DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on November 25, 2025 has been entered.
Application Status
Applicant’s remarks, and amendments to the claims and specification filed November 25, 2025 are acknowledged. Claims 22 and 35 were amended, and claim 44 was introduced. Claims 22-41, and 43-44 are pending and under examination herein.
Applicant’s remarks and amendments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow. Any rejection or objection not reiterated herein has been overcome by amendment.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosures of the prior-filed provisional applications, Application Nos. 62/140,454 and 62/210,451, fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Specifically, Application No. 62/140,454 does not disclose the subject matter of claims 23-24, 29, or 32-34. Specifically, SEQ ID NOs: 52, 58-61 recited in claims 23-24, 29, and 32-34 are first disclosed in Application No. 62/210,451 filed August 27, 2015. In addition, neither of Application Nos. 62/140,454 nor 62/210,451 disclose the subject matter of claims 26-27. SEQ ID NO: 71 recited in claims 26-27 is first disclosed in Application No. PCT/US2016/025426 filed March 31, 2016. Accordingly, the effective filing date of claims 23-24, 29, and 32-34 is August 27, 2015 (the filing date of Application No. 62/210,451), and the effective filing date of claims 26-27 is March 31, 2016 (the filing date of Application No. PCT/US2016/025426).
Notice to Joint Inventors
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim Rejections - 35 USC § 103 – Liu in view of Tan
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 22-25, 28-31, 35-37, 40, and 44 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record) in view of Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record). The rejections of claims 22-25, 28-31, 35-37, and 40 are maintained from the prior action with modification necessitated by Applicant’s amendments. The rejection of claim 44 is new and necessitated by Applicant’s amendments to the claims.
Regarding claims 22, 25 , and 44, Liu teaches a system for insertion of a donor nucleic acid into a genome ([0133]-[0136]; [0244]). Regarding (a), Liu teaches the system comprises a fusion protein comprising a first protein domain comprising a Cas9 protein lacking nuclease activity, hereinafter referred to as dCas9 (“A nuclease-inactivated Cas9 protein… “dCas9” protein (for nuclease “dead” Cas9)”, [0042]-[0043]; [0007]). Liu teaches the dCas9 specifically binds a target DNA in the presence of a guide RNA (“fusion protein comprising two domains are provided: (i) a protein capable of specifically binding a target nucleic acid (e.g., a nuclease-inactivated Cas9, as described herein)”, [0098]; [0007]; [0053]). Liu teaches the fusion protein comprises a second, active protein domain, e.g., a recombinase ([0006]; [0016]; [0062]), which is linked to the first protein domain via a linker ([0007]; [0051]). Liu teaches an XTEN linker (“FokI-L8”, Fig. 12A), which when disposed between the dCas9 and the second protein domain supports the highest expected fusion protein activity ([0169]). Liu provides working examples of dCas9 proteins fused to a second protein domain via the optimal XTEN linker (i.e., wherein the second protein domain is FokI) ([0166]-[0171]; Fig. 12A-B). The optimal XTEN linker of Liu has the amino acid sequence “SGSETPGTSESATPES” (Fig. 12A; [0094]), which is 100% identical to the amino acid sequence of instant SEQ ID NO: 61. See attached alignment.
Regarding (b), Liu teaches the system comprises a gRNA (at least [0122]).
Regarding (c), Liu teaches the system comprises a polynucleotide comprising a donor nucleic acid for insertion (“a nucleic acid comprising a donor sequence to be inserted is also provided, e.g., to a cell”, [0133]). Liu’s polynucleotide comprises sequences which are homologous to the genomic sequences flanking, i.e., on either side of, the target site ([0133]).
Liu teaches that Cas9 and recombinases are powerful tools which afford targeted genome modification in vitro and in vivo, and which may be harnessed for gene therapy purposes ([0002]; [0073]). Liu strongly suggests that improving target specificity is a pre-requisite for use of the proteins in clinical applications, and for high-efficiency genomic manipulations in basic research ([0003]). Liu teaches that the system advantageously improves the specificity of the fusion protein domains for their intended target, e.g., by “splitting the activities (e.g., target binding and target cleaving) of a nuclease between two or more proteins” ([0076]), and allows targeting of essentially any site specified by the gRNA ([0006]; [0079]).
Liu does not teach that:
the second protein domain comprises an integrase, and that the polynucleotide comprises a first IRS (claim 22); or
that the second protein domain is an HIV-1 integrase (claim 25).
Tan also teaches a system for insertion of a donor nucleic acid into a genome (“we constructed and purified various fusion proteins consisting of HIV-1 integrase and the polydactyl zinc finger protein E2C. The fusion proteins retained their integration activity and ability to bind specifically to the E2C-binding site”, pg. 1302, right col.; Abstract; Figs. 1A, 4-5). Regarding (a), Tan teaches the system comprises a fusion protein comprising a first protein domain comprising the polydactyl zinc finger protein, E2C (pg. 1302, right col.; Fig. 1A). Tan teaches the fusion protein comprises a second protein domain comprising HIV-1 integrase, which is linked to the first protein domain (pg. 1302, right col.; Fig. 1A). Regarding (c), Tan teaches the system comprises a polynucleotide comprising a donor nucleic acid for insertion, and a first integrase recognition site (IRS) (Fig. 4A; “U5 viral DNA,” “U5 double-stranded oligonucleotide… as the donor… substrate[]”, Fig. 4A description; results described pg. 1305-1310).
