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 .
Claims 1, 5, 10-12, 19, 25, 27, 40, 42, 55-56, 68-69, 76, 79-80, 85, and 91 are pending and under consideration.
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. The effective filing date of the instant application is 08/24/2020.
Information Disclosure Statement
Receipt of information disclosure statements on 10/01/2023, 01/25/2024, and 05/24/2024 is acknowledged. The signed and initialed PTO-1449‘s have been mailed with this action.
Drawings
The drawings filed 09/15/2023 are acceptable.
Claim Objections
Claim 40 is objected to because of the following informalities:
With regard to claim 40, which recites “the Cas9 fusion protein comprises the Cas fused to a DNA replication ATP-dependent helicase/nuclease (DNA2) or a fragment thereof,” the recitation of a Cas fused to DNA2 is grammatically inconsistent with the recitation of instant claim 25. Instant claim 25 recites a Cas nuclease fusion (bolded emphasis added; see line 2). It would be remedial to update the instant claim language to be internally consistent such that claim 40 recites that the fusion protein “comprises the Cas nuclease fused to a DNA replication ATP-dependent helicase/nuclease (DNA2) or a fragment thereof” (bolded emphasis added).
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 12, 19, 27, and 42 are 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.
Claims 12, 19, 27, and 42 are drawn to a set of polypeptide sequences corresponding to a Cas9 nuclease, a Cas12 nuclease, a human Exo1, and a DNA replication ATP-dependent helicase/nuclease, respectively. Each of the instant claims recites “a polypeptide sequence that is at least 75% identical” to the claimed SEQ ID NOs (7-23, 55-57, 1-2, and 4-5, respectively). The rejected claims thus comprise a set of polypeptide sequences with a large number of variable residues, all variations of which must correspond to a functional enzyme, be it Cas9 nuclease, Cas12 nuclease, human Exo1, or DNA replication ATP-dependent helicase/nuclease.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification discloses the instantly claimed SEQ ID NOs as well as experimentation with the same. However, no description is provided of a set of polypeptide sequences with at least 75% identity to the instantly claimed SEQ ID NOs.
As set forth above, SEQ ID NOs: 7-23, 55-57, 1-2, and 4-5 are respectively polypeptide sequences corresponding to a Cas9 nuclease, a Cas12 nuclease, a human Exo1, and a DNA replication ATP-dependent helicase/nuclease, all of which are enzymes. As is known to those of skill in the art, the active site of any enzyme is crucial to its function, as its shape and charge properties enable the enzyme to bind to a single type of substrate molecule (reviewed in Robinson, 2015; see page 4, paragraphs 2-3). The instant specification is silent as to the essentiality of any particular residues or motifs, such as those that determine the active site(s) of each claimed enzyme.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of the instantly claimed SEQ ID NOs. The results are not necessarily predictive of any polypeptide sequence with at least 75% identity to the claimed SEQ ID NOs. Thus, it is impossible for one to extrapolate from the examples described herein those polypeptide sequences that would necessarily meet the structural/functional characteristics of the rejected claims.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe a set of polypeptide sequences with at least 75% identity to the claimed SEQ ID NOs.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 12, 19, 27, and 42.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 40 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 40 recites the limitation "the Cas9 fusion protein" in line 1. There is insufficient antecedent basis for this limitation in the claim. Instant claim 40 depends from instant claim 1, which does not recite any Cas9 fusion protein.
Claim Rejections - 35 USC § 103
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, 5, 10, 11, 68, 69, 79, 80, 85, and 91 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010.
