner therefore concludes the combined teachings of Baszczynski and Yadav render the claimed eukaryotic host cell prima facie obvious. Ans. 12. Specifically, the Examiner finds a person of ordinary skill in the art would have known that: (i) amplifying geminiviral replicons results in enhanced gene targeting; (ii) similar to site-specific recombinase, rolling circle replication could be used to release and amplify geminiviral replicons; and (iii) using an integrated master proreplicon and rolling circle replication to release and amplify replicon copies has advantages over release via site-specific recombinase because it provides an heritable master proreplicon that can be induced to replicate at the desired times in the plant host cell or its progeny. Id. at 12—13. Furthermore, the Examiner concludes that a person of ordinary skill in the art would have been motivated to use Yadav’s proreplicon and rolling circle replication, instead of site-specific recombinase, in the host plant cell of Baszczynski with the reasonable expectation of generating high copy replicon numbers for enhanced integration into the targeted locus in the genome of the host plant cell without the need for infection in every generation. Ans. 13. We are not persuaded by Appellants’ arguments. Baszczynski teaches that it was well known in the art that T-DNA can be integrated into the genome of a plant cell. F. 1 (“The transferred T-DNA is then targeted to the nucleus and integrated into the plant genome”). Baszczynski teaches that the gene expression cassette, or replicon, is transferred to a plant cell via T- DNA-bearing A gro b a cl eri z/ m - m e d i at e d transfer; the replicon is then excised 13 Appeal 2015-006247 Application 10/467,639 from the T-DNA and can then be integrated directly into the genome of the plant at a specific locus. F.2; F.3. Baszczynski further teaches the expression cassette encoding the site-specific recombinase may be supplied on the T-DNA in cis to the viral DNA; in trans on a plant chromosome or extrachromosomal replicon; or may be transferred to the plant near to the time of agro-mediated transformation. F.4. However, Baszczynski is silent with regard to whether the inserted replicon is heritable. Yadav teaches a binary transgenic viral expression system consisting of (1) a heritable proreplicon lacking a functional replication gene for autonomous episomal replication and (2) a heritable chimeric trans-acting replication gene comprising a regulated plant promoter operably-linked to a viral replication protein coding sequence. F.5; F.10. Yadav teaches that both components of the system are chromosomally integrated. FF6. One component is a chimeric trans-acting replication gene in which the coding sequence of the geminivirus replication protein(s) is placed under the control of a tissue or development-specific and/or inducible promoter. Id. The second component is a proreplicon, which is unable to replicate by itself but does so in the presence of viral replication protein(s). Id. Yadav further teaches that “[rjegulated expression of a chromosomally integrated chimeric replication gene will result in the release and replication of the replicon from a chromosomally integrated master copy of the proreplicon.'1'’ F.7. Yadav teaches that “[t]he second component of the system ... consists of a regulated promoter operably-linked to the coding region for a viral replication protein. Expression of the viral replication protein results in the release and replication of the replicon from the proreplicon,” and that the 14 Appeal 2015-006247 Application 10/467,639 released replicon is capable of episomal replication, independent of chromosomal replication. F.8; F.9. We therefore agree with the Examiner that the combination of the cited prior art references teaches or suggests: DNA replication initiated at the replication initiator sequence results in the regeneration of the gene targeting cassette for subsequent rounds of DNA replication to produce multiple copies of the reproducible sequence, wherein the gene targeting cassette remains integrated into the genome during replication; and wherein at least a portion of one of the copies of the reproducible sequence mediates a heritable genetic change in the target sequence in the genome of the host cell by recombination with the target sequence, as recited by claim 1. Both Baszczynski and Yadav explicitly teach embodiments in which the gene expression cassette is integrated into the chromosome. FI; F.5; FF6. Appellants argue, inter alia, that the proreplicon of Yadav must be excised from the genome for replication (see App. Br. 10), but this is contrary to the teachings of Yadav, which teaches that the replicon is formed from the master copy (i.e., the proreplicon), which remains integrated in the chromosome. F.7. We consequently agree with the Examiner’s conclusion