DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/19/2025 has been entered.
Applicant’s amendments to the claims and arguments filed on December 19, 2025 have been received and entered. Claims 1, 4, 8, 11 have been amended, while claims 3, 7, 10, 15-58 have been canceled. New claims 63-64 have been added. Claims 1-2, 4-6, 8-9, 11-14, 59-63 and 64 are pending in the instant application.
Election/Restrictions
Applicant’s election of claims 1-15 in the reply filed on January 21, 2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Priority
This application is a 371 of PCT/US2020/054745 filed on 10/08/2020, which claims priority from US provisional application no 62/913,092 filed on 10/09/2019.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 12/19/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Claims 1-2, 4-6, 8-9, 11-14, 59-63 and 64 are under consideration.
Withdrawn-Claim Rejections - 35 USC § 112
Claims 7 and 15 were rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Maintained & New-Claim Rejections - 35 USC § 103-in modified form
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 1-2, 4-5, 8-9, 11-12, 14, 59, 61, 63 and 64 are also rejected under 35 U.S.C. 103 as being unpatentable over in view of Padidam (USPGPUB 20180346934, dated 12/6/2018filed on 6/6/2018, EFD2/2/2005), Inniss et al (Biotechnology and Bioengineering, 2017, 114(8), 1837-1846) as evidenced by Jusiak (ACS Synth. Biol. 2019, 8, 16−24) and (Xu et al (BMC Biotechnology 2013, 13:87, 1-17).
Claims are directed to a mouse comprising within its genome a first attP Bxb1 attachment site comprising the sequence of SEQ ID NO: 1 and a second attP* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1,
or a first attB Bxb1 attachment site comprising the sequence of SEQ ID NO: 2 and a second attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 2.
With respect to claims 1, 8, 14, Padidam teaches transgenic animal whose genome comprising a first recombination attachment site and a second recombination attachment site, wherein the recombinase is selected from the group consisting of an A118 recombinase, a SF370.1 recombinase, a SPβc2 recombinase, a ϕRv1 recombinase, and a Bxb1 recombinase (see par. 145, 167, 172). Padidam further teaches embryo (para 167), wherein the animal is produced by introducing a single cell embryo comprising a nucleic acid construct comprising a first recombination site capable of recombining with a second recombination site found within the genome of the organism from which the cell was derived and a nucleic acid fragment of interest, in a manner such that the nucleic acid fragment of interest is stably integrated into the DNA of germline cells of the animal (see para. 172, 212), wherein animal is mouse or rat (see para. 168).
Regarding claims 2, 5, 9-10, Padidam teaches that the animal of claim 1, further comprising a polynucleotide encoding Bxb1 recombinase (para 205). It is further disclosed that the first Bxb1 attachment site is an attP site; the second Bxb1attachment site is an attB site (para212). Padidam teaches that the first and second Bxb1 attachment sites are separated from each other by 50 to 500 nucleotides (see para. 32) as recombination sites typically include left and right arm separated by a core or spacer region (see para. 212). Padidam discloses a 52 bp synthetic oligonucleotide sequence containing the attP site of Bxb1 recombinase (SEQ ID NO: 16 that has 100% sequence identity to SEQ ID NO: 1), and a 46bp synthetic oligonucleotide sequence containing the attB site (SEQIDNO:17 that has 100% sequence identity to SEQ ID NO: 2) of Bxb1 recombinase were cloned in that order between the CMV promoter and luciferase gene (see para. 212). Xu who teaches wild type Bxbl attachment site is selected from an attP site and/or attB site, wherein an attP sequence that has 100% sequence identity to SEQ ID NO: 1 and/or an attB sequence that has 100% sequence identity to SEQ ID NO: 2 (table 2).
Padidam contemplated among various embodiment a mouse or mouse embryo whose genome comprises a first recombination attachment site and a second recombination attachment site, wherein recombinase is selected from a group of recombinases including Bxb1 recombinase, however, the reference differs from claimed invention by not disclosing
(i) a first attP Bxb1 attachment site comprising the sequence of SEQ ID NO: 1 and a second attP* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1,
or a first attB Bxb1 attachment site comprising the sequence of SEQ ID NO: 2 and a second attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 2 and
(ii) an attP Bxb1 attachment site comprising a GT dinucleotide pairing comprising SEQ IDNO: 1 and a modified attP* Bxb1 attachment site comprises a GA dinucleotide pairing of SEQ ID NO: 7.