Tan teaches that retroviral integration, e.g., that mediated by HIV-1 integrase, can be exploited for genetic engineering and gene therapy (pg. 1301; pg. 1312, left col.). Like Liu, Tan teaches that a major challenge for its use in such applications is the nonspecific nature of the integration (pg. 1301, right col.). Tan teaches that fusions of integrase to “a sequence specific DNA-binding protein” allows for greater control of the site-specificity of integration (pg. 1301, right col.). However, Tan teaches that “[a] major limitation of the strategy is that the DNA-binding sequences of the previously tested fusion proteins are defined and fixed and may not necessarily be localized to a desired chromosomal site,” and “[i]n addition, these DNA-binding proteins can recognize multiple DNA variants of their consensus binding sequence, or the number of nucleotides required for specific protein-DNA interaction is insufficient for specifying a unique site within a mammalian genome” (pg. 1301, right col.). Thus, Tan’s system, like Liu’s attempts to overcome the challenges related to a lack of target specificity, and the inability to target any desired target site.
Regarding the second protein domain, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have fused the dCas9 protein and XTEN linker of Liu to the HIV-1 integrase taught by Tan, in order to prepare a system for insertion of a donor nucleic acid into a genome. It would have amounted to a simple substitution of two known second, active protein domains, by known means to yield predictable results. The skilled artisan would have had a reasonable expectation of success in preparing a dCas9-HIV-1 integrase fusion protein for insertion of a donor nucleic acid into a genome because Liu and Tan demonstrate that dCas9 and HIV-1 integrase, respectively, perform their expected functions as part of fusion proteins, and as evidenced by both Liu and Tan, preparing and optimizing fusion protein to achieve a desired result (i.e., genomic integration) was well within the purview of the skilled artisan (see working examples of Liu and Tan). The skilled artisan, recognizing the advantages of the dCas9 taught by Liu for improving target specificity and versatility of the HIV-1 integrase fusion protein taught by Tan, would have been motivated to fuse the dCas9 protein and XTEN linker of Liu to the HIV-1 integrase of Tan. The skilled artisan would have reasonably expected to achieve the benefits discussed by Liu and Tan, and improve the potential of Tan’s fusion protein for genetic engineering and gene therapy applications.
Regarding the first integrase recognition site (IRS), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have adapted the polynucleotide of Liu, to include a first IRS taught by Tan, in order to effectively insert the donor nucleic acid comprised by the polynucleotide using the system rendered obvious in paragraph 11 above. It would have amounted to modifying a known polynucleotide, by known means, to yield predictable results. The skilled artisan would have had a reasonable expectation of success in modifying the polynucleotide of Liu, using the teachings of Tan, because the sequences required for integration via HIV-1 integrase were well known in the art as evidenced by Tan, and synthesis of polynucleotides with a desired sequence was well within the purview of the skilled artisan as evidenced by both Liu and Tan. The skilled artisan would have recognized that the HIV-1 integrase taught by Tan requires specific sequences for integration of a donor nucleic acid, and therefore, would have been motivated to modify the polynucleotide of Liu to include a first IRS.
Regarding claims 23-24, Liu teaches the dCas9 comprises the amino acid sequence of SEQ ID NO: 52 (see alignment of record between Liu SEQ ID NO: 5 and instant SEQ ID NO: 52).
Regarding claims 28-29, Liu teaches that the XTEN linker has a length of 16 amino acids (Fig. 12A; [0094]). As stated above, the optimal XTEN linker of Liu has the amino acid sequence “SGSETPGTSESATPES” (Fig. 12A; [0094]), which is 100% identical to the amino acid sequence of instant SEQ ID NO: 61. See attached alignment.
Regarding claim 30, Liu teaches gRNAs comprising guide sequences (referred to as a “spacer” in Liu) with lengths in the claimed range (see for example, Fig. 12B, “gRNA spacer length (bp)” “14, 20, 25”).
Regarding claim 31, an “att site” is interpreted as a site which is recognized by HIV-1 integrase for integration into the genome (specification, [00163]). The first IRS taught by Tan is a “U5” site, which is an att site (“PCR-based assay for distribution and frequency of integration events,” and “In vitro assays for integrase activity”, pg. 1304-1305; “B2-1” and “C220”, Table 1; Fig. 4A).
Regarding claim 35, Liu teaches the fusion protein further comprises a nuclear localization signal ([0107]-[0116]; [0166]-[0173]).
Regarding claims 36-37, Liu teaches the system further comprises an in vitro human cell comprising the fusion protein, the gRNA, and the polynucleotide ([0118]; “the methods can be performed on a cell or tissue in vivo or ex vivo,” [0127]; “any of the methods… can be performed in vivo or in vitro,” [0132]-[0133]; “HEK293 cells,” [0173]).
Regarding claim 40, as stated above in paragraph 7, Liu’s polynucleotide comprises sequences which are homologous to the genomic sequences flanking, i.e., on either side of, the target site ([0133]). Thus, Liu teaches a polynucleotide comprising a 5’ arm of homology and a 3’ arm of homology.
