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 .
Claim Objections
Claims 133 and 136 are objected to because of the following informalities:
In claim 133, human in parenthesis in penultimate line is considered to be a claim limitation and should not appear in parenthesis.
In claim 136, “the cleavage site is after the purification tag” should be stated as “the cleavage site is C-terminal to the cleavage site.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 133 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.
Regarding claim 133, the phrase "e.g." (considered equivalent to such as) renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Here, it is unclear if “e.g. a nucleoplasmin bipartite NLS, a c-myc NLS” is a required limitation of the claim.
Claim 133 recites the limitation "the SV40 virus large T-antigen" in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of claim features that provides any literal nor inherent antecedent basis for "the SV40 virus large T-antigen" such that it is unclear what claim element is being referenced.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 123-131, 133 and 138 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gill et al. (U.S. 2018/0371497 A1).
Gill, abstract, states:
Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.
“Embodiments of the invention also relate to methods and compositions related to knocking out genes, editing genes, altering genes, amplifying genes, and repairing particular mutations.” Gill, para. 0151]. The preceding is understood as a description of any of the described nucleic acid-guided nucleases can be present is some generic composition.
“A targetable nuclease complex can comprise a nucleic acid-guided nuclease of SEQ ID NO: 7 and a compatible guide nucleic acid. A targetable nuclease complex can comprise a nucleic acid-guided nuclease of SEQ ID NO: 7 and a compatible guide nucleic acid comprising any one of SEQ ID NO: 88, 93, 94, or 95” Gill, para. [0076].
“In some embodiments, a vector encodes a nucleic acid-guided nuclease comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineered nuclease comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the engineered nuclease comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 111); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:112)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:113) or RQRRNELKRSP (SEQ ID NO:114); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 115); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:1 116) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:117) and PPKKARED (SEQ ID NO:118) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:119) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:120) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:121) and PKQKKRK (SEQ ID NO:122) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:123) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 124) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 125) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 126) of the steroid hormone receptors (human) glucocorticoid.” Gill, para. [0085].
SEQ ID NO: 7 of Gill is identical to recited SEQ ID NO: 1.
“Codon optimized MAD1-MAD20 were cloned into an expression construct comprising a constitutive or inducible promoter (eg., proB promoter SEQ ID NO: 83, or pBAD promoter SEQ ID NO: 81 or SEQ ID NO: 82) and an optional 6X-His tag (SEQ ID NO: 182) (eg., FIG. 2). The generated MAD1-MAD20 expression constructs are provided as SEQ ID NOs: 61-80, respectively” Gill, para. [0248].
The above is considered to be an anticipatory disclosure of a composition comprising a nucleic acid-guided nuclease comprising a Type Va CRISPR nuclease polypeptide having SEQ ID NO: 1 comprising at least three, four, five, or six nuclear localization signals (NLS) all at or near the N-terminus of the polypeptide, wherein such NLS can be NLS of SV40 virus large T-antigen.
Regarding claim 138, the term “spacer” as used in the specification does not appear to be a reference to a PAM sequence but rather the sequence of a guide nucleic acid that hybridizers with a target sequence. “[R]ecognize a 5' T-rich PAM located immediately upstream from the
target nucleotide sequence, the orientation determined using the non-target strand (i.e., the strand
not hybridized with the spacer sequence) as the coordinate.” Specification, para. [0042]. “In certain embodiment the composition further comprises a second polynucleotide coding for a gNA or portion thereof, wherein the gNA, e.g., gRNA, comprises a spacer sequence that targets a target nucleotide sequence within a polynucleotide.” Specification, para. [0057]. “Editing cassettes as depicted in FIG. 3 were generated to assess the functionality of the MAD nucleases and corresponding guide nucleic acids. Each editing cassette comprises an editing sequence and a promoter operably linked to an encoded guide nucleic acid. The editing cassettes further comprises primer sites (P1 and P2) on flanking ends. The guide nucleic acids comprised various scaffold sequences to be tested, as well as a guide sequence [i.e. gNA] to guide the MAD nuclease to the target sequence for editing [i.e., such guide sequence is a spacer sequences that targets a target nucleic acid within a polynucleotide]. The editing sequences comprised a PAM mutation and/or codon mutation relative to the target sequence. The mutations were flanked by regions of homology (homology arms or HA) which would allow recombination into the cleaved target sequence.” Gill, para [0251]. Such guide sequences are necessarily compatible with the MAD (Type V Crispr nucleases) as to meet the features of claim 138.
