Valton, Julien et al.Download PDFPatent Trials and Appeals BoardMar 27, 202014235759 - (D) (P.T.A.B. Mar. 27, 2020) 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. 14/235,759 05/07/2014 Julien Valton DI2011-04US1 5698 76392 7590 03/27/2020 ARRIGO, LEE, GUTTMAN & MOUTA-BELLUM LLP 2200 Pennsylvania Ave NW Suite 400E WASHINGTON, DC 20037 EXAMINER WEILER, KAREN S ART UNIT PAPER NUMBER IPLA NOTIFICATION DATE DELIVERY MODE 03/27/2020 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): SAL@ARRIGO.US legaladmin@arrigo.us scott@arrigo.us PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE ____________ BEFORE THE PATENT TRIAL AND APPEAL BOARD ____________ Ex parte JULIEN VALTON, ALEXANDRE JUILLERAT, and PHILIPPE DUCHATEAU ____________ Appeal 2019-004044 Application 14/235,759 Technology Center IP00 ____________ Before ERIC B. GRIMES, DEBORAH KATZ, and ULRIKE W. JENKS, Administrative Patent Judges. JENKS, Administrative Patent Judge. DECISION ON APPEAL Pursuant to 35 U.S.C. § 134(a), Appellant1,2 appeals from Examiner’s decision to reject claims 40–42, 45, 49, 53, 55, and 57. We have jurisdiction under 35 U.S.C. § 6(b). We AFFIRM. 1 We use the word “Appellant” to refer to “applicant” as defined in 37 C.F.R. § 1.42(a). Appellant identifies the real party in interest as Cellectis. Appeal Br. 3. 2 An oral hearing was held on March 2, 2020. Appeal 2019-004044 Application 14/235,759 2 STATEMENT OF THE CASE Claims 40–42, 45, 49, 53, 55, and 57 are on appeal, and can be found in the Claims Appendix of the Appeal Brief. Claim 40, reproduced below, is illustrative of the claimed subject matter: 40. A method of generating and assembling polynucleotides comprising arrays of at least two transcription activator-like effector (TALE) DNA binding repeat polynucleotide modules which each encodes for 30 to 42 amino acids, comprising the steps of: a) Generating “n” polynucleotide building blocks, each comprising at least: one TALE DNA binding repeat polynucleotide module; a single cleavage site for a first type IIS restriction enzyme A, placed on one side of the polynucleotide module; a single cleavage site for a second type IIS restriction enzyme B, placed on the other side of the polynucleotide module; wherein A and B can produce compatible cohesive ends; wherein cleavage of each of said polynucleotide building blocks with restriction enzyme A results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that can be re- ligated with a polynucleotide building block cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A and/or B; wherein cleavage of each of said polynucleotide building blocks with restriction enzyme B results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that can be re- ligated with a polynucleotide building block cleaved by restriction enzyme A without restoring a sequence cleavable by restriction enzyme A and/or B; Appeal 2019-004044 Application 14/235,759 3 b) Generating “n” polynucleotides linked to a solid phase by: b1) linking each of the polynucleotide building blocks generated according to step a) to a solid phase so that the cleavage site for the first restriction enzyme A is placed on the side of the polynucleotide module that is linked to the solid phase and b2) cleaving said polynucleotide building block with restriction enzyme B; c) Generating one C-terminal polynucleotide building block comprising at least: one TALE DNA binding repeat polynucleotide module; a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotide module; a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotide module; wherein cleavage of said C-terminal polynucleotide building block with restriction enzyme A results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that can be re- ligated with a polynucleotide building block of step a) cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A; and wherein cleavage of said C-terminal polynucleotide building block with restriction enzyme B results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that cannot be re- ligated with a polynucleotide building block of step a) cleaved by restriction enzyme A and/or B; d) Cutting said one C-terminal polynucleotide building block of c) with restriction enzyme A; e) Ligating the resulting C-terminal polynucleotide module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotide module; f) Cutting the resulting new immobilized polynucleotide with restriction enzyme A, thus producing a new Appeal 2019-004044 Application 14/235,759 4 polynucleotide having a free end compatible with the cohesive ends resulting from cleavage of a polynucleotide building block of step a) with restriction enzyme B; g) Ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotide module. Appeal Br. 24–27 (Claims Appendix). REFERENCE(S) The prior art relied upon by Examiner is: Name Reference Date Cease et al. (“Cease”) US 5,827,704 Oct. 