Claim Rejections - 35 USC § 103 – Liu and Tan in view of GenBank AEA11266.1
Claims 26-27 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record) and Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record) as applied to claims 22-25, 28-31, 35-37, 40, and 44 in further view of GenBank AEA11266.1 (integrase, partial [Human immunodeficiency virus 1], GenBank: AEA11266.1, available 30 March 2011; of record). The rejections that follow are maintained from the prior action.
The teachings of Liu and Tan are described above in paragraphs 7-19 and applied as to claims 22-25, 28-31, 35-37, 40, and 44 above. Neither Liu nor Tan teach that the fusion protein’s HIV-1 integrase comprises the amino acid sequence of SEQ ID NO: 71. Indeed, while Tan teaches that the HIV-1 integrase is 288 amino acids in length (“DNA constructs”, pg. 1302; Fig. 1 description), Tan does not appear to teach the sequence of the HIV-1 integrase used in the fusion proteins.
GenBank AEA11266.1 teaches the sequence of HIV-1 integrase. An alignment between GenBank AEA11266.1’s sequence and instant SEQ ID NO: 71, illustrates that the sequences are 100% identical (see alignment of record).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have prepared the dCas9-HIV-1 integrase fusion protein rendered obvious in paragraph 11 above, using the HIV-1 integrase sequence taught by GenBank AEA11266.1. It would have amounted to substituting a generic HIV-1 integrase sequence, for a known amino acid sequence of HIV-1 integrase, by known means to yield predictable results. The obviousness of fusing the dCas9 protein of Liu to the HIV-1 integrase taught by Tan, via the optimal XTEN linker, in order to prepare a system for insertion of a donor nucleic acid into a genome is described above in paragraph 11 and applied here. The skilled artisan would have had a reasonable expectation of success in preparing the fusion protein with the sequence taught by GenBank because the sequence was known, and methods of preparing fusion proteins with specific, desired sequences was well-known in the art as evidenced by both Tan and Liu. The skilled artisan, seeking to prepare the fusion protein rendered obvious in paragraph 11 above, would have been motivated to use the known sequence of HIV-1 integrase taught by GenBank because Tan fails to provide the actual sequence of the HIV-1 integrase used in fusion proteins.
Claim Rejections - 35 USC § 103 – Liu and Tan in view of GenBank AAL65138.1
Claim 33 is rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record) and Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record) as applied to claims 22-25, 28-31, 35-37, 40, and 44 in further view of GenBank AAL65138.1 (methionine-initiated HIV-1 integrase [synthetic construct], GenBank: AAL65138.1, available 17 January 2002; of record). The rejection that follows is maintained from the prior action.
The teachings of Liu and Tan are described above in paragraphs 7-19 and applied as to claims 22-25, 28-31, 35-37, 40, and 44 above. Liu also teaches that prior to their disclosure, dCas9 had not been fused to an active, second domain ([0166]). Accordingly, Liu employs routine experimentation, using known linker sequences, to arrive at the most efficacious arrangements for the fusion proteins ([0166]-[0172]). Liu teaches that the arrangement of the fusion protein components, as well as the sequence and length of the linkers between each domain influence the activity of the fusion proteins ([0168]-[0172]; Fig. 11-12). Liu’s highest performing working example comprises the following structure: NLS-GGS-FokI-XTEN-dCas9 ([0173]). Liu teaches the sequences of each of the aforementioned components (“NLS Domain” is Liu SEQ ID NO: 318, [0093]; “GGS” corresponds to the simple “GGS” linker, [0172]; “FokI Cleavage Domain” is Liu SEQ ID NO” 6; “XTEN Linker” is Liu SEQ ID NO: 16; “dCas9 (D10A and H840A)” is Liu SEQ ID NO: 5, [0042]). The sequences of Liu, in the optimal arrangement determined by Liu through routine experimentation, results in the amino acid sequence below, wherein the NLS is italicized, FokI domain is underlined, and each linker is bolded:
MAPKKKRKVGIHRGVPGGSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFSGSETPGTSESATPESMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
Neither Liu nor Tan teach the sequence of the HIV-1 integrase present in the fusion protein represented in instant SEQ ID NO: 58. Indeed, while Tan teaches that HIV-1 integrase is 288 amino acids in length (“DNA constructs”, pg. 1302; Fig. 1 description), Tan does not appear to teach the sequence of HIV-1 integrase used in the fusion proteins.
GenBank AAL65138.1 teaches the sequence of a methionine-initiated HIV-1 integrase. An alignment between GenBank AAL65138.1’s sequence and instant SEQ ID NO: 58, illustrates that the sequences of the HIV-1 integrases are 100% identical (see alignment of record).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have prepared a dCas9-HIV-1 integrase fusion protein using Liu’s highest performing structure, and using the known sequence of HIV-1 integrase taught by GenBank. It would have amounted to substituting a known HIV-1 integrase sequence, into a known, optimized fusion protein structure, by known means to yield predictable results. The obviousness of fusing the dCas9 protein of Liu to the HIV-1 integrase taught by Tan, via the XTEN linker, in order to prepare a system for insertion of a donor nucleic acid into a genome is described above in paragraph 11 and applied here. The obviousness of substituting the generic amino acid sequence of HIV-1 integrase taught by Tan, for a known HIV-1 integrase sequence taught by GenBank is described above in paragraph 22 and applied here with respect to GenBank AAL65138.1. The skilled artisan would have arrived at the sequence below, wherein the FokI domain is substituted with the HIV-1 integrase sequence taught by GenBank AAL65138.1 (underlined). As shown in the alignment of record, the fusion protein below is 98.4% identical to the fusion protein represented by instant SEQ ID NO: 58, with no substitutions, mismatches, indels, or gaps.