Inniss cures the deficiency by disclosing a Bxbl recombinase-mediated cassette exchange (RMCE) system comprising two Bxbl attp attachment sites in the genome, while a donor polynucleotide comprising a sequence of interest flanked by attB isconstructed for insertion of the sequence of interest in the CHO cell genome (see page 1838 Fig. 1). Inniss discloses that "Bxbl integrase is very efficient in catalyzing recombination between its attP and attB sites in mammalian cells. The in vitro data shows that altering the GT dinucleotide in the center of the attP site disrupts its ability to recombine with the wild-type attB site, while making compensatory mutations on attB can restore the function of the attP: attB pair. Inniss paired the wild-type GT sequence with a GA mutant that proved orthogonal to the wild-type GT sequence to construct the Bxbl RMCE system "( page 1838, col. 1, paragraph 1, Figure 1A) (limitation of claims 1, 4, 8, 11, 63 and 64). The use of wild-type GT sequences in the RMCE landing pad and the corresponding donor DNA, respectively, paired with the GA mutant, avoids integration of the plasmid backbone into the landing pad as GT and GA are orthogonal. Inniss shows that the Bxbl RMCE system is specific and highly efficient (see page 1845, col. 1, para. 1). This is further evidenced by Jusiak who reported Bxb1-GA point mutant sites do not cross react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. Jusiak tested the efficiency and orthogonality of ϕC31 and Wβ integrases, and show that Wβ has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines (abstract).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art to combine the teachings of prior art to modify the mouse of Padidam to insert donor nucleic acid that is flanked by attp/attb Bxb1 attachment sites using serine-integrase mediated integration as disclosed in Inniss/ Jusiak, with a reasonable expectation of success, to improve the serine integrase system efficiency in a mouse and/or mouse embryo, as instantly claimed, before the effective filing date of the instant application. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated select Bxb1 attachment sites over other as prior art recognized that Bxb1 integrase yielded more recombinants and displayed less damage to the recombination sites as compared to other serine integrase including φC31 integrase (see Xu). Further, prior art also recognized the advantage of altering the GT dinucleotide in the center of the bxb1 attachment site with a GA mutant that is orthogonal to the wild-type GT sequence to construct the Bxbl RMCE system to prevent integration of the plasmid backbone into the landing pad as GT and GA are orthogonal. Absent evidence of any unexpected and/or superior results, one of ordinary skill in the art would have been expected to have a reasonable expectation of success in using Bxb1 attachment site because prior art successfully reported inserting a first Bxb1 attachment site and a second modified Bxb1 attachment to insert gene of interest from the teaching of Inniss. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396).
Claims 6, 8, 13 , 60 and 62 are also rejected under 35 U.S.C. 103 as being unpatentable over in view of Padidam (USPGPUB 20180346934), Inniss et al (Biotechnology and Bioengineering, 2017, 114(8), 1837-1846) as evidenced by Jusiak (ACS Synth. Biol. 2019, 8, 16−24), Xu et al (BMC Biotechnology 2013, 13:87, 1-17) as applied above and further in view of Schillit et al (Curr Protoc Hum Genet. 2017 91: 15.10.1–40).
The teaching of Padidam, Inniss as evidenced by Xu haven been described above and relied in same manner here. The combination of references differs from claimed invention by not disclosing a mouse or mouse embryo whose genome comprises a first and a second Bxb1 attachment sites or are within a safe harbor locus .