With regard to claim 1, which recites “a method for introducing an edit into a genomic locus of a plurality of cells, the method comprising: contacting the plurality of cells with: a) a Cas fusion protein complex comprising a Cas fusion protein complexed with a guide polynucleotide configured to bind to the genomic locus of a cell of the plurality of cells; and b) a repair template comprising at least 5000 base pairs (bp) in length, thereby introducing the edit into the genomic locus of the plurality of cells with the repair template, wherein the edit comprises a homology directed repair (HDR),” Shaul discloses a recombinant system and method of use of the same for improving genome editing via homologous recombination (abstract; page 3, lines 10-14). The recombinant system disclosed in Shaul comprises fusion polypeptides (or proteins) formed through translating chimeric nucleic acid constructs disclosed therein (page 23, lines 29-33; page 24, lines 7-13) and comprising a DNA editing agent such as Cas9 fused to a polypeptide capable of increasing homologous recombination for purposes of improving genome editing via homologous recombination (page 1, lines 15-18; page 3, lines 1-5; page 4, lines 9-10) as well as at least one guide RNA (sgRNA) sequence to complex with the Cas9 and recruit the sgRNA/Cas9 complex to the targeted genomic sequence (page 4, lines 11-12; page 15, lines 17-26). Example 2 of Shaul discloses that fusion (or chimeric) Cas9 facilitated greater levels of genome editing in human cell lines at the PSMB6 locus (specifically knocking in YFP), as quantified by FACS analysis of 100,000 transfected cells (i.e. a plurality of cells contacted with the fusion protein set forth above) analyzed per sample (page 64, line 4-page 65, line 23). Thus, Shaul discloses a method for introducing an edit into a genomic locus of a plurality of cells, said method comprising contacting the cells with a Cas fusion protein complex directed to the targeted genomic site by a guide polynucleotide specifying the targeted genomic site, as instantly claimed.
While Shaul does disclose insertion templates with a range of possible lengths, these insertions are disclosed to have a maximum length of 5,000 nucleotides, which does not fully satisfy the claimed repair template comprising at least 5,000 base pairs. However, this deficiency is cured by Wang et al., 2015, which discloses successful homology directed repair-mediated gene knock-in of 5.8 and 7.4 kb fragments (abstract; figure 1). Both of these fragments are larger than 5,000 base pairs, as instantly claimed.
With regard to claim 5, which recites “at least 50% of the plurality of cells [of the method of claim 1] remain viable after introducing the edit into the genomic locus of the plurality of cells with the repair template,” given that Shaul and Wang et al., 2015 collectively disclose the composition of the method of claim 1, as set forth above, the composition of the method of instant claim 1 (as claimed therein) and of Shaul and Wang et al., 2015 must be structurally identical and must therefore have identical functions, meaning the genome editing outcomes of each composition must also be identical. See MPEP § 2114 and § 2173.05(g).
With regard to claim 10, which recites “the Cas fusion protein [of the method of claim 1] comprises a Cas nuclease fused to an exonuclease or a fragment thereof,” as set forth above, Shaul discloses a recombinant system for improving genome editing via homologous recombination (abstract; page 3, lines 10-14), said recombinant system comprising fusion polypeptides (or proteins) (page 23, lines 29-33; page 24, lines 7-13) that themselves comprise a DNA editing agent such as Cas9 fused to a polypeptide capable of increasing homologous recombination for purposes of improving genome editing via homologous recombination (page 1, lines 15-18; page 3, lines 1-5; page 4, lines 9-10). This polypeptide capable of increasing homologous recombination is disclosed to be selected from a group of polypeptides comprising an exonuclease (page 19, lines 6-9), as instantly claimed.
With regard to claim 11, which recites “the Cas fusion protein [of the method of claim 1] comprises a Cas9 nuclease or a Cas12 nuclease,” as set forth above, the recombinant system of Shaul comprises fusion polypeptides (or proteins) (page 23, lines 29-33; page 24, lines 7-13) that themselves comprise a DNA editing agent such as Cas9 (as instantly claimed) fused to a polypeptide capable of increasing homologous recombination for purposes of improving genome editing via homologous recombination (page 1, lines 15-18; page 3, lines 1-5; page 4, lines 9-10). Thus, Shaul discloses a Cas9 fusion protein, as instantly claimed.