The skilled artisan would have had a reasonable expectation of success in preparing the dCas9-HIV-1 integrase fusion protein below for insertion of a donor nucleic acid into a genome because I) Liu demonstrates that this specific arrangement, out of other working examples, was the optimal arrangement for the predicted activity of the fusion protein, and II) GenBank teaches a known HIV-1 integrase sequence that matches the sequence length used in the fusion proteins of Tan, and which includes a start codon. The skilled artisan, recognizing that prior to Liu’s disclosure, dCas9 proteins had not been fused to a second, active protein domain, would have been motivated to prepare the fusion protein using the optimal structures and sequences taught by Liu. In doing so, the skilled artisan would have reasonably expected to achieve an optimally functioning fusion protein.
MAPKKKRKVGIHRGVPGGSMFLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRNPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDSGSETPGTSESATPESMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
Claim Rejections - 35 USC § 103 – Liu, Tan, and GenBank AAL65138.1, in view of Terpe
Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record), Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record), and GenBank (methionine-initiated HIV-1 integrase [synthetic construct], GenBank: AAL65138.1, available 17 January 2002; of record) as applied to claims 22-25, 28-31, 33, 35-37, 40, and 44, in further view of Terpe (Terpe, 2003, Appl Microbiol Biotechnol, 60:523-533; of record). The rejection that follows is maintained from the prior action.
The teachings of Liu, Tan, and GenBank are described above in paragraphs 7-19, and 23-26, and applied as to claims 22-25, 28-31, 33, 35-37, 40, and 44 above. Liu also teaches the sequence of a 3x FLAG Tag ([0092], corresponding to Liu SEQ ID NO: 321). Liu teaches that the fusion proteins may be purified, by methods which are well-known in the art ([0058]; [0070]). Liu does not provide a specific location for the 3x FLAG Tag within the fusion protein. However, Terpe teaches that a “FLAG-tag can be located at the C- or N-terminus of the protein” (pg. 526, left col.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have prepared the fusion protein rendered obvious in paragraph 26 above, with the 3x FLAG Tag taught by Liu at the N-terminus as taught by Terpe. It would have amounted to combining a known sequence of a protein tag, with an obvious fusion protein sequence, by known means to yield predictable results. The obviousness of preparing the fusion protein 98.4% identical to the fusion protein of instant SEQ ID NO: 58 is described in paragraph 26 above and applied here. Combining the fusion protein above with the 3x FLAG Tag of Liu at the N-terminus as taught by Terpe, the skilled artisan would have arrived at the sequence below. As shown in the alignment of record, the fusion protein below is 100% identical to the fusion protein represented by instant SEQ ID NO: 58, with zero substitutions, mismatches, indels, or gaps. The skilled artisan would have had a reasonable expectation of success in preparing the dCas9-HIV-1 integrase fusion protein rendered obvious above with the 3x FLAG Tag taught by Liu, because as evidenced by Terpe the 3x FLAG Tag system is well-known in the art, and may be applied to either the C- or N-terminus of a protein. The skilled artisan would have been motivated to prepare the fusion protein with a 3x FLAG Tag because Liu teaches that the fusion proteins may be purified, by methods which are well-known in the art, and the skilled artisan would know that a 3x FLAG Tag facilitates purification of fusion proteins.
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPGGSMFLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRNPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDSGSETPGTSESATPESMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
Claim Rejections - 35 USC § 103 – Liu and Tan in view of Chow
Claims 41 and 43 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record) and Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record) as applied to claims 22-25, 28-31, 35-37, 40, and 44 above, in further view of Chow (Chow, WO 97/20038, published 5 June 1997; of record). The rejections that follow are maintained from the prior action.
The teachings of Liu and Tan are described above in paragraphs 7-19 and applied as to claims 22-25, 28-31, 35-37, 40, and 44 above. Neither Liu nor Tan teach:
that the system further comprises a reverse transcriptase (claim 41); or
that the polynucleotide further comprises a second IRS, wherein the donor nucleic acid is located between the first IRS and the second IRS (claim 43).