Schillit teaches transgenesis using mice comprising attP sites and attB sites recognized by the serine integrase that are routinely used to insert donor plasmid through PhiC31-integrase using CRISPR/Cas mediated integration into a Rosa26 locus (see figure 15.10.1, Table 15.10.1; 15010.3 and figures) (limitation of claims 59-62).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art to combine the teachings of prior art to modify the mouse of Padidam to insert donor nucleic acid that is flanked by attp/attb Bxb1 attachment sites using serine-integrase using CRISPR/Cas mediated integration into a Rosa26 locus as disclosed in Schillit and Inniss/ Jusiak, with a reasonable expectation of success, to improve the serine integrase system efficiency in a mouse and/or mouse embryo, as instantly claimed, before the effective filing date of the instant application. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated select Bxb1 attachment sites over other as prior art recognized that Bxb1 integrase yielded more recombinants and displayed less damage to the recombination sites as compared to other serine integrase including φC31 integrase (see Xu). Further, prior art also recognized the advantage of inserting donor nucleic acid that is flanked by recombination attachment sites within Rosa26 locus because such would exhibit ubiquitous and high expression that is consistent between transgenic littermates and from generation to generation. Further, prior art also recognized the advantage of altering the GT dinucleotide in the center of the bxb1 attachment site with a GA mutant that is orthogonal to the wild-type GT sequence to construct the Bxbl RMCE system to prevent integration of the plasmid backbone into the landing pad as GT and GA are orthogonal. Absent evidence of any unexpected and/or superior results, one of skill in the art would have been expected to have a reasonable expectation of success in using Bxb1 attachment site because prior art successfully reported inserting a first Bxb1 attachment site and a second Bxb1 attachment to insert gene of interest using CRISPR/Cas9 mediated homologous recombination as evident from the teaching of Inniss. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396).
Claims 1-2, 4-6, 8-9, 11-14, 59-63 and 64 are also rejected under 35 U.S.C. 103 as being unpatentable over in view of Tasic (PNAS, 2011, 108, 7902-7907 or US2012124686) as evidenced by Schillit et al (Curr Protoc Hum Genet. 2017 91: 15.10.1–40), Inniss et al (Biotechnology and Bioengineering, 2017, 114(8), 1837-1846)/ Jusiak (ACS Synth. Biol. 2019, 8, 16−24) and further in view of Xu et al (BMC Biotechnology 2013, 13:87, 1-17).
Regarding claims 1-2, 8-9, Tasic teaches a mouse embryo and transgenic mouse whose genome comprises a first phiC31 attachment site and a second a second phiC31 attachment site and further comprising a nucleic acid encoding phiC31 integrase (see fig. 1).
With respect to claims 6, 13 and 62, Tasic teaches discloses a mouse comprising multiple attP sites inserted within the Rosa26 locus. These mice serve as embryo donor that are injected with a recombinase and a vector comprising a gene of interest flanked by attB sites (see fig. 1). This is further supported by Schillit who teaches transgenesis using mice comprising attP sites and attB sites recognized by the PhiC31 serine integrase that are routinely used to insert donor plasmid through PhiC31-integrase using CRISPR/Cas mediated integration into a Rosa26 locus (see figure 15.10.1, Table 15.10.1; 15010.3 and figures) (limitation of claims 59-62). Tasic differs from claimed invention by not disclosing use of
(i) a first attP Bxb1 attachment site comprising the sequence of SEQ ID NO: 1 and a second attP* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1,
or a first attB Bxb1 attachment site comprising the sequence of SEQ ID NO: 2 and a second attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 2 and
(ii) an attP Bxb1 attachment site comprising a GT dinucleotide pairing comprising SEQ IDNO: 1 and a modified attP* Bxb1 attachment site comprises a GA dinucleotide pairing of SEQ ID NO: 7.