With regard to claim 68, which recites “the genomic locus [targeted by the method of claim 1] encodes a gene associated with cancer selected from the group consisting of cadherin and catenin,” Shaul discloses that the system taught therein may be used to treat or prevent a cancerous disease in a subject by targeting a gene of interest associated with onset or progression of the cancerous disease, such as CTNNB1 (beta-catenin) (page 47, lines 28-33). Thus, Shaul discloses targeting a catenin gene associated with cancer, as instantly claimed.
With regard to claim 69, which recites “the genomic locus [targeted by the method of claim 1] encodes a gene selected from the group consisting of an oncogene or a tumor suppressor gene,” Shaul discloses that the system taught therein may be used to treat or prevent a cancerous disease in a subject by targeting a gene of interest associated with onset or progression of the cancerous disease, such as KRAS (page 47, lines 28-33), which is known to be an oncogene (reviewed in Jančík et al., 2010). Thus, Shaul discloses targeting an oncogene, as instantly claimed.
With regard to claim 79, which recites “the Cas fusion protein [of the method of claim 1] increases a rate of HDR in the plurality of the cells compared to a rate of HDR induced by a second Cas protein in a plurality of cells lacking the Cas fusion protein,” as set forth above regarding instant claim 5, given that Shaul and Wang et al., 2015 collectively disclose the composition of the method of claim 1, as set forth above, the composition of the method of instant claim 1 (as claimed therein) and of Shaul and Wang et al., 2015 must be structurally identical and must therefore have identical functions, meaning the genome editing outcomes of each composition must also be identical. See MPEP § 2114 and § 2173.05(g). In further support of this, Shaul discloses at example 2 that genomic knock-in driven by chimeric or fusion Cas9 occurred at much higher rates in transfected cells than occurred in cells transfected with wild-type Cas9 (i.e. cells lacking the Cas fusion protein). Per Shaul, chimeric or fusion Cas9 showed a 1.7-3.3 fold improvement over the genomic knock-in mediated by wild-type Cas9 (page 65, lines 4-6 and lines 13-15). Thus, Shaul discloses a Cas9 fusion protein that increases the rate of HDR in a plurality of cells compared to the rate of HDR mediated by wild-type Cas9, as instantly claimed.
With regard to claim 80, which recites “the Cas fusion protein [of the method of claim 79] increases the rate of HDR in the plurality of the cells by at least 10% or more compared to the rate of HDR induced by a second Cas protein in the plurality of cells lacking the Cas fusion protein,” as set forth above, Shaul discloses that chimeric or fusion Cas9 showed a 1.7-3.3 fold improvement over the genomic knock-in mediated by wild-type Cas9 (page 65, lines 4-6 and lines 13-15). A 1.7-3.3 fold improvement corresponds to a 70%-230% improvement in homology directed repair rates when the method utilizes a chimeric or fusion Cas9 rather than a wild-type Cas9. This percentage improvement satisfies the limitation of increasing the rate of HDR at least 10%.
With regard to claim 85, which recites “the Cas fusion protein [of the method of claim 1] decreases an endogenous p53 activity in the plurality of the cells compared to an endogenous p53 activity induced by a second Cas protein in a plurality of cells lacking the Cas fusion protein,” as set forth above, Shaul discloses that chimeric or fusion Cas9 showed a 1.7-3.3 fold improvement over the genomic knock-in mediated by wild-type Cas9 (page 65, lines 4-6 and lines 13-15). Furthermore, Shaul discloses that the system taught therein may be used to treat or prevent a cancerous disease in a subject by targeting a gene of interest associated with onset or progression of the cancerous disease, such as the p53 family (page 47, lines 28-33).
While Shaul does not specifically disclose reduction of endogenous p53 activity, given that Shaul and Wang et al., 2015 collectively disclose the composition of the method of claim 1, as set forth above, the composition of the method of instant claim 1 (as claimed therein) and of Shaul and Wang et al., 2015 must be structurally identical and must therefore have identical functions, meaning the genome editing outcomes of each composition must also be identical. See MPEP § 2114 and § 2173.05(g).