Regarding claim 131, as set forth above Gill is understood as having an anticipatory disclosure of 5 NLSs near an N-terminus of a nuclease having recited SEQ ID NO: 1. SEQ ID NO: 1 has over 80% identity to SEQ ID NO: 111 (1395 amino acid residues, Qy) as shown in the following alignment such that any polypeptide having one or more (e.g. 5) NLSs fused to an N-terminus of recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) has at least 80% identity to SEQ ID NO: 1 (1263 positions matching 1395 amino acid residues is about 90% identity):
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That is, any kind or quantity of amino acid sequence fused to the termini of a polypeptide having recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) maintains at least 80% identity to SEQ ID NO: 111.
Claim(s) 123-131, 133, and 138-139 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gill et al. (WO 2021/067788 A1) (published 04/08/2021). The Sequence Listing for WO 2021/067788 A1 is also available in U.S. 2022/0136014 A1.
Gill, abstract, teaches:
The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR- Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and/or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.
Gill in the claims states:
1. An engineered, non-naturally occurring system comprising:
(a) a targeter nucleic acid comprising:
(i) a spacer sequence designed to hybridize with a target nucleotide sequence; and
(ii) a targeter stem sequence; and
(b) a modulator nucleic acid comprising a modulator stem sequence complementary' to the targeter stem sequence, wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids [i.e. a dual gRNA], and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA.
29. The engineered, non-naturally occurring system of any one of claims 1-28, wherein the Cas nuclease comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1.
SEQ ID NO: 1 of Gill is identical to recited SEQ ID NO: 1. As such, the “Cas nuclease” of Gil is a Type Va CRISPR nuclease having recited SEQ ID NO: 1.
Gill further states:
[0074] In certain embodiments, the engineered Cas protein comprises one or more nuclear localization signal (NLS) motifs. In certain embodiments, the engineered Cas protein comprises at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motifs. Non-limiting examples of NLS motifs include: the NLS of SV4Q large T-antigen, having the amino acid sequence of PKKKRKV (SEQ ID NO: 23); the NLS from nucleoplasmin, e.g, the nucleoplasmin bipartite NLS having the amino acid sequence of KRP AATKK AGQAKKKK (SEQ ID NO: 24); the c-myc NLS, having the amino acid sequence of PAAKRVKLD (SEQ ID NO: 25) or RQRRNELKRSP (SEQ ID NO: 26); the hRNPAl M9 NLS, having the amino acid sequence of NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27); the importin-a IBB domain NLS, having the amino acid sequence of
RMRIZFKN GKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28); the myoma T protein NLS, having the amino acid sequence of VSRKRPRP (SEQ ID NO: 29) or PPKKARED (SEQ ID NO: 30); the human p53 NLS, having the amino acid sequence of PQPKKKPL (SEQ ID NO: 31); the mouse c-abl IV NLS, having the amino acid sequence of SALIKKKKKMAP (SEQ ID NO: 32); the influenza virus NS1 NLS, having the amino acid sequence of DRLRR (SEQ ID NO: 33) or PKQKKRK (SEQ ID NO: 34); the hepatitis virus d antigen NLS, having the amino acid sequence of RKLKKKIKKL (SEQ ID NO: 35); the mouse Mxl protein NLS, having the amino acid sequence of REKKKFLKRR (SEQ ID NO: 36); the human poly(ADP-ribose) polymerase NLS, having the amino acid sequence of KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 37); the human glucocorticoid receptor NLS, having the amino acid sequence of RKCLQAGMNLEARKTKK (SEQ ID NO: 38), and synthetic NLS motifs such as PAAKKKKLD (SEQ ID NO: 39).
[0075] In general, the one or more NLS motifs are of sufficient strength to drive accumulation of the Cas protein in a detectable amount in the nucleus of a eukaryotic cell.
The strength of nuclear localization activity may derive from the number of NLS motifs) in the Cas protein, the particular NLS motif(s) used, the position(s) of the NLS motif(s), or a combination of these factors. In certain embodiments, the engineered Cas protein comprises at least 1 (e.g, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motif(s) at or near the N-terminus (e.g., within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus).