However, before the effective filing date of instant application, the use of Bxb1, alone or in combination with PhiC31, to insert large DNA segment in a safe harbor of a mammalian cell was known in prior art. Inniss cures the deficiency by disclosing a Bxbl recombinase-mediated cassette exchange (RMCE) system comprising two Bxbl attp attachment sites in the genome, while a donor polynucleotide comprising a sequence of interest flanked by attB is constructed for insertion of the sequence of interest in the CHO cell genome (see page 1838 Fig. 1). Inniss discloses that "Bxbl integrase is very efficient in catalyzing recombination between its attP and attB sites in mammalian cells. The in vitro data shows that altering the GT dinucleotide in the center of the attP site disrupts its ability to recombine with the wild-type attB site, while making compensatory mutations on attB can restore the function of the attP: attB pair. Inniss paired the wild-type GT sequence with a GA mutant that proved orthogonal to the wild-type GT sequence to construct the Bxbl RMCE system "( page 1838, col. 1, paragraph 1, Figure 1A) (limitation of claims 1, 4, 8, 11, 63 and 64). The use of wild-type GT sequences in the RMCE landing pad and the corresponding donor DNA, respectively, paired with the GA mutant, avoids integration of the plasmid backbone into the landing pad as GT and GA are orthogonal. Inniss shows that the Bxbl RMCE system is specific and highly efficient (see page 1845, col. 1, para. 1). Xu provide the requisite guidance with respect to Bxbl attachment site is selected from an attP site and/or attB site, wherein an attP sequence that has 100% sequence identity to SEQ ID NO: 1 and/or an attB sequence that has 100% sequence identity to SEQ ID NO: 2 (table 2).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art to combine the teachings of prior art to replace the PhiC31 attachment site as disclosed in Tasic/ Schillit with attp/attb Bxb1 attachment sites using serine-integrase mediated integration as disclosed in Inniss, with a reasonable expectation of success, to improve the serine integrase system efficiency in a mouse and/or mouse embryo, as instantly claimed, before the effective filing date of the instant application. Said modification amounting to combining prior art elements according to known methods to yield predictable results. Other limitation of first and second attachment site are separated from each other by 50 to 500 bp would be obvious modification of known use of Bxb1 attachment sites disclosed in Xu. One of ordinary skill in the art would be motivated select Bxb1 attachment sites over other as prior art recognized that Bxb1 integrase yielded more recombinants and displayed less damage to the recombination sites as compared to other serine integrase including φC31 integrase (see Xu). Further, prior art also recognized the advantage of altering the GT dinucleotide in the center of the bxb1 attachment site with a GA mutant that is orthogonal to the wild-type GT sequence to construct the Bxbl RMCE system to prevent integration of the plasmid backbone into the landing pad as GT and GA are orthogonal. Absent evidence of any unexpected and/or superior results,One of skill in the art would have been expected to have a reasonable expectation of success in using Bxb1 attachment site because prior art successfully reported landing pad vectors containing a first Bxb1 attachment site, TK expression unit, and an SV40 promoter driving expression of EGFP followed by a second Bxb1 attachment to insert gene of interest using CRISPR/Cas9 mediated homologous recombination. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396).
Response to arguments
Applicant disagrees with the rejection arguing that the amended claims he cited combination of references does not teach or suggest a mouse or mouse embryo comprising the newly added elements of amended claim 1. Padidam, Inniss, and Xu each describe bulk in vitro cell editing and rely on antibiotic selection to enrich for edited clones, 6 a process that is incompatible with editing mouse embryos. Schilit, by contrast, describes the generation of "seed mice" using CRISPR/Cas9-mediated genome editing, to precisely insert a defined attB or attP attachment site at a selected genomic locus. Schilit describes PhiC31-mediated transgene integration only as a secondary approach that incorporates vector backbone sequences into the genome and therefore requires an additional excision step.⁷ This disclosure confirms that Schilit relies on CRISPR/Cas9 for precise targeted genome modification, while PhiC31 introduces technical drawbacks rather than enabling clean targeted integration. Accordingly, a skilled artisan starting with Padidam and considering Schilit, Inniss, and Xu would expect that the cited combination would not yield animals suitable for generating edited embryos at high efficiency. Further, use of antibiotic selection of edited cells-a technique incompatible with embryo editing. The skilled artisan would further expect that applying the cited references would result in undesirable integration of vector backbone sequences into the embryonic genome, as described in Schilit. Applicants’ arguments have been fully considered, but are not found persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Applicants have further engaged in selective reading of the teachings of Padidam and Schilit to formulate the grounds for not teaching or teaching away. It should be noted that the ultimate goal of using a Bxb1 “landing pad” in a safe harbor locus, then use Bxb1 integrase to swap in a donor transgene carrying cognate Bxb1 attachment sites or use two heterologous attachment sites that could be used such that the donor backbone is excluded from the final integration product.