With regard to claim 91, which recites “the second Cas protein [of the method of claim 79] is a wild type Cas9 nuclease,” as set forth above, Shaul discloses that chimeric or fusion Cas9 showed a 1.7-3.3 fold improvement over the genomic knock-in mediated by wild-type Cas9 (page 65, lines 4-6 and lines 13-15), as instantly claimed.
Given that Shaul discloses a method for increasing rates of HDR as compared to those achieved with wild-type Cas9, said method comprising guiding a fusion polypeptide comprising Cas9 and another polypeptide that enhances HDR (such as an exonuclease) to a targeted genomic locus (such as beta-catenin or KRAS) specified by a user-designed sgRNA, and that Wang et al., 2015 discloses HDR insertion templates greater than 5000 bp (5.8 and 7.4 kb), it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the longer HDR insertion templates disclosed in Wang et al., 2015 in the system disclosed in Shaul to predictably knock in larger sequences, effectively facilitating insertion of a wider range of sequences at user-specified loci, including sequences with therapeutic benefits (i.e. for treating cancer). One would have been motivated to make such a modification in order to receive the expected benefit of facilitating insertion of a wider range of sequences at user-specified loci, including sequences with therapeutic benefits (i.e. for treating cancer).
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010 as applied to claim 11 above, and further in view of US 10,266,850 B2 (hereinafter Doudna; as cited in the IDS filed 10/01/2023).
The combined disclosures of Shaul, Wang et al., 2015, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the Cas9 sequence of instant claim 12.
With regard to claim 12, which recites “the Cas9 nuclease [of the method of claim 11] comprises a polypeptide sequence that is at least 75% identical to any one of SEQ ID NOs: 7-23,” Doudna discloses that SEQ ID NO: 1285 taught therein corresponds to a naturally occurring Cas9 endonuclease that can be used as a site-directed modifying polypeptide (column 46, lines 62-66). As shown in Appendix I, SEQ ID NO: 1285 of Doudna is 100% identical to instant SEQ ID NO: 7. Thus, Doudna discloses a Cas9 nuclease comprising a polypeptide sequence that is at least 75% identical to SEQ ID NO: 7, as instantly claimed.
Given that Shaul and Wang et al., 2015 collectively disclose a method for increasing rates of HDR, said method comprising a Cas fusion polypeptide and that Doudna discloses a Cas9 nuclease comprising SEQ ID NO: 1285 (which is 100% identical to instant SEQ ID NO: 7), it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the Cas9 nuclease polypeptide sequence taught in Doudna in the system collectively disclosed by Shaul and Wang et al., 2015 to predictably insert user-specified sequence(s) at user-specified genomic loci via Cas9 fusion protein-mediated HDR. One would have been motivated to make such a modification in order to receive the expected benefit of inserting user-specified sequence(s) at user-specified genomic loci via Cas9 fusion protein-mediated HDR.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010 as applied to claim 1 above, and further in view of US 9,790,490 B2 (hereinafter Zhang), as evidenced by Swarts and Jinek, 2018.
The combined disclosures of Shaul, Wang et al., 2015, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the Cas12 sequence of instant claim 19.
With regard to claim 19, which recites “the Cas12 nuclease [of the method of claim 1] comprises a polypeptide sequence that is at least 75% identical to any one of SEQ ID NOs: 55-57,” Zhang discloses that SEQ ID NO: 1110 taught therein corresponds to a Cas-Cpf1 ortholog capable of mediating CRISPR-driven HDR (column 4, lines 5-27; column 46, lines 62-66). It will be appreciated by those of ordinary skill in the art that the term “Cpf1” is synonymous with the Cas12 nuclease Cas12a (reviewed in Swarts and Jinek, 2018; see in particular the abstract). As shown in Appendix II, SEQ ID NO: 1110 of Doudna is 100% identical to instant SEQ ID NO: 56. Thus, Zhang discloses a Cas12 nuclease comprising a polypeptide sequence that is at least 75% identical to SEQ ID NO: 56, as instantly claimed.