Chow, like Liu and Tan, teaches a system for integration of a donor nucleic acid into a genome (pg. 1, lines 11-18). Chow teaches the system comprises a fusion protein comprising a sequence specific DNA binding domain (i.e., “LexA”), fused to HIV-1 integrase (pg. 25, lines 23-30; Fig. 4; Table 3). Chow teaches a polynucleotide (“Donor DNA”) that comprises “end sequences of about 15-35 nucleotides derived from the U5 or U3 ends of the retroviral long terminal repeat (LTR)” (pg. 9, lines 8-21). Chow teaches that the sequence of the LTR corresponds to the retrovirus from which the integrase component of the fusion protein is obtained; for example, in the case of a fusion protein comprising HIV-1 integrase, “the sequence of the ends of the donor DNA will be constructed so as to mimic either the U5 or U3 end of the HIV-1 LTR” (pg. 9, lines 14-26). Chow teaches that “a gene for therapeutic purposes (e.g., cystic fibrosis gene), or a reporter gene for selection (e.g., neomycin resistant gene)” (pg. 9, lines 26-30) may be located between the two end sequences of the polynucleotide.
Chow also teaches that the system further comprises a reverse transcriptase, which allows for the use of “replication-defective retroviruses, which contain the donor RNA and the fusion protein” such that following infection, “the donor RNA will be made into cDNA by the viral reverse transcriptase… enter the nucleus and integrate into a specific site determined by the specificity of the DNA-binding moiety of the fusion protein” (pg. 59, line 26 to pg. 60, line 6).
Regarding claim 41, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the system rendered obvious in paragraph 11 above, with a replication-defective retrovirus as taught by Chow, and therefore, to include a reverse transcriptase as taught by Chow. It would have amounted to combining a known system, with a known delivery means and reverse transcriptase, by known means to yield predictable results. The skilled artisan would have had a reasonable expectation of success in combining the system with a replication-defective retrovirus and reverse transcriptase as taught by Chow, because Chow teaches that such delivery means and machinery are compatible with a system substantially identical to the one rendered obvious in paragraph 11 above. The skilled artisan would have been motivated to combine the system with a replication-defective retrovirus and a reverse transcriptase as taught by Chow, because viruses are known means of delivering the system components to cells as evidenced by both Chow and Liu ([0023]; [0136]).
Regarding claim 43, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have adapted the polynucleotide in the system rendered obvious in paragraph 0 above, to include U5 and U3 sites specific for HIV-1 integrase flanking the donor nucleic acid for insertion as taught by Chow. It would have amounted to modifying a known polynucleotide, by known means, to yield predictable results. The skilled artisan would have had a reasonable expectation of success in modifying the polynucleotide using the teachings of Chow, because both Tan and Chow teach a system which utilizes a fusion protein comprising an HIV-1 integrase, and because the sequences required for integration via HIV-1 integrase were well known in the art as evidenced by Tan and Chow. The skilled artisan would have recognized that the HIV-1 integrase in the fusion protein rendered obvious in paragraph 0 above requires specific sequences for integration of a donor nucleic acid, and therefore, would have been motivated to modify the polynucleotide to include the sequences flanking the donor nucleic acid as taught by Chow.
Claim Rejections - 35 USC § 103 – Liu, Tan, and Chow, in view of Matsuda
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record), Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record), and Chow (Chow, WO 97/20038, published 5 June 1997; of record) as applied to claims 22-25, 28-31, 35-37, 40-41, and 43-44, in further view of Matsuda (Matsuda et al., October 1998, Journal of Virology, Vol. 72, No. 10, p. 8396-8402; of record). The rejection that follows is maintained from the prior action.
The teachings of Liu, Tan, and Chow are described above in paragraphs 7-19 and 30-34, and applied as to claims 22-25, 28-31, 35-37, 40-41, and 43-44 above. Neither Liu, Tan, or Chow teach that the polynucleotide comprises a first att site comprising the nucleotide sequence of SEQ ID NO: 59, and a second att site comprising the nucleotide sequence of SEQ ID NO: 60 (claim 31).
Matsuda teaches the sequences of HIV-1 integrase U5 and U3 sites (“U5att (WT)” and “U3att(WT)”, Fig. 1). As shown in the alignments of record, the U3 and U5 sites taught by Matsuda are 100% identical to SEQ ID NOs: 59 and 60, respectively.
It would have been obvious to of ordinary skill in the art before the effective filing date of the claimed invention to have prepared the polynucleotide comprising U3 and U5 sites specific for HIV-1 integrase rendered obvious in paragraph 34 above, using the specific U3 and U5 sequences taught by Matsuda. It would have amounted to substituting generic U3 and U5 sequences taught by Chow, for specific, known sequences of HIV-1 integrase U3 and U5 sites, by known means to yield predictable results. The obviousness of including U5 and U3 sites specific for HIV-1 integrase flanking the donor nucleic acid for insertion as taught by Chow is described above in paragraph 34 and applied here. The skilled artisan would have had a reasonable expectation of success in preparing the polynucleotide with the sequences taught by Matsuda because the sequences were known sites recognized by HIV-1 integrase, and methods of preparing polynucleotides with specific, desired sequences was well-known in the art. The skilled artisan, seeking to prepare the polynucleotide rendered obvious in paragraph 34 above, would have been motivated to use the known sequences taught by Matsuda because Chow fails to provide the actual sequences of the U3 and U5 sites specific for HIV-1 integrase, which facilitate integration of a donor nucleic acid into a genome.