In this context, as previously indicated, Inniss teaches use of wild-type GT sequences in the RMCE landing pad and the corresponding donor DNA, respectively, paired with the GA mutant, that would inherently avoid integration of the plasmid backbone into the landing pad since GT and GA are orthogonal. Inniss explicitly states that the Bxbl RMCE system is specific and highly efficient. The teaching of Inniss is further supported by Jusiak who reported a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells . It is further disclosed that Bxb1-GA point mutant sites do not cross react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. To the extent that Inniss/ Jusiak describe the point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus, the rejection is applicable to the instant case.
In view of foregoing, it is apparent that, one of ordinary skill in the art seeking to integrate large piece of DNA with high efficiency and excluding plasmid backbone into the landing pad would be motivated use Bxbl attachment site and can reasonably be expected to improve insertion efficiency and accuracy in mouse embryos, with reasonable expectation of success, particularly since prior art teaches mice with multiple attP sites in tandem.
Applicant continue to argue that the claims are supported by data demonstrating that two Bxb1 attachment sites-such as an attB Bxb1 attachment site together with a modified attB* Bxb1 attachment site, or an attP Bxb1 attachment site together with a modified attP* Bxb1 attachment site-improves mouse embryo editing efficiency by three- to four-fold relative to a single Bxb1 attachment site. Applicants’ arguments have been fully considered, but are not found persuasive.
In response to applicant’s argument that the instant application provides evidence of the surprising efficiency of the two landing pads system, it should be noted that Inniss teaches the use of heterologous Bxb1 attachment sites to integrate a transgene in CHO cells (see pages 1837 and 1838, figures 1) and shows the superior results with Bxb1 attachment site. Inniss teaches use of wild-type GT sequences in the RMCE landing pad and the corresponding donor DNA, respectively, paired with the GA mutant, that would inherently avoid integration of the plasmid backbone into the landing pad since GT and GA are orthogonal. Inniss explicitly states that the Bxbl RMCE system is specific and highly efficient. The teaching of Inniss is further supported by Jusiak who reported a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells . It is further disclosed that Bxb1-GA point mutant sites do not cross react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites.
In view of foregoing, the relevance of Applicants' arguments with respect to superiority of using two sites over one site is not apparent. Further, unexpected results have to be commensurate with the scope of the invention. "Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980)." Example of instant application discloses uses a CRISPR/Cas9 and an oligonucleotide donor to insert a second attP* (modified) site -240 bp away from the original attP site at a safe harbor locus (rosa26). In the modified site, the dinucleotide pairing was changed from nucleotide sequence comprising SEQ IDN O: 1 containing GT to sequence set forth in SEQ ID NO: 7 containing GA. The addition of the second site allows for exclusion of the vector backbone without first having to convert the donor construct into minicircles (FIG. 1B). In the instant case, claims are not so limited. There is no evidence on record that a mouse comprising within its genome a first attP Bxb1 attachment site comprising the sequence of SEQ ID NO: 1 and a second attP* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 separated by several kb apart at any locus would exhibit improved mouse embryo editing efficiency by three- to four-fold relative as argued by applicant.
On pages 7-8 of the applicant’s argument, applicant assert that Tasic employs identical cognate sites on the donor plasmid and identical attachment sites in the genome, such that cassette exchange results in either integration of the transgene of interest or integration of the bacterial backbone. As a result, only half of the insertion events contain the desired sequence, while the remaining half incorporate plasmid backbone. Accordingly, the high editing efficiency achieved by the claimed mouse or mouse embryo, without incorporation of vector backbone sequences, could not have been predicted based on Tasic. Applicants’ arguments have been fully considered, but are not found persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Applicants have further engaged in selective reading of the teachings of Tasic to formulate the grounds for teaching away. In the instant case, Tasic teaches the use of multiple sites and suggests the use of noncompatible pairs of attB and attP sites to control for the direction of insertion. Schillit discloses a landing pad comprising JT15 and Lox2272 sites for integration of a transgene within the Rosa26 locus (see figure 15.10.1). Thus, the concept of using two sites over one site is already taught in Schillit and cannot be regarded as unexpected superior results. Further, Innis teaches that Bxb1 has improved efficiency and fidelity. Inniss explicitly states that the Bxbl RMCE system is specific and highly efficient. The teaching of Inniss is further supported by Jusiak who reported a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells . To the extent that Inniss/ Jusiak describe the point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus, the rejection is applicable to the instant case. Therefore, one of ordinary skill in the art would combine the teaching of Schillit with Innis to arrive at claimed mouse or embryo. Inniss confirms the higher performance of Bxb1 over commonly used RMCE system. Inniss teaches use of wild-type GT sequences in the RMCE landing pad and the corresponding donor DNA, respectively, paired with the GA mutant, that would inherently avoid integration of the plasmid backbone into the landing pad since GT and GA are orthogonal. Thus, the skilled person would be aware of the improved efficiency of Bxb1 and of the possible drawback when using CHO cells would foresee advantages of two sites and Bxb1 when using cells other than CHO by selecting most efficient recombinase, with reasonable expectation of success.