Given that Shaul and Wang et al., 2015 collectively disclose a method for increasing rates of HDR, said method comprising a Cas fusion polypeptide and that Zhang discloses a Cas12 nuclease capable of mediating CRISPR-driven HDR and comprising SEQ ID NO: 1110 (which is 100% identical to instant SEQ ID NO: 56), it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the Cas12 nuclease polypeptide sequence taught in Zhang in the system collectively disclosed by Shaul and Wang et al., 2015 to predictably insert user-specified sequence(s) at user-specified genomic loci via Cas12 fusion protein-mediated HDR. One would have been motivated to make such a modification in order to receive the expected benefit of inserting user-specified sequence(s) at user-specified genomic loci via Cas12 fusion protein-mediated HDR.
Claims 25 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010 as applied to claims 1 and 10 above, and further in view of Karanja et al., 2012.
The combined disclosures of Shaul, Wang et al., 2015, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the human Exo1-Cas fusion protein of instant claim 25 or the DNA2-Cas fusion protein of instant claim 40.
With regard to claims 25 and 40, which respectively recite “the Cas fusion protein comprises the Cas nuclease fused to a Human Exo1 (hExo1)” or “a DNA replication ATP-dependent helicase/nuclease (DNA2) or a fragment thereof,” Karanja et al., 2012 discloses that human DNA2 and Exo1 are both implicated in mediating HDR and are somewhat (but not completely) functionally redundant (abstract; page 3994, column 1, paragraphs 1 and 2). For example, cells depleted of DNA2 exhibit reduced HDR activity (figure 5), and both DNA2 and Exo1 were found to influence the frequency of HDR versus non-homologous end joining (page 3994, column 1, paragraph 3). Thus, Karanja et al., 2012 discloses that both DNA2 and human Exo1 facilitate higher HDR activity.
Given that Shaul and Wang et al., 2015 collectively disclose a method for increasing rates of HDR, said method comprising a fusion polypeptide, itself comprising a DNA editing agent such as Cas9 fused to a polypeptide capable of increasing homologous recombination, and that Karanja et al., 2012 discloses that both DNA2 and human Exo1 facilitate higher HDR activity, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize either DNA2 or human Exo1 (as disclosed in Karanja et al., 2012) as the fusion polypeptide component of a polypeptide capable of increasing homologous recombination (as collectively disclosed by Shaul and Wang et al., 2015) to predictably increase homologous recombination in cells contacted with said fusion polypeptide. One would have been motivated to make such a modification in order to receive the expected benefit of increasing homologous recombination in cells contacted with said fusion polypeptide.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015 and Karanja et al., 2012, as evidenced by Jančík et al., 2010 as applied to claim 25 above, and further in view of GenPept Entry NP_001306153.1.
The combined disclosures of Shaul, Wang et al., 2015, Karanja et al., 2012, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the human Exo1 polypeptide sequence of instant claim 27.
With regard to claim 27, which recites “the hExo1 [of the method of claim 25] comprises a polypeptide sequence that is at least 75% identical to SEQ ID NO: 1 or SEQ ID NO: 2,” GenPept entry NP_001306153.1 (indicated to have replaced the prior version XP_005273407.1 on January 30, 2016-see page 2) discloses the sequence of human exonuclease 1 isoform c. Sequence NP_001306153.1 is 99.7% identical to instant SEQ ID NO: 1, as shown in Appendix III. Thus, GenPept entry NP_001306153.1 discloses an hExo1 that comprises a polypeptide sequence that is at least 75% identical to SEQ ID NO: 1, as instantly claimed.
Given that Shaul, Wang et al., 2015, and Karanja et al., 2012 collectively disclose a method for increasing rates of HDR, said method comprising a Cas-hExo1 fusion polypeptide and that GenPept entry NP_001306153.1 discloses a known hExo1 that is 99.7% identical to instant SEQ ID NO: 1, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the hExo1 polypeptide sequence taught in GenPept entry NP_001306153.1 in the system collectively disclosed by Shaul, Wang et al., 2015, and Karanja et al., 2012 to predictably insert user-specified sequence(s) at user-specified genomic loci via Cas-hExo1 fusion protein-mediated HDR at increased rates. One would have been motivated to make such a modification in order to receive the expected benefit of inserting user-specified sequence(s) at user-specified genomic loci via Cas-hExo1 fusion protein-mediated HDR at increased rates.