The above is considered to be an anticipatory disclosure of a composition comprising a nucleic acid-guided nuclease composition comprising a Type Va CRISPR nuclease polypeptide having SEQ ID NO: 1 comprising at least three, four, five, or six nuclear localization signals (NLS) all at or near the N-terminus of the polypeptide, wherein such NLS can be NLS of SV40 virus large T-antigen.
As set forth in the abstract of Gill, “The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR- Associated (Cas) nuclease, such as a type V-A Cas nuclease,” which anticipates the features of claims 138 and 139. Specifically, “Type V-.A, type V-C, and type V-D CRISPR-Cas systems naturally include a Cas nuclease and a single guide RNA (i.e., crRNA). By splitting the single guide RNA into two different nucleic acids, the engineered system describe herein provides better flexibility and tunability.” Gill, para. [0007]. This description of Gill along with claim 1 of Gill is a description of a dual guide RNA (gNA/gRNA) having a spacer sequence that targets a target nucleotide sequence within a polynucleotide that is compatible with Type V CRISPR nuclease (i.e. nuclease having recited SEQ ID NO: 1).
Regarding claim 131, as set forth above Gill is understood as having an anticipatory disclosure of 5 NLSs near an N-terminus of a nuclease having recited SEQ ID NO: 1. SEQ ID NO: 1 has over 80% identity to SEQ ID NO: 111 (1395 amino acid residues, Qy) as shown in the following alignment such that any polypeptide having one or more (e.g. 5) NLSs fused to an N-terminus of recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) has at least 80% identity to SEQ ID NO: 1 (1263 positions matching 1395 amino acid residues is about 90% identity):
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That is, any kind or quantity of amino acid sequence fused to the termini of a polypeptide having recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) maintains at least 80% identity to SEQ ID NO: 111.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
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(s) ) 123-133 and 138 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gill et al. (U.S. 2018/0371497 A1) as evidenced by Pollard et al. (A Novel Receptor-Mediated Nuclear Protein Import Pathway, Cell 86, 1996, 985-94).
Gill, abstract, states:
Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.
“Embodiments of the invention also relate to methods and compositions related to knocking out genes, editing genes, altering genes, amplifying genes, and repairing particular mutations.” Gill, para. 0151]. The preceding is understood as a description of any of the described nucleic acid-guided nucleases can be present is some generic composition.
“A targetable nuclease complex can comprise a nucleic acid-guided nuclease of SEQ ID NO: 7 and a compatible guide nucleic acid. A targetable nuclease complex can comprise a nucleic acid-guided nuclease of SEQ ID NO: 7 and a compatible guide nucleic acid comprising any one of SEQ ID NO: 88, 93, 94, or 95” Gill, para. [0076].
“In some embodiments, a vector encodes a nucleic acid-guided nuclease comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineered nuclease comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the engineered nuclease comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 111); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:112)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:113) or RQRRNELKRSP (SEQ ID NO:114); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 115); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:1 116) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:117) and PPKKARED (SEQ ID NO:118) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:119) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:120) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:121) and PKQKKRK (SEQ ID NO:122) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:123) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 124) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 125) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 126) of the steroid hormone receptors (human) glucocorticoid.” Gill, para. [0085].
SEQ ID NO: 7 of Gill is identical to recited SEQ ID NO: 1.
“Codon optimized MAD1-MAD20 were cloned into an expression construct comprising a constitutive or inducible promoter (eg., proB promoter SEQ ID NO: 83, or pBAD promoter SEQ ID NO: 81 or SEQ ID NO: 82) and an optional 6X-His tag (SEQ ID NO: 182) (eg., FIG. 2). The generated MAD1-MAD20 expression constructs are provided as SEQ ID NOs: 61-80, respectively” Gill, para. [0248].
The above is considered to be a direct suggestion teaching and motivation that the various MAD nucleases, including SEQ ID NO: 7 of Gill identical to recited SEQ ID NO: 1, be fused at or near an N-terminus of any appropriate number of NLSs including at least three, four, five, or six nuclear localization signals (NLS) all at or near the N-terminus of the polypeptide, wherein such NLS can be NLS of SV40 virus large T-antigen. For this reason, an ordinarily skilled artisan at time of filing would have been motivated to form a fusion protein of a CRISPR nuclease being SEQ ID NO: 7 of Gill identical to recited SEQ ID NO: 1 to exactly or more of three, four, five, or six nuclear localization signals (NLS) all at or near the N-terminus of the polypeptide since Gill directly suggests that the same is appropriate and beneficial for allowing “accumulation of the nucleic acid-guided nuclease in a detectable amount in the nucleus of a eukaryotic cell.” Gill, para. [086].
anticipatory disclosure of a composition comprising a nucleic acid-guided nuclease comprising a Type Va CRISPR nuclease polypeptide having SEQ ID NO: 1 comprising at least three, four, five, or six nuclear localization signals (NLS) all at or near the N-terminus of the polypeptide, wherein such NLS can be NLS of SV40 virus large T-antigen.