Therefore, in view of the fact patterns of the instant case, and the ground of rejection outlined by the examiner, applicants’ arguments are not compelling and do not overcome the rejection of record.
Claim Rejections - 35 USC § 112-written description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 5-6, 8-9, 12-14, 59-63 and 64 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claim embraces a first attP Bxb1 attachment site comprising the sequence of SEQ ID NO: 1 and a second attP* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 or a first attB Bxb1 attachment site comprising the sequence of SEQ ID NO: 2 and a second attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 2. Claim 64 encompasses two Bxb1 attachment sites identical to each other except for dinucleotide bases that confer specificity. Claims are also directed to an attP Bxb1 attachment site comprising a GT dinucleotide pairing and a modified attP* Bxb1 attachment site comprises a GA dinucleotide pairing; or an attB Bxb1 attachment site comprising a GT dinucleotide pairing and a modified attB* Bxb1 attachment site comprising a GA dinucleotide pairing.
Vas-Cath Inc. v. Mahurkar, 19USPQ2d 1111 (Fed. Cir. 1991), clearly states that ''applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the 'written description' inquiry, whatever is now claimed.'' Vas-cath Inc. v. Mahurkar, 19USPQ2d at 1 117. The specification does not ''clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.'' Vas-cath Inc. v. Mahurkar, 19USPQ2d at 1116.
The DNA sequences of all the attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 or 2 respectively , other than SEQ ID NO: 7 or SEQ ID NO: 8 encompassed within the genus of attP* or attB* modified sequence have not been disclosed. Based upon the prior art there is expected to be sequence variation among the genus of attP* or attB* modified sequence relative to SEQ ID NO: 1 or 2. Ghosh (PNAS, 2008, 105, 3238-3243) teaches that the central dinucleotide is the sole determinant of attL Versus attR identity in ΘRv1excision. It is noted that use of a nonpalindromic central dinucleotide is the common feature that not only ensures integrative recombination yields recombinant products with the sites aligned in the appropriate configuration, but also distinguishes between attL and attR for excisive recombination. Ghosh concludes that nonpalindromic central dinucleotide is the sole determinant of attL versus attR identity in both the Bxb1 and ΘRv1 systems (see page 3241, col. 1, para. 1-2). Singh (PLoS Genetics, 2013, 9, e1003490, 1-14) reported that attP and attB are functionally symmetrical such that the central dinucleotide is the sole determinant of integration polarity, and the sequences of both sites are partially symmetric, although outside of an 8 bp common core there is only limited sequence similarity (Figure 1B). Singh reported that there are two critical site components one is the outermost flanks of attP that are required for Int binding and for recombination, but which also prevent usage as attB. The second is a key discriminator position at positions -15 and +15 where a T:A/A:T base pair is required for both Int binding and recombination as an attB site, but which interferes with attP functionality. The identities of attP and attB are mutually exclusive, but they can be interconverted with mutations in the critical discriminator and flanking motifs (see page 1, col. 2, para. 1 to page 2, col. 1). Singh teaches most of the single substitutions in the attP flanks (223, 221, 220, 219, +19, +20, +21, +23) are deleterious for recombination, even though Int binding to most of these substrates is only mildly affected (Figure 3B, 3C) (see page 2, col. 2, para. 4). In the instant case, the specification fails to provide any description of two Bxbl attachment sites that are identical except for the dinucleotide bases other than SEQ ID NO: 7 or 8 that confer specificity. With this strategy, it is not necessary to convert the donor plasmid into minicircles prior to delivery. Rather, recombination between the dual heterologous attachment sites (engineered/modified e.g., using alternative dinucleotide base pairs) functions to exclude the plasmid backbone from integration into the landing pad (see fig. 1). The art teaches using a modified attP* or attB* sites in Bxb1-mediated recombination alongside wild-type attP or attB landing pads can lead to highly unpredictable integration outcomes because these systems rely on strict, sequence-specific pairing. Bxb1 integrase operates via precise synaptic alignment, and mutating either the core