Claim 42 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015 and Karanja et al., 2012, as evidenced by Jančík et al., 2010 as applied to claim 40 above, and further in view of GenPept Entry NP_001073918.2.
The combined disclosures of Shaul, Wang et al., 2015, Karanja et al., 2012, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the DNA2 polypeptide sequence of instant claim 42.
With regard to claim 27, which recites “the DNA2 [of the method of claim 40] comprises a polypeptide sequence that is at least 75% identical to SEQ ID NO: 4 or SEQ ID NO: 5,” GenPept entry NP_001073918.2 (indicated to have replaced the prior version NP_001073918.1 on January 29, 2011-see page 3) discloses the sequence of human DNA replication ATP-dependent helicase/nuclease DNA2. Sequence NP_001073918.2 is 100% identical to instant SEQ ID NO: 4, as shown in Appendix IV. Thus, GenPept entry NP_001073918.2 discloses a DNA2 that comprises a polypeptide sequence that is at least 75% identical to SEQ ID NO: 4, as instantly claimed.
Given that Shaul, Wang et al., 2015, and Karanja et al., 2012 collectively disclose a method for increasing rates of HDR, said method comprising a Cas-DNA2 fusion polypeptide and that GenPept entry NP_001073918.2 discloses a known DNA2 that is 100% identical to instant SEQ ID NO: 4, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the DNA2 polypeptide sequence taught in GenPept entry NP_001073918.2 in the system collectively disclosed by Shaul, Wang et al., 2015, and Karanja et al., 2012 to predictably insert user-specified sequence(s) at user-specified genomic loci via Cas-DNA2 fusion protein-mediated HDR at increased rates. One would have been motivated to make such a modification in order to receive the expected benefit of inserting user-specified sequence(s) at user-specified genomic loci via Cas-DNA2 fusion protein-mediated HDR at increased rates.
Claims 55 and 56 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010 as applied to claim 1, and further in view of Paquet et al., 2016.
The combined disclosures of Shaul, Wang et al., 2015, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the mutated PAM sequence of instant claims 55 and 56.
With regard to claims 55 and 56, which respectively recite “the repair template [of the method of claim 1] comprises a mutated protospacer adjacent motif (PAM) sequence located at the immediate 3’ end of a cleavage site, wherein said mutated PAM sequence comprises 5’-NCG-3’ or 5’-NGC-3’,” and further that “the mutated PAM sequence is not cleaved by the Cas fusion protein,” Paquet et al., 2016 discloses that mutating the PAM site canonically recognized by Cas9 to 5’-NGCG-3’ increased HDR accuracy in both iPS cells and HEK293 two- to tenfold (page 125, column 1, paragraph 1-page 125, column 2, paragraph 2; figure 1; extended data figure 2). This mutation is referred to as a “Cas blocking mutation” because wild-type Cas9 cannot recognize, bind to, and cleave its intended target without the PAM sequence it is known to utilize, meaning re-editing following a targeted editing event can be blocked by mutating the PAM sequence to 5’-NGCG-3’ (page 125, column 1, paragraph 1). Thus, Paquet et al., 2016 discloses a Cas blocking mutation that is an altered PAM site comprising 5’-NGC-3’, as instantly claimed. This altered PAM site is disclosed to increase HDR accuracy and prevent re-editing following a targeted editing event.
Given that Shaul and Wang et al., 2015 collectively disclose a method for increasing rates of HDR, said method comprising a Cas fusion polypeptide and that Paquet et al., 2016 discloses that mutating the PAM sequence recognized by a Cas endonuclease such that the activity of the Cas endonuclease is subsequently blocked increases HDR accuracy and prevents re-editing, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to further engineer the HDR-enhancing system collectively disclosed by Shaul and Wang et al., 2015 to comprise a mutated PAM sequence that blocks Cas activity following successful editing (as disclosed in Paquet et al., 2016) to predictably increase HDR accuracy and unintended prevent re-editing of the targeted locus. One would have been motivated to make such a modification in order to receive the expected benefit of increasing HDR accuracy and unintended prevent re-editing of the targeted locus.