Regarding claim 138, the term “spacer” as used in the specification does not appear to be a reference to a PAM sequence but rather the sequence of a guide nucleic acid that hybridizers with a target sequence. “[R]ecognize a 5' T-rich PAM located immediately upstream from the
target nucleotide sequence, the orientation determined using the non-target strand (i.e., the strand
not hybridized with the spacer sequence) as the coordinate.” Specification, para. [0042]. “In certain embodiment the composition further comprises a second polynucleotide coding for a gNA or portion thereof, wherein the gNA, e.g., gRNA, comprises a spacer sequence that targets a target nucleotide sequence within a polynucleotide.” Specification, para. [0057]. “Editing cassettes as depicted in FIG. 3 were generated to assess the functionality of the MAD nucleases and corresponding guide nucleic acids. Each editing cassette comprises an editing sequence and a promoter operably linked to an encoded guide nucleic acid. The editing cassettes further comprises primer sites (P1 and P2) on flanking ends. The guide nucleic acids comprised various scaffold sequences to be tested, as well as a guide sequence [i.e. gNA] to guide the MAD nuclease to the target sequence for editing [i.e., such guide sequence is a spacer sequences that targets a target nucleic acid within a polynucleotide]. The editing sequences comprised a PAM mutation and/or codon mutation relative to the target sequence. The mutations were flanked by regions of homology (homology arms or HA) which would allow recombination into the cleaved target sequence.” Gill, para [0251]. Such guide sequences are necessarily compatible with the MAD (Type V Crispr nucleases) as to meet the features of claim 138.
Regarding claim 131, as set forth above Gill is understood as having an anticipatory disclosure of 5 NLSs near an N-terminus of a nuclease having recited SEQ ID NO: 1. SEQ ID NO: 1 has over 80% identity to SEQ ID NO: 111 (1395 amino acid residues, Qy) as shown in the following alignment such that any polypeptide having one or more (e.g. 5) NLSs fused to an N-terminus of recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) has at least 80% identity to SEQ ID NO: 1 (1263 positions matching 1395 amino acid residues is about 90% identity):
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That is, any kind or quantity of amino acid sequence fused (or not fused) to the termini of a polypeptide having recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) maintains at least 80% identity to SEQ ID NO: 111.
Regarding claim 132, “When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.” Gill, para. [0086]. As such, at the time of filing an ordinarily skilled artisan would have been motivated to include any two of the specific NLS sequences set forth in para. [0086] to be appropriately fused to SEQ ID NO: 7 of Gill (recited SEQ ID NO: 1) at or near an N-terminus as discussed above.
Pollard, abstract, evidences that “We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants.” Fig. 4C of Pollard shows an M9 NLS sequence that is consistent with SEQ ID NO: 115 of the specification for hnRNPA1 M9 NLS. In contrast, Pollard, page 985, left col., identifies SV40 large T antigen-type NLS as a classical NLS operating through a different mechanism than the M9 mutant (consisting with SEQ ID NO: 115 of specification).
As such, selection from para. [0086] Gill of “NLS of the SV40 virus large T-antigen” and “hRNPA1 M9 NLS” results in at least two of the NLSs have different nuclear localization mechanisms. An ordinarily skilled artisan would have been motivated to select SV40 NLS and hnRNPA1 NLS since Gill instructs that any two NLSs as described can be selected.
Claim(s) ) 123-138 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gill et al. (U.S. 2018/0371497 A1) as evidenced by Pollard et al. (A Novel Receptor-Mediated Nuclear Protein Import Pathway, Cell 86, 1996, 985-94) further in view of Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
The rejections of claims 123-133 and 138 under 35 U.S.C. 102 and 103 as stated above for those claims are incorporated herein by reference.