that a person or ordinary skill in the art would have found the claims on appeal obvious over the combined cited prior art. Issue 2 Appellants argue claim 59 separately. App. Br. 10. Claim 59 recites 15 Appeal 2015-006247 Application 10/467,639 “The eukaryotic host cell of claim 1, wherein the reproducible sequence is an inverted repeat sequence so that the copies of the reproducible sequence anneal to one another to form double stranded DNA.” Id. at 19. Appellants contend that the cited prior art neither teaches nor suggests “wherein the reproducible sequence is an inverted repeat sequence.” Id. at 11. Findings of Fact F.14: Mason teaches: “As used herein, a Tong intergenic region’ (LIR) refers to a noncoding region that contains sequences capable of forming a hairpin structure, including a conserved 9-base sequence (TAATATTAjC) found in all geminiviruses.” Mason col. 4,11. 57— 60. Analysis Appellants argue that the inverted repeat recited in claim 59 is the “reproducible sequence,” which is homologous to a target sequence in the genome of the host. App. Br. 11. Appellants contend that, in these circumstances, the released single stranded copy of the reproducible sequence (encoding the two inverted reproducible sequences) of Applicants' claim 59 is able to anneal to form a double stranded DNA molecule. Id. According to Appellants, the long intergenic region (“LIR”) region of the virus, as taught by Mason, that is essential for virus replication is not “a reproducible sequence homologous to a target sequence in the genome of the host cell,” as recited in claim 59. Id. 16 Appeal 2015-006247 Application 10/467,639 The Examiner responds that, although Baszczynski, Yadav, and Hodges, do not specifically teach an inverted repeat as recited in claim 59, their geminivirus proreplicon comprises all cis-acting viral sequences required for replication and therefore, the replicon must necessarily contain a LIR which is essential for viral replication. Final Act. 6 (citing Mason col. 3,11. 10—17). The Examiner finds that, since a LIR has a hairpin structure, the replicons of Baszczynski, Yadav, and Hodges include an inverted repeat. Id. The Examiner further finds: (1) the claims do not require homology over the entire length of the reproducible sequence. Homology over short homology sequences are encompassed by the instant claims; (2) the claims do not require the entire reproducible sequence be an inverted repeat. Consistent with the instant specification, the only requirement for a nucleic acid to be considered an inverted repeat is to comprise a portion of inverted repeat sequences that could form a hairpin (citing Spec. 13,11. 10-20); and (3) the claims do not require the inverted repeat portion of the reproducible sequence be the sequence that is homologous to the target sequence in the genome of the host cell. Ans. 22—23. Thus, there is no requirement for the LIR to be homologous to a target sequence in the genome of the plant host cell. Id. at 23. We are persuaded by Appellants’ argument. Mason explicitly teaches: a LIR refers to a noncoding region that contains sequences capable of forming a hairpin structure, including a conserved 9-base sequence (TAATATTAjC) found in all geminiviruses. F.14. Appellants do not dispute that both Baszczynski and Yadav teach geminiviruses, and, because Mason teaches that all geminiviruses contain an LIR, and LIRs are capable 17 Appeal 2015-006247 Application 10/467,639 of forming hairpins, then, by the rules of base pairing, the LIR must contain reverse sequences capable of annealing to each other. However, claim 59 requires that the “the reproducible sequence is an inverted repeat sequence.” Appellants’ claim 1 defines the “reproducible sequence” as being homologous to a target sequence in the genome of the host cell. This is not equivalent to, or suggestive of, the noncoding LIR of the geminivirus that is essential for replication, and we reverse the Examiner’s rejection of claim 59. Issue 3 Appellants argue that a person of ordinary skill in the art would have neither motivation to combine the references nor a reasonable expectation of success in doing so. App. Br. 11. Analysis Appellants dispute the Examiner’s conclusion that that it would have been obvious to one of skill in the art that rolling circle replication could be used to generate high copy numbers of geminivirus replicons in the plant host cell of Baszczynski for enhanced integration into the genome of the host cell. App. Br. 11 (citing Final Act 4). Appellants acknowledge Yadav teaches that it was known in the art that that geminiviruses “replicate via double-stranded DNA intermediate by ‘rolling circle replication.’” App. Br. 12 (Yadav, col. 2,11. 36—39). Nevertheless, Appellants assert, one of skill in the art would not be motivated to combine Yadav with Baszczynski, regardless of whether rolling circle amplification or another type of amplification was used. Id. at 12. 