Claim Rejections - 35 USC § 103 – Liu and Tan in view of Maruyama
Claims 38-39 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu et al., US 2015/0071898 A1, published March 12, 2015; of record) and Tan (Tan et al., February 2004, Journal of Virology, Vol. 78, No. 3, p. 1301-1313; of record) as applied to claims 22-25, 28-31, 35-37, 40, and 44 in further view of Maruyama (Maruyama et al., 23 March 2015, Nature Biotechnology, Vol. 33, No. 5, pg. 538-546; of record). The rejections that follow are maintained from the prior action.
The teachings of Liu and Tan are described above in paragraphs 7-19 and applied as to claims 22-25, 28-31, 35-37, 40, and 44 above. Liu also teaches that the system can be used to modify genes, e.g., those associated with disease ([0056]), or as part of a therapeutic approach (Example 2, [0226]-[0230]). For example, Liu teaches inactivating a CCR5 allele in cells ([0230]). Neither Liu or Tan teach:
that the donor nucleic acid comprises a stop codon (claim 38); or
that the donor nucleic acid encodes a first coding exon of a gene or a portion of the first coding exon, wherein the first coding exon comprises the stop codon (claim 39).
Maruyama teaches a polynucleotide (“Targeting template (dsDNA)”) comprising a donor nucleic acid comprising a stop codon (“Stop cassette”, Fig. 1A). Maruyama demonstrates that polynucleotides comprising a donor nucleic acid can be inserted into multiple desired sites with a Cas9 and suitable gRNA, including the first coding exon or a 3’UTR of a gene (see, for example, the donor nucleic acids and target sites in Figs 1D, or Fig. 2A).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the generic donor nucleic acid in the system rendered obvious in paragraph 0 above, with a donor nucleic acid encoding a first coding exon of a gene or a portion thereof, wherein the first coding exon comprises a stop codon, as taught by Maruyama. The skilled artisan would have had a reasonable expectation of success in modifying the donor nucleic acid to encode a first coding exon of a gene or a portion thereof and a stop codon, because Maruyama teaches multiple donor nucleic acids which encode sequences from various regions of different genes, and which include stop codons. The skilled artisan would have recognized the utility of the stop codon-containing donor nucleic acid of Maruyama for the purposes of inactivating a disease allele, e.g., like a CCR5 allele taught by Liu, and would have been motivated to modify the generic donor nucleic acid to one comprising the first coding exon of a gene or a portion thereof, and a stop codon, because as demonstrated by Maruyama such donor nucleic acids can be successfully inserted into a desired genomic location, and would be predicted to result in a premature, inactivating stop codon at the target gene.
Response to Remarks - 35 USC § 103
Applicant’s remarks regarding the § 103 rejections over Liu and Tan have been thoroughly reviewed. Applicant cites MPEP 2143(I)(A) (“The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements…”). Examiner acknowledges these requirements, but notes that the obviousness rejection in paragraph 11 above applies a substitution-based rationale, rather than a combination-based rationale (“It would have amounted to a simple substitution of two known second, active protein domains, by known means to yield predictable results.”). Examiner has met the requirements described in MPEP 2143(I)(B) to apply a substitution-based rationale with respect to the fusion protein domains.
Applicant’s argues that “there would have been no reasons for a person of ordinary skill in the art to modify the dCas9-FokI fusion protein of Liu with an integrase domain of the E2C-integrase (E2C-Int) fusion protein of Tan because the fusion proteins of the systems of Liu[] and Tan are so significantly different from one another, with regard to their structures, mechanisms of actions, and functions that the principle of operation of the prior art would have to substantively change upon making the combination to arrive at the claimed invention.” These arguments are not convincing. The skilled artisan would have recognized that the structures, mechanism of actions, functions, and principle of operation of the prior art references is essentially the same – a fusion protein comprising a site-specific DNA binding domain and a second domain (i.e., FokI or recombinase in Liu, or HIV-1 integrase in Tan) facilitates insertion of a donor nucleic acid into a genome. The skilled artisan would have recognized that a second domain could be fused to a DNA binding domain (e.g., a zinc finger protein, dCas9 protein, etc.) by means well known in the art, with a reasonable expectation that the fused domains would retain their separate functions for the purposes of site-specific integration. The rejection also provides motivation for substituting the fusion protein domains (“The skilled artisan, recognizing the advantages of the dCas9 taught by Liu for improving target specificity and versatility of the HIV-1 integrase fusion protein taught by Tan, would have been motivated to fuse the dCas9 protein and XTEN linker of Liu to the HIV-1 integrase of Tan.”).
Applicant also argues that “neither Liu nor Tan provide any expectation that an integrase domain would have had activity in the system of Liu modified with the integrase domain of Tan.” This argument has been addressed previously (paragraph 47 of the prior action). Means of making functional fusion proteins were known as evidenced by Tan and Liu. Tan provides a fusion protein with a functional integrase domain. Liu provides a fusion protein with a functional dCas9 DNA binding domain. There is no evidence of record that the skilled artisan could not have prepared a fusion protein with a dCas9 DNA binding domain and a functional integrase domain, or that the skilled artisan would not have expected each domain to be functional when fused using known means in the art.