Regarding claims 134-137, as discussed above, Gill teaches that it is appropriate to fuse a nuclease having recited SEQ ID NO: 1 (SEQ ID NO: 7) with several NLS sequences (including near the N-terminus) and a 6x-His tag, which is a purification tag. “Codon optimized MAD1-MAD20 were cloned into an expression construct comprising a constitutive or inducible promoter (eg., proB promoter SEQ ID NO: 83, or pBAD promoter SEQ ID NO: 81 or SEQ ID NO: 82) and an optional 6X-His tag (SEQ ID NO: 182) (eg., FIG. 2). The generated MAD1-MAD20 expression constructs are provided as SEQ ID NOs: 61-80, respectively” Gill, para. [0248].
The specific constructs described by Gill, including SEQ ID NO: 67 comprising recited SEQ ID NO: 1, appears to have 6x-His tag C-terminal. However, CRISPR nuclease constructs having the following components in a single polypeptide:
A CRISPR nuclease;
Several NLS domains; and
A 6x-His purification tag.
For example, Staahl teaches polypeptides containing Cas9 CRISPR nuclease polypeptides as a fusion construct. Fig. 1 of Staahl teaches constructs having the following structure:
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More specific, Fig. 1b of Staahl shows constructs of a Cas9 CRISPR nuclease with several SV40 NLS domains fused C-terminal and/or N-terminal with optional presence of GFP.
Staahl provides:
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From on-line version of paper:
“The recombinant Streptococcus pyogenes Cas9 used in this study carries two C-terminal SV40 nuclear localization sequences. The protein was expressed with an N-terminal hexahistidine tag and maltose binding protein in Escherichia coli Rosetta 2 cells (EMD Millipore, Billerica, MA) from plasmids based on pMJ915 (Addgene plasmid # 69090). N-terminal nuclear localization sequence peptide arrays and sfGFP modifications were cloned into the plasmid using Gibson DNA assembly technique (Supplementary Table 1). The His tag and maltose binding protein were cleaved by TEV protease, and Cas9 was purified by the protocols described previously.”
In order for the His tag to be cleaved by TEV protease as taught, there must necessarily be a cleavage site for TEV. That is, “cleaved by TEV protease” means cleaved at a cleavage site. Since the His-tag is N-terminal, the cleavage site is near the His-tag that is near the N-terminus of the nuclease polypeptide. It is clear that after removal of the His-tag, the remaining construct containing the NLS domains and CRISPR Cas9 nuclease remains.
From Supplementary Table 1 of Staahl:
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That is, the 6x-His purification tag is N-terminal to “TEVscar,” which is a remnant of a TEV cleavage site, followed by NLS and Cas9 nuclease in that order from N-terminus to C-terminus of the polypeptide.
It is recognized that Staahl employs a different CRISPR nuclease, being Cas9, than the MAD7 nuclease discussed above with respect to Gill. Regardless, Gill and Staahl teaches that the following are well-known components of CRISPR nuclease polypeptides made for modification of nuclear DNA:
A CRISPR nuclease;
Several NLS domains; and
A 6x-His purification tag.
Regardless of the different identity of CRISPR nuclease between Gill and Staahl, an ordinarily skilled artisan would have recognized that:
It is desirable for a His-Tag to be removable by presence of a cleavage site wherein inclusion of a His-tag as C-terminal or N-terminal is a design choice wherein Staahl teaches that inclusion of a His-tag N-terminal followed by a cleavage sequence is expected to be successful.
Plural NLS sequences followed by a CRISPR nuclease, whether recited SEQ ID NO: 1 or Cas9, can be ordered N-terminal to C-terminal within an appropriate polypeptide.
MPEP 2144.04(V) sets forth that rearrangement of parts, without any new or unexpected effect, is an obvious matter of design choice. Here, both Gill and Staahl teach that the critical elements of a polypeptide for DNA modification is 1) a CRISPR nuclease, 2) Several NLS domains, and 3) preferably a purification tag (e.g. 6x-His). Staahl teaches that the following arrangement of elements, from N-terminus to C-terminus in a polypeptide, is expected to be successful in producing a useful CRISPR:
6x-His purification tag—TEV cleavage site—multiple NLS—CRISPR nuclease.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to form a polypeptide with a MAD7 nuclease having recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) in a polypeptide having the same structure as taught above in connection with Staahl, since Staahl teach that the same is a proper arrangement of a CRISPR nuclease, several NLS domains and a 6x-His purification tag expected to be successful for the purpose of modification of nuclear DNA wherein the arrangement of such elements including the inclusion of a cleavage site for removal of a purification tag is a matter of design choice. Upon incorporation of a MAD7 nuclease having recited SEQ ID NO: 1 (SEQ ID NO: 7 of Gill) in a polypeptide having the same structure as taught above in connection with Staahl the features of claims 134 and 135 are reached.