18 Appeal 2015-006247 Application 10/467,639 Appellants argue that the proreplicon of Yadav includes duplicated viral sequences, including the viral origin of replication. App. Br. 12 (citing Yadav, Fig. 1; col. 10,11. 43—64). Appellants contend Baszczynski teaches that, for replicative release, two origins of replication sequences must be present to initiate and complete the replication process and that tandem dimers of viral DNA are often difficult to construct and are unstable in recombination proficient host cells. Id. (citing, e.g., Baszczynski, col. 2,11. 3—13). According to Appellants, Baszczynski further teaches that replicative release (as well as mobilization by recombination) is a complex biochemical process that may be attenuated by a number of factors which in turn affect the efficiency of viral excision. Id. Therefore, Appellants argue, Baszczynski teaches that having two copies of a rolling circle replication virus (as described by Yadav) is predicted to be unstable. Id. Appellants contend that a person of ordinary skill in the art would not be motivated to combine Yadav with Baszczynski and would not have had a reasonable expectation of success in combining the teachings of Yadav and Baszczynski. Id. The Examiner admits that Baszczynski teaches viral replication and mobilization is complex and can be attenuated by a number of factors and that viral DNA tandem dimers are often difficult to construct and are unstable. Ans. 24. The Examiner finds, however, that Yadav teaches successful construction and use of stably integrated and heritable viral DNA tandem dimers as master copies from which to successfully mobilize and amplify replicons by using a replication factor and rolling circle replication. Id. The Examiner finds Yadav provides evidence that viral DNA tandem dimers can be constructed and that they are stable in the plant host cells. Id. 19 Appeal 2015-006247 Application 10/467,639 The Examiner finds both Baszczynski and Yadav teach the use of dicotyledonous plant host cells. Ans. 24 (citing, e.g., Yadav, cols. 15—16,11. 64—2). The Examiner finds combining the teachings of Baszczynski and Yadav would entail using the proreplicon/replication factor system in a dicotyledonous plant cell (already proven successful by Yadav), and further modifying the proreplicon by introducing an homology sequence mediating recombination with a target sequence found in the genome of the dicotyledonous plant cell, as taught by Baszczynski. Id. The Examiner therefore finds that the only remaining variable would be introducing the homology sequence. Id. The Examiner finds Appellants do not adduce any evidence suggesting that the introduction of a homology sequence into Yadav’s proreplicon would adversely affect its stability. Id. The Examiner finds that, in fact, Yadav teaches that the proreplicon could comprise portions that are homologous to sequences found in the plant host cell genome. Id. at 24—25 (citing Yadav col 6,11. 19-25, col. 9,11. 50-61). The Examiner concludes that, based on the guidance of Yadav, a person of ordinary skill in the art would have reasonably expected to be successful in modifying the teachings of Baszczynski by using Yadav's proreplicon system, and would have been motivated to do so to obtain high copy numbers. We agree with the Examiner. Appellants adduce no credible evidence to demonstrate why the teachings of Baszczynski and Yadav could not be successfully combined with a reasonable expectation of success. Both references teach successful insertion of gene expression cassettes into eukaryotic plant cells for site-specific recombinase-mediated mobilization of viral replicons from T-DNA. Although the passage cited by Appellants 20 Appeal 2015-006247 Application 10/467,639 states that: “[b]oth mechanisms of mobilization are complex biochemical processes that may be attenuated by a number of factors which in turn affect the efficiency of viral excision. Furthermore, tandem dimers of viral DNA are often difficult to construct and are unstable in recombination proficient host cells,” Baszczynski is referring in this context to prior art studies over which the invention of Baszczynski provides a significant improvement. See Baszczynski col. 2,11. 9—14. Consequently we agree with the Examiner that a person of ordinary skill in the art would have been motivated to combine the teachings of Baszczynski and Yadav with a reasonable expectation of success. DECISION The Examiner’s rejection of claims 1, 2, 4—8, 15, 60, 62, and 63 as unpatentable under 35 U.S.C. § 103(a) is affirmed. The Examiner’s rejection of claim 59 as unpatentable under 35 U.S.C. §103 (a) is reversed. 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). See 37 C.F.R. § 1.136(a)(l)(iv). AFFIRMED-IN-PART 21 Copy with citationCopy as parenthetical citation