Applicant states that “the sole working example of Liu relates to an obligate dimeric Cas9 system, in which a dCas9 was fused to a FokI restriction cleavage domain that would cleave DNA only when two distinct FokI-dCas9:gRNA complexes bound to adjacent half sites.” Applicant argues that the “FokI domain of the dCas9-FokI fusion protein of Liu is [] unrelated to an integrase activity of the integrase domain of the E2C-Int fusion protein of Tan” because of it “requires dimerization for nuclease activity.” These arguments are not convincing. As stated in the prior action, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). The rejection is not based on any single working example of Liu, nor is the rejection based on any alleged mechanistic similarities (or dissimilarities) of the heterologous domain of Liu (i.e., FokI) to the heterologous domain of Tan (i.e., HIV-1 integrase) The rejection relies on the teachings of Liu and Tan as whole. Based on Liu, the skilled artisan would have understood that functional fusion proteins comprising dCas9 and a second domain, e.g., recombinase, FokI, could be prepared. The skilled artisan would have understood that functional fusion proteins comprising a DNA binding domain and an HIV-1 integrase could be prepared based on Tan. Liu and Tan provide evidence that the respective DNA binding domains and second domains perform their expected function when parts of a fusion protein. Together, the skilled artisan would have had the means (as described in further detail below), and substantial motivation (see paragraphs 10-11 above) to prepare a dCas9-HIV-1 integrase fusion protein.
Applicant argues that “the E2C domain of the E2C-Int fusion protein of Tan is unrelated to the dCas9 domain of the dCas9-FokI fusion protein of Liu… [because] the mechanisms of action with regard to targeting nucleic acid sequences are completely unrelated between the E2C-Int fusion protein of Tan and the dCas9 domain of the dCas9-FokI fusion protein of Liu.” This argument is not convincing. First, the E2C domain of Tan and the dCas9 domain of Liu are related; both Liu and Tan describe that E2C and dCas9 act as DNA binding domains (Tan: “One class of DNA-binding proteins… are the synthetic proteins derived from… zinc finger proteins… One example of such a polydactyl protein is E2C,” pg. 1302, left col.; Liu: “fusion proteins comprising two domains are provided: (i) a protein capable of specifically binding a target nucleic acid (e.g., a nuclease-inactivated RNA programmable nuclease such as a nuclease-inactivated Cas9, as described herein)… In some embodiments, domain (i) of the aforementioned fusion protein comprises a DNA binding domain, for example a DNA binding domain of a zinc finger or TALE protein,” [0098]). The skilled artisan would have certainly recognized this level of “relatedness,” but also would have been well aware of the mechanistic differences between various DNA binding proteins. See description of zinc finger proteins, dCas9 proteins, and TALEs in Liu (at least [0042]; [0071]-[0072]; [0067]-[0068]). These mechanistic differences would not have deterred the skilled artisan from arriving at the instantly claimed fusion protein. On the contrary, the mechanistic differences between E2C and dCas9 were the very basis for the motivation to arrive at the instant invention in the rejection above (“The skilled artisan, recognizing the advantages of the dCas9 taught by Liu for improving target specificity and versatility of the HIV-1 integrase fusion protein taught by Tan, would have been motivated to fuse the dCas9 protein and XTEN linker of Liu to the HIV-1 integrase of Tan.”).
Applicant provides that “the dCas9 domain of the dCas9-FokI fusion protein of Liu is 1367-residues long and thus substantially larger than the 184-residue E2C domain of the E2C-Int fusion protein of Tan.” Applicant asserts, therefore, that “modifying the dCas9-FokI fusion protein of Liu with the integrase domain of the E2C-Int fusion protein of Tan would result in a drastic change in context for the integrase domain.” Applicant submits that “protein folding as well as the location or occlusion of active sites would be so substantially different for an integrase domain in a large, modified fusion protein compared to the integrase domain of the much smaller E2C-Int fusion protein that an activity… could not have been readily predicted….” These arguments are not convincing. First, Applicant has not provided any evidence that the skilled artisan would predict substantial differences in protein folding and/or occlusion of active sites when preparing the fusion protein rendered obvious above, e.g., structural studies which illustrate that dCas9 or HIV-1 integrase could not be fused to a separate domain, or would be incompatible with one another. The prior art evidence further described below also does not support Applicant’s opinion.
Examiner agrees that the “context” of the integrase domain changes relative to Tan, i.e., it is fused to a different DNA binding domain. However, swapping different DNA binding domains of fusion proteins was well known in the art. Indeed, Liu explicitly discusses using a zinc finger protein or dCas9 as a DNA binding domain of a fusion protein (“fusion proteins comprising two domains are provided: (i) a protein capable of specifically binding a target nucleic acid (e.g., a nuclease-inactivated RNA programmable nuclease such as a nuclease-inactivated Cas9, as described herein)… In some embodiments, domain (i) of the aforementioned fusion protein comprises a DNA binding domain, for example a DNA binding domain of a zinc finger or TALE protein,” [0098]). The skilled artisan would have recognized that Tan’s zinc finger protein, E2C, and dCas9 perform the same function in their respective fusion proteins, and therefore, would have predicted a fusion protein with either E2C or dCas9 would have DNA binding capability.