Regarding claim 136, as set forth above, Gill directly states that 5 NLS domains can be present all of which are present at or near an N-terminus. In view of such a direct teaching, an ordinarily skilled artisan at time of filing would have been motivated to include 5 NLS domains in any polypeptide construct consistent with the teachings of Gill.
Regarding claim 137, as discussed above, SEQ ID NO: 7 of Gill (recited SEQ ID NO: 1) has at least 80% identity to recited SEQ ID NO: 111 regardless of the nature or quantity of sequence that may or may not be fused to the N-terminus of SEQ ID NO: 7 of Gill (recited SEQ ID NO: 1).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 123-139 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 46-75 of copending Application No. 17/506,572 in view of Gill et al. (U.S. 2018/0371497 A1) ) and Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
Patented or copending claims are referred to as reference claims. All rejections under 35 U.S.C. 102 and 103 stated above relying upon Gill and Staahl are incorporated herein by reference.
Reference claim 46 states:
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Further, SEQ ID NO: 1 as recited in the reference claims is identical to recited SEQ ID NO: 1. The “separate nucleic” acids as recited in the reference claims is understood as dual gRNA having the features directly recited in claims 138 and 139.
The reasons why an ordinarily skilled artisan at the time of filing would have been motivated to form a composition/system being a Type V CRISPR nuclease having recited SEQ ID NO: 1 with several (i.e. 2, 3, 4, or 5) NLS near the N-terminus of the same polypeptide, as well as the remaining features of the rejected claims are stated above, is set forth above. In particular, NLS sequences allows for a nuclease to access the nuclease of a cell of DNA modification. As such, an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the reference claims to have all of the features of the rejected claims for this reason as stated above with reference to the teachings of Gill and Staahl.
This is a provisional nonstatutory double patenting rejection.
Claim 123-139 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-27 of U.S. Patent No. 12,270,044 in view of Gill et al. (U.S. 2018/0371497 A1) ) and Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
Patented or copending claims are referred to as reference claims. All rejections under 35 U.S.C. 102 and 103 stated above relying upon Gill and Staahl are incorporated herein by reference.
Reference claims recite:
1. An engineered, non-naturally occurring system comprising:
(a) a targeter nucleic acid comprising, from 5′ to 3′:
(i) a targeter stem sequence;
(ii) a spacer sequence designed to hybridize with a target nucleotide sequence; and
(iii) an optional additional nucleotide sequence;
(b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,
wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, wherein the targeter nucleic acid, the modulator nucleic acid, or both, comprises a chemically modified RNA, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A Cas nuclease; and
(c) the Type V-A Cas nuclease.
Further, SEQ ID NO: 1 as recited in the reference claims is identical to recited SEQ ID NO: 1. The “separate nucleic” acids as recited in the reference claims is understood as dual gRNA having the features directly recited in claims 138 and 139.
The reasons why an ordinarily skilled artisan at the time of filing would have been motivated to form a composition/system being a Type V CRISPR nuclease having recited SEQ ID NO: 1 with several (i.e. 2, 3, 4, or 5) NLS near the N-terminus of the same polypeptide, as well as the remaining features of the rejected claims are stated above, is set forth above. In particular, NLS sequences allows for a nuclease to access the nuclease of a cell of DNA modification. As such, an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the reference claims to have all of the features of the rejected claims for this reason as stated above with reference to the teachings of Gill and Staahl.
Claim 123-139 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12 of U.S. Patent No. 12,319,932 in view of Gill et al. (U.S. 2018/0371497 A1) ) and Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
Patented or copending claims are referred to as reference claims. All rejections under 35 U.S.C. 102 and 103 stated above relying upon Gill and Staahl are incorporated herein by reference.