Claim 76 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/003311 A1 (hereinafter Shaul) in view of Wang et al., 2015, as evidenced by Jančík et al., 2010 as applied to claim 1, and further in view of Papapetrou and Schambach, 2016.
The combined disclosures of Shaul, Wang et al., 2015, and Jančík et al., 2010 are described above and applied as before. However, these disclosures do not teach the safe harbor site of instant claim 76.
With regard to claim 76, which recites “the genomic locus [of the method of claim 1] comprises a safe harbor site (SHS),” while Shaul discloses a number of intended genomic targets, they do not specifically disclose that any genomic targets are safe harbor sites. Papapetrou and Schambach, 2016 disclose that genomic safe harbor sites are sites in the genome that are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function predictably and do not cause alterations of the host genome posing a risk to the host cell or organism (abstract). While no truly validated safe harbor sites have yet been identified (abstract), their selection is governed by a conceptual framework embracing criteria such as excluding target regions that are in close proximity (i.e. within 50 kb) of coding or non-coding genes or are within 300 kb of genes known to play a role in cancer and microRNA genes (page 678, column 2, paragraph 2). Based on these criteria and empirical data, AAVS1, CCR5, and other intragenic regions may be acceptable safe harbor sites for research applications, but there is clearly a much higher burden of proof of safety needed for clinical applications (page 679, column 1, paragraph 1). Thus, Papapetrou and Schambach, 2016 disclose that although no true genomic safe harbor site has yet been validated for clinical use, any clinical application of gene therapy such as knocking in exogenous sequences at specified loci will have to identify, validate, and subsequently target a genomic safe harbor site in order to safely and effectively deliver the therapeutic sequence(s) to the patient.
Given that Shaul and Wang et al., 2015 collectively disclose a method for increasing rates of HDR, said method comprising a Cas fusion polypeptide and that Papapetrou and Schambach, 2016 disclose that genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function predictably and do not cause alterations of the host genome that would pose a risk to the host, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to further target the HDR-enhancing system collectively disclosed by Shaul and Wang et al., 2015 to a genomic safe harbor site (as disclosed in Papapetrou and Schambach, 2016) to predictably insert therapeutic sequence(s) at a genomic site that is likely to express at robust levels in the host without endangering the host. One would have been motivated to make such a modification in order to receive the expected benefit of inserting therapeutic sequence(s) at a genomic site that is likely to express at robust levels in the host without endangering the host.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Sarah E Allen whose telephone number is (571)272-0408. The examiner can normally be reached M-Th 8-5, F 8-12.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL, Ph.D./Primary Examiner, Art Unit 1637
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1003
736
media_image1.png
Greyscale
PNG
media_image2.png
180
754
media_image2.png
Greyscale
Appendix I - Instant SEQ ID NO: 7 vs. Doudna SEQ ID NO: 1285
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media_image3.png
791
760
media_image3.png
Greyscale
PNG
media_image4.png
179
744
media_image4.png
Greyscale
PNG
media_image5.png
998
734
media_image5.png
Greyscale
Appendix II - Instant SEQ ID NO: 56 vs. Zhang SEQ ID NO: 1110
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media_image6.png
701
751
media_image6.png
Greyscale
PNG
media_image7.png
1007
753
media_image7.png
Greyscale
PNG
media_image8.png
179
747
media_image8.png
Greyscale
Appendix III - Instant SEQ ID NO: 1 vs. NP_001306153.1
PNG
media_image9.png
162
742
media_image9.png
Greyscale
PNG
media_image10.png
170
745
media_image10.png
Greyscale
PNG
media_image11.png
999
750
media_image11.png
Greyscale
Appendix IV - Instant SEQ ID NO: 4 vs. NP_001073918.2
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media_image12.png
392
756
media_image12.png
Greyscale