27, 1998 Joung et al. (“Joung”)3 US 2014/0274812 Al Sept. 18, 2014 Schatz et al. (“Schatz”) US 2006/0194202 Al Aug. 31, 2006 REJECTION(S) Appellant requests review of the following grounds of rejection made by Examiner: I. Claims 40–42, 45, 49, 55 and 57 under 35 U.S.C. § 103(a) as unpatentable over Joung in view of Schatz. II. Claims 40–42, 45, 49, 53, 55 and 57 under 35 U.S.C. § 103(a) as unpatentable over Joung in view of Schatz and further in view of Cease. OBVIOUSNESS OVER JOUNG AND SCHATZ Since both of these rejections rely upon Joung and Schatz for teaching “a polynucleotide building block with a TALE DNA binding 3 Examiner’s rejection relies on the earliest filing date of Joung’s provisional application No. 61/508,366 filed on July 15, 2011, herein referred to as “J-Provisional”. Appeal 2019-004044 Application 14/235,759 5 repeat polynucleotide module and two different, but compatible restriction sites on each side of the module,” the same issue is dispositive for both rejections, so we will consider the rejections together. Findings of Fact FF1. Joung teaches the assembly of an elongated polynucleotide sequence containing TALE repeat sequences. Figure 1 of Joung’s provisional application, reproduced below, shows “[a]n exemplary method of assembling a TALE repeat domain array” by the addition of preassembled sequences to the C-terminal end TALE sequence. J- Provisional 8:13. Appeal 2019-004044 Application 14/235,759 6 Figure 1 of Joung’s provisional application, reproduced above, shows the following steps: (1) attachment of a single biotinylated PCR product encoding one single N-terminal TALE repeat domain[] to a solid support (in the example shown here, a magnetic streptavidin coated bead is used but other solid supports might also be utilized as well as other ways of tethering the initial DNA fragment to the solid support), (2) creation of an overhang at the 3' end of the anchored DNA (e.g., using a Type IIS restriction enzyme), (3) ligation of a second fragment containing four TALE repeat domain[s], (4) additional cycles of steps (2) and (3) to create a long array, (5) in the final cycle performing ligation of a piece of DNA encoding one, two, three, or four TALE repeat domains depending upon the length of the desired final array, and (6) release of the extended DNA encoding the TALE repeats from the solid support (e.g., by using a Type IIS restriction enzyme whose site is built in at the 5' end of the initial biotinylated DNA product). The final fragment can then be prepared for ligation to an appropriate expression plasmid. See J-Provisional 8:13–26 (formatting added); Joung ¶ 59; see Ans. 8. FF2. Joung teaches that “ligatable ends can be produced by cutting with a restriction endonuclease (e.g., a type II or type IIS restriction endonuclease) or by ‘chewing back’ the end using an enzyme (or enzymes) with exonuclease and polymerase activities in the presence of one or more nucleotides.” J-Provisional 10:12–15; Joung ¶ 65; see Ans. 8. Appeal 2019-004044 Application 14/235,759 7 FF3. Joung’s example 1 teaches assembly of TALE repeat arrays using streptavidin coated magnetic beads. The assembly method contains the following steps: (1) amplification of α-type TALE repeat to insert biotin and digestion of the amplified fragment with BsaI HF4 (a type IIS restriction endonuclease) and purification. J-Provisional 48:1–12; Joung ¶ 193. (2) cutting a plasmid containing a four TALE repeat domain subarray unit (βγδϵ) with BbsI and purifying the fragment. J-Provisional 48:13–22; Joung ¶ 194. (3) ligating the biotin containing α-subunit polynucleotide and the βγδϵ-subunit containing polynucleotide to create a nucleic acid containing αβγδϵ TALE subunits. J-Provisional 48:23–49:5; Joung ¶ 195. (4) attaching the ligated αβγδϵ polynucleotide to streptavidin containing beads, washing, and digesting the bead bound polynucleotide with BsaI HF. J-Provisional 48:23–49:5; Joung ¶ 195. (5) extending the bead bound nucleic acid by adding TALE repeat domain subarray unit (βγδϵ) and ligating to create a polynucleotide containing TALE subunits αβγδϵβγδϵ. J-Provisional 49:6–25; Joung ¶ 196. This cycle is repeated one more time to create a bead bound polynucleotide containing TALE subunits αβγδϵβγδϵβγδϵ. Finally, a TALE subarray 4 Appellant does not dispute that BsaI is a type IIS endonuclease. See Appeal Br. 22. Appeal 2019-004044 Application 14/235,759 8 containing βγδ is added to the bead bound polynucleotide. J- Provisional 49:6–25; Joung ¶ 195. (6) digesting the bead bound polynucleotide with BsaI HF to create a 3' end for cloning into an expression vector and digesting the bead bound polynucleotide with BbsI to cleave the 5' end to release the product from the bead. J-Provisional 49:26–50:3; Joung ¶ 197. FF4. Schatz teaches methods of producing nucleic acid sequences by stringing together elongation