The skilled artisan would have also been well versed in means to prepare functional fusion proteins, using domains of different sizes, different linker sequences, and/or domain positioning to support each domain’s independent function. Indeed, Tan prepares a zinc finger protein (E2C)-HIV-1 integrase fusion, and Liu prepares functional dCas9-FokI fusions, which Applicant’s remarks indicate have domains of different sizes. Liu and Tan both illustrate that the domains in the fusion proteins retain their respective functions when combined, as when separate. The evidence supporting this conclusion is described in paragraph 47 of the prior action and applied here. The skilled artisan also would have had knowledge of many other fusion proteins in the art, including other dCas9 fusions, e.g., dCas9 fused to VP64 or KRAB domains described by Doudna (Doudna and Charpentier, 25 November 2014, Science, Vol. 346, Issue 6213; pg. 1258096-6, left col.). Liu also teaches various linker sequences and domain arrangements for dCas9 fusion proteins, and demonstrates that the structure of dCas9 fusion proteins can be optimized using routine experimentation (i.e., modifying the arrangement of the fusion partners, and sequence of linkers, ([0169]).
Taken together, the skilled artisan would have had extensive knowledge of various DNA binding domains, e.g., zinc finger proteins, dCas9, etc., which they would have believed to be interchangeable for the purposes of DNA binding. The skilled artisan also would have had extensive knowledge of means to prepare functional fusion proteins. The skilled artisan, therefore, could have prepared a dCas9-HIV-1 integrase fusion protein, beginning with the optimized dCas9 fusion protein structure which Liu illustrates is supportive of genomic modification. Coupled with the demonstrated functionality of Tan’s and Liu’s fusion proteins, the skilled artisan would have predicted that substituting the two second domains would result in a fusion protein which has both DNA binding and HIV-1 integrase capabilities.
Applicant also alleges that “there was a belief that certain oligonucleotides inhibited the function of proteins, such as integrases.” Applicant cites Radomska which “discloses certain aptamers, such as short oligonucleotides, inhibited certain proteins, including HIV integrase.” Applicant also cites de Soultrait, Mouscadet, and Snasel which also disclose inhibition of HIV-1 integrase by certain oligonucleotides.” It is not entirely clear how Applicant intends these references to support non-obviousness of the instant claims over Liu and Tan. Given Applicant’s earlier arguments related to the mechanistic differences between the two DNA binding domains, Examiner assumes that Applicant is attempting to argue that the skilled artisan’s knowledge of certain prior art oligonucleotides that may inhibit HIV-1 integrase, would teach away from fusing an HIV-1 integrase domain with a dCas9 domain which requires a gRNA.
This argument is not convincing. The teachings of Radomska, de Soultrait referred to by Applicant relate to “tetraplex aptamer[s],” “triple helix mediated inhibition,” and “oligonucleotides known as guanosine quartets.” Snasel (Snasel et al., 2001, Eur. J. Biochem. 268, pg. 980-986) describes modified double-stranded oligonucleotides derived from HIV-1 LTRs, that competitively inhibit HIV-1 action on substrate LTRs (Abstract; Results, pg. 981-982; Discussion). Mouscadet (Mouscadet et al., 1994, The Journal of Biological Chemistry, Vol. 269, No. 34, pg. 21635-21638) describes the use of “oligonucleotide-intercalator conjugates” to promote formation of a “short DNA triplex” that inhibits HIV-1 integrase (Abstract; Fig. 1; pg. 21635). The skilled artisan would know the structure of a gRNA, and know the structure formed by a gRNA at a target locus. See Fig. 1 and paragraph [0059] of Liu, for example. None of the references cited by Applicant describe an oligonucleotide which the skilled artisan would perceive a resembling the structure of a gRNA, or the structure of a gRNA/Cas9 complex at a target locus. The skilled artisan, reading Radomska, de Soultrait, Snasel, and Mouscadet, would have recognized major structural differences between gRNAs and the HIV-1-inhibiting oligonucleotides, e.g., gRNAs are single-stranded, form a double-stranded substrate with a target site, are non-intercalating, etc. So, while the skilled artisan may have been aware of the aforementioned HIV-1 inhibitors, there is no convincing evidence of record that mere knowledge of HIV-1 inhibitors which are tetraplex aptamers, guanosine quartets, double-stranded oligonucleotides derived from HIV-1 LTRs, or which produce triple-helix mediated inhibition, would have steered the skilled artisan away from making a dCas9-HIV-1 integrase fusion protein for the purposes of site-specific integration.
Finally, regarding the rejections in further view of GenBank I, GenBank II, Terpe, Chow, Matsuda, and Maruyama, Applicant submits that none of the references remedy the aforementioned deficiencies of Liu and Tan described above. Examiner notes that these references were not cited to remedy any alleged deficiency of Liu and Tan. Applicant’s remarks regarding the alleged deficiencies of Liu and Tan are addressed in the preceding paragraphs and applied herein with respect to the rejections in further view of GenBank I, GenBank II, Terpe, Chow, Matsuda, and Maruyama.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JENNA L PERSONS whose telephone number is (703)756-1334. The examiner can normally be reached M-F: 9-5pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, JENNIFER A DUNSTON can be reached at (571) 272-2916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/JENNA L PERSONS/Examiner, Art Unit 1637
/Soren Harward/Primary Examiner, TC 1600