dinucleotide or the flanking half-sites can disrupt enzyme binding, alter recombination directionality, or cause cross-reactivity. The art teaches a modified attP* or modified attB* site that includes a point mutation at key discriminator positions may no longer be recognized by wild-type Bxb1. There is no evidence on the record of an attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 at any site or region other than centrally located dinucleotide as set forth in SEQ ID NO: 7 and 8 respectively. The structures of the attP* or attB* as set forth in SEQ ID NO: 7 or 8 does not provide any reliable information about the structure of modification at any other Bxb1 attachment site within the genus. There is no evidence on the record that embraced modified attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 as broadly recited had known structural relationships with mutation at any other Bxb1 attachment site other than centrally located dinucleotide. The art indicated that point mutations at key discriminator positions would no longer be recognized by wild-type Bxb1, or it may or may not act as an orthogonal site. The claimed invention as a whole is not adequately described if the claims require essential or critical elements or motifs which are not adequately described in the specification and which is not conventional in the art as of applicants effective filing date.
Possession may be shown by actual reduction to practice, clear depiction of the invention in a detailed drawing or by describing the invention with sufficient relevant identifying characteristics such that a person skilled in the art would recognize that the inventor had possession of the claimed invention. Pfaff v. Wells Electronics. Inc., 48 USPQ2d 1641, 1646 (1998). In the instant case, the claimed embodiments of all the attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 or 2 respectively , other than SEQ ID NO: 7 or SEQ ID NO: 8 encompassed within the genus of attP* or attB* modified sequence or any two Bxb1 attachment sites identical to each other except for dinucleotide bases that confer specificity lack a written description. The specification fails to describe what DNA molecules fall into this genus. The skilled artisan cannot envision the detailed chemical structure of the encompassed attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 or 2 or , and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of isolation. Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993) and Amgen lnc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016 (Fed. Cir. 1991).
One cannot describe what one has not conceived. See Fiddes v. Baird, 30 UsPQ2d 1481, 1483. In Fiddes, claims directed to mammalian FGF'S were found to be unpatentable due to lack of written description for that broad class. The specification provided only the bovine sequence.
In view of the above considerations one of skill in the art would not recognize that applicant was in possession of the necessary common features or attributes possessed by member of the genus of l the attP* or attB* Bxb1 attachment site modified relative to the sequence of SEQ ID NO: 1 or 2 respectively, other than the SEQ ID NO: 7 or SEQ ID NO: 8. Therefore, Applicant was not in possession of the genus the genus as encompassed by the claims. University of California v. Eli Lilly and Co., 43 USPQ2d 1398, 1404, 1405 held that to fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that ''the inventor invented the claimed invention.''
Withdrawn-Double Patenting
Claims 1-15, 59-62 were provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 7-9, 11-12 and 13 of copending Application No. 18019999 and further in view of Xu et al (BMC Biotechnology 2013, 13:87, 1-17). Applicant’s amendments to the claims obviates the basis of the rejection.
Conclusion
No claims allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Luo et al (US 20120124686) teaches introducing a site-specific recombinase and a targeting construct, containing a first recombination site and the polynucleotide sequence of interest, into the mammalian cell. The genome of the cell contains a second recombination site and recombination between the first and second recombination sites is facilitated by the site-specific, uni-directional recombinase. The result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the mammal. This inserted sequence is then also transmitted to the progeny of the mammal.
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/ANOOP K SINGH/ Primary Examiner, Art Unit 1632
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