blocks of nucleic acids. Schatz’s method contains the following steps: (a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang, b) immobilising the first oligonucleotide to the surface via the modification, c) providing a second at least partially double-stranded oligonucleotide whereby the oligonucleotide comprises a recognition site or a part thereof for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang, d) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product, e) cutting the immobilised ligation product with the first type IIS restriction enzyme thus releasing an elongated oligonucleotide having an overhang, f) providing a further at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be specifically coupled to a surface, whereby the oligonucleotide contains a recognition site for a further type IIS restriction enzyme and a single-stranded overhang, Appeal 2019-004044 Application 14/235,759 9 g) immobilising the further at least partially double-stranded oligonucleotide on a surface via its modification, h) combining the elongated oligonucleotide with the immobilised further oligonucleotide, and ligating them via their overhangs forming a further ligation product, i) cutting the resulting further ligation product with the further type IIS restriction enzyme releasing an elongated oligonucleotide having an overhang, and j) optionally, repeating steps f) to i). Schatz ¶¶ 21–30, see also ¶¶ 93–101, see Figure 1. Principle of Law “If the claim extends to what is obvious, it is invalid under § 103.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 419 (2007). Analysis Examiner finds that Joung teaches generating a polynucleotide using building blocks comprising TALE repeat domains. Final Act. 3; FF1–FF3. Examiner finds that Joung teaches a “PCR product containing a single N- terminal TALE repeat domain having type IIS restriction enzyme sites at the 5' and 3' ends.” Final Act. 3 (citing ¶¶ 59, 65). Examiner acknowledges that “the assembly process of Joung [] involves successive ligation of TALE repeat modules to the growing ‘C-terminal’ end of the repeat array (distal to the support), rather than insertion of TALE repeat modules at the ‘N-terminal’ end of the repeat array (proximal to the support), as per instant claim 40.” Final Act. 4; FF1–FF3. Examiner relies on Schatz for teach[ing] ligating an immobilized ‘N-terminal’ repeat module to an elongated ‘C-terminal’ repeat module array (e.g. releasing the first ligation product (elongated oligonucleotide) by cleavage with a type IIS enzyme and ligating it to an Appeal 2019-004044 Application 14/235,759 10 immobilized further oligonucleotide, which in turn can be released and ligated to another immobilized further oligonucleotide. Final Act. 4 (citing Schatz ¶¶ 25–30); FF4. Examiner concludes that one of ordinary skill in the art would have been motivated to apply Schatz’s reverse solid phase synthesis (RSPS) approach using Joung’s TALE nucleic acid building blocks because the RSPS method results in an almost complete removal of side products. Final Act. 4–5 (citing Schatz ¶ 116). Appellant contends that example 5 of Joung is not prior art to Appellant’s claims (Appeal Br. 13, Reply Br. 2–3) and that example 1 of Joung’s provisional application does not teach many aspects of the claimed invention (Appeal Br. 13). Specifically, Appellant contends that Joung does not teach the limitation ‘wherein cleavage of each of said polynucleotide building blocks with restriction enzyme A results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A and/or B’ is not discussed anywhere in the Office Action. Appeal Br. 16, 17. Appellant contends that there is no specific mention of a second enzyme site that is compatible with the first. Id. at 20. Appellant contends that the disclosure in Joung is not sufficient to establish that the TALE polynucleotide modules contain different but comparable restriction enzyme sites on each side of the module. Id. at 22. Although we agree with Appellant that example 5 is not disclosed in Joung’s provisional application, we, however, do not agree with Appellant’s contention that example 1 of Joung is missing the disputed Appeal 2019-004044 Application 14/235,759 11 aspects of the claimed invention. In the Answer, Examiner directs attention to passages in Joung that are supported in Joung’s provisional application that disclose adding multiple TALE repeat domain subarray unit (βγδϵ) into a growing polynucleotide chain. See FF1–FF3. Specifically, Examiner directs our attention to paragraph 59 and 65 of Joung, which are fully supported in Joung’s provisional application, for teaching the cyclic assembly process. See Ans. 8 (“These paragraphs describe the cyclic assembly (i.e. cutting and ligation) process to create a long array, and that the ligatable ends can be produced using type IIS restriction enzymes.”). Examiner finds that the “growth of the polynucleotide [in Joung] takes place in the direction corresponding to N terminus → C terminus of the encoded polypeptide.” Ans. 6. Examiner identifies “[e]nzyme ‘A’ of claim 40 corresponds to the type IIS restriction endonuclease BbsI of the working examples [including example 1], and enzyme ‘B’ of claim 40 corresponds to the type IIS restriction endonuclease BsaI of the working examples [including example 1].” Ans. 9; FF3. Joung teaches cutting a plasmid containing TALE repeat domain subarray unit (βγδϵ) with BbsI and purifying the fragment. FF3 (step 2). In the first cycle the purified TALE repeat domain subarray unit (βγδϵ) is added to the α-subunit containing biotin and the two units are ligated. FF3 (step 3). The ligated product containing TALE subunits αβγδϵ polynucleotide is mixed with streptavidin containing beads, washed, and the bound polynucleotide is digested with BsaI HF (FF3 (step 4)), thereby, creating a ligatable C-terminal end to receive another TALE repeat domain subarray unit (βγδϵ). FF3 (step 2). Because the same TALE subarray unit (βγδϵ) can be added one or more times to the attached array, we agree with Appeal 2019-004044 Application 14/235,759 12 Examiner that this reasonably indicates that 3′ and 5′ ends cut with different enzymes produce cohesive ends. Ans. 9. We are not persuaded by Appellant’s argument that example 1 does not teach a polynucleotide building block with a TALE DNA binding repeat polynucleotide having two different but compatible restriction sites. See Appeal Br. 21. The plasmid carrying the subarray unit (βγδϵ) meets the building block limitation. See FF3 (steps 2, 4, 5). The building block plasmid contains the BbsI and BsaI HF type IIS restriction endonuclease sites. Id. Because the subarray unit (βγδϵ) is added multiple times the ends created by the restriction endonuclease BbsI and BsaI HF are cohesive. Here, the digestion with BbsI creates an N-terminus in the subarray unit (βγδϵ) that is ligated to the C-terminus created by the BsaI HF digestion. We are not persuaded that more information is required in order to demonstrate that the BbsI and BsaI HF cleavage products create cohesive ends. Here, Joung’s growing polynucleotide chain indicates that it has an end that is acceptable for ligating the next subarray unit (βγδϵ) to form the chain. Examiner further explains that “the extended polynucleotide can not retain an ‘internal’ site that is recognized by the first [and second] type IIS restriction endonuclease because such a recognition site would result in cleavage 3' of the α repeat (releasing the ligated fragment) at each cycle, precluding polynucleotide growth.” Ans. 9. The second type IIS restriction endonuclease (FF3 (step 4)) is also lost during the growth of the polynucleotide chain. Examiner finds that Joung’s teaching of creating an extended polynucleotide chain reasonably suggests “that the recognition sequence of the first type IIS enzyme (i.e. “B”) is removed by digestion and Appeal 2019-004044 Application 14/235,759 13 not reformed during ligation, as additional cycles of cleavage and ligation could not otherwise create a long array.” Ans. 9. We find no error with Examiner’s explanation that Joung’s teaching of adding a multiple TALE subarray unit (βγδϵ) reasonably suggests that a type IIS enzyme (i.e. “B” = BsaI) site is not part of the elongated array because this enzyme is used to prepare a cohesive end to which another TALE subarray unit (βγδϵ) is ligated in order to grow the chain. See Ans. 9, 11 (“the use of BsaI (i.e. “B”) to cleave the new 3' end of the extended polynucleotide evidences the presence of BsaI at the 3' end of each TALE repeat sub-array unit, and its absence at the site of ligation of the a [sic] unit with the initial TALE repeat sub-array unit”); FF3. Examiner explains that the library of TALE repeat subarrays functions as the ‘building blocks’. Cleavage of each TALE repeat sub-array plasmid with BbsI results in a 5' end that is compatible for ligation with the 3' end of the growing polynucleotide, which is produced by cleavage with BsaI. Notably, the growing polynucleotide comprises multiple TALE repeat sub-arrays [(βγδϵ)]; the new 3' end formed at each cycle corresponds to the 3' end of the most recently added TALE repeat sub-array [(βγδϵ)]. Ans. 12. We are not persuaded by Appellant’s contention that there is insufficient disclosure in Joung’s provisional application to suggest building blocks having “compatible restriction sites on each side of the module.” Appeal Br. 22. Examiner relies on Joung’s teaching of “digestion of the TALE repeat sub-array plasmids with BbsI for 2 hrs, thereby indicating [] the presence of the BbsI recognition sequence in the cloned repeat unit.” Ans. 12–13. Examiner notes that “a BsbI [sic] recognition site is not present in the pUC cloning vector,” thus, the BbsI recognition site is part of the TALE polynucleotide sequence inserted into the plasmid. Appeal 2019-004044 Application 14/235,759 14 Ans. 12. Examiner notes that the presence of a BsaI recognition sequence in the cloned repeat unit of the library plasmids is not explicitly described, but is readily apparent in view of the TALE assembly method exemplified in example 1. Ans. 13; FF3. In particular, after adding a TALE repeat sub- array to the 3' end of the growing polynucleotide, the ‘new’ 3' end is cleaved with BsaI. Ans. 13. We agree with Examiner’s position that, even though, the disclosure in Joung’s provisional application does not recite the vector sequences, Joung’s teaching that the subarray unit (βγδϵ) is ligated to form the growing polynucleotide chain sufficiently supports the position that the digested polynucleotide ends are compatible. See FF3. Thus, notwithstanding Appellant’s contentions to the contrary, knowledge of the specific sequences of the plasmids containing TALE are not necessary to establish that the resulting enzyme cleavage products have ligatable ends. Here, the evidence further supports that the cleavage site for enzymes A and B are not present in the ligated product forming the elongated polynucleotide chain, because if such enzyme recognition and cleavage sites were present the polynucleotide, the chain would not grow. One skilled in the art must be presumed to know something about the art apart from what the references disclose. In re Jacoby, 309 F.2d 513, 516 (CCPA 1962); In re Sovish, 769 F.2d 738, 743 (Fed. Cir. 1985); see also KSR, 550 U.S. at 418 (An analysis under 35 U.S.C. § 103 “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.”). At the time of the invention, one of ordinary skill in the art would have been aware of Appeal 2019-004044 Application 14/235,759 15 the mechanics of the Golden Gate cloning protocol. The Golden Gate protocol provides that a DNA fragment of interest can be subcloned with very high efficiency in one step and one tube from one plasmid to another []. The principle of the cloning strategy is based on the ability of type IIs restriction enzymes to cut outside of their recognition site. Two DNA ends can be designed to be flanked by a type IIs restriction site such that digestion of the fragments removes the enzyme recognition sites and generates ends with complementary 4 nt overhangs; such ends can be ligated seamlessly, creating a junction that lacks the original site. . . . This property allows cloning to be performed using a one-step restriction-ligation. This strategy was shown to result in the conversion of more than half of all input plasmids present into the desired recombinant product in just a 30 minutes restriction ligation. Subcloning was also found to be very efficient when two and three inserts were subcloned, but the total amount of recombined plasmid was lower. Engler5 2 (emphasis added), see Spec. 3. Here, example 1 of Joung teaches that the same βγδϵ subarray unit is added in multiple rounds to the polynucleotide attached to the bead, and with each round the polynucleotide chain grows. FF3 (steps 2 and 5), see also FF1. Even if Joung does not disclose the sequence of the plasmids containing the polynucleotide building blocks, one of ordinary skill in the art would ensure that digestion with the type IIs restriction results in ends that are compatible for ligation purposes in order to grow the polynucleotide chain. Accordingly, we are not persuaded by Appellant’s contention that Joung’s example 1 provides insufficient information to establish that Joung teaches a polynucleotide 5 Engler et al., Golden Gate Shuffling: A. One-Pot DNA Shuffling Method Based on Type Us Restriction Enzymes, 4 PloS ONE eSSS3, 1–9 (2009) (“Engler”) submitted Jan. 28, 2014 with Information Disclosure Statement. Appeal 2019-004044 Application 14/235,759 16 building block having two different restriction sites on each side of the module that contain compatible ends. Considering the totality of the cited evidence and arguments, we conclude that the preponderance of the evidence supports Examiner’s conclusion of obviousness with respect to claim 40, and Appellant has not provided sufficient rebuttal evidence that outweighs the evidence supporting Examiner’s conclusion. As Appellant does not argue the claims separately, claims 41, 42, 45, 49, 53, 55, and 57 fall with claim 40. 37 C.F.R. § 41.37 (c)(1)(iv). DECISION SUMMARY In summary: Claims Rejected 35 U.S.C. § Reference(s)/Basis Affirmed Reversed 40–42, 45, 49, 55, 57 103 Joung, Schatz 40–42, 45, 49, 55, 57 40–42, 45, 49, 53, 55, 57 103 Joung, Schatz, Cease 40–42, 45, 49, 53, 55, 57 Overall Outcome 40–42, 45, 49, 53, 55, 57 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). See 37 C.F.R. § 1.136(a)(1)(iv). AFFIRMED Copy with citationCopy as parenthetical citation