Reference claims recite:
A nuclease complex composition comprising a synthetic split guide nucleic acid (gNA) comprising: (i) a targeter nucleic acid comprising: (a) a spacer sequence capable of hybridizing with a target nucleotide sequence, and (b) a targeter stem sequence; and (ii) a modulator nucleic acid comprising: (a) a modulator stem sequence complementary to the target stem sequence, and (b) a 5′ sequence; wherein the targeter stem sequence and the modulator stem sequence comprise four or five nucleotides that base pair with each other, and wherein the modulator and targeter nucleic acids are separate nucleic acids, and the split gNA is capable of binding to and forming a nucleic acid-guided nuclease complex, wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A Cas nuclease, and the Type V-A nuclease, wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides and/or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, wherein the modulator nucleic acid comprises a 2′-O-methoxy modification to the 5′ nucleotide, and wherein the nuclease complex retains activity compared to a nuclease complex with the same split gNA without the modifications.
The “split guide nucleic acid” as recited in the reference claims is understood as dual gRNA having the features directly recited in claims 138 and 139 wherein discussion of crRNA in the claim indicates that guide nucleic acid should be RNA.
The reasons why an ordinarily skilled artisan at the time of filing would have been motivated to form a composition/system being a Type V CRISPR nuclease having recited SEQ ID NO: 1 with several (i.e. 2, 3, 4, or 5) NLS near the N-terminus of the same polypeptide, as well as the remaining features of the rejected claims are stated above, is set forth above. In particular, NLS sequences allows for a nuclease to access the nuclease of a cell of DNA modification. As such, an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the reference claims to have all of the features of the rejected claims for this reason as stated above with reference to the teachings of Gill and Staahl.
Claim 123-139 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12 of U.S. Patent No. 12,270,043 in view of Gill et al. (U.S. 2018/0371497 A1) ) and Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
Patented or copending claims are referred to as reference claims. All rejections under 35 U.S.C. 102 and 103 stated above relying upon Gill and Staahl are incorporated herein by reference.
Reference claims recite:
A nuclease complex composition comprising a synthetic guide nucleic acid (gNA) comprising: (i) a targeter nucleic acid comprising: (a) a spacer sequence capable of hybridizing with a target nucleotide sequence, and (b) a targeter stem sequence; and (ii) a modulator nucleic acid comprising: (a) a modulator stem sequence complementary to the targeter stem sequence, and (b) a 5′ sequence; wherein the targeter stem sequence and the modulator stem sequence each comprise 4-10 nucleotides that base pair with each other, and the gNA is capable of binding to a CRISPR Type VA nucleic acid-guided nuclease and forming a nucleic acid-guided nuclease complex, wherein the targeter and modulator nucleic acids are separate polynucleotides, and wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, and wherein the modulator nucleic acid comprises at least one modified nucleotide and at least two modified internucleotide linkages within the first 10 nucleotides from the 5′ end, wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A Cas nuclease, and wherein the nuclease complex retains activity compared to a nuclease complex with the same gNA without the modifications, and the Type V-A nuclease.
The “split guide nucleic acid” as recited in the reference claims is understood as dual gRNA having the features directly recited in claims 138 and 139 wherein discussion of crRNA in the claim indicates that guide nucleic acid should be RNA.
The reasons why an ordinarily skilled artisan at the time of filing would have been motivated to form a composition/system being a Type V CRISPR nuclease having recited SEQ ID NO: 1 with several (i.e. 2, 3, 4, or 5) NLS near the N-terminus of the same polypeptide, as well as the remaining features of the rejected claims are stated above, is set forth above. In particular, NLS sequences allows for a nuclease to access the nuclease of a cell of DNA modification. As such, an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the reference claims to have all of the features of the rejected claims for this reason as stated above with reference to the teachings of Gill and Staahl.
Claims 123-139 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 46-75 of copending Application No. 17/506,572 in view of Gill et al. (U.S. 2018/0371497 A1) ) and Staahl et al. (Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nature Biotechnol. 35, 2017, 431-34).
Patented or copending claims are referred to as reference claims. All rejections under 35 U.S.C. 102 and 103 stated above relying upon Gill and Staahl are incorporated herein by reference.
The reference claims recite:
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Tables 1, 2 and 3 of the specification of Application No. 17/506,572 are crRNA sequences. A guide nucleic acid sequence with an additional modulator nucleic acid as in reference claim 5 is considered to be a guide RNA (gNA) that meets the features directly recited in claim 138 and 139.
The reasons why an ordinarily skilled artisan at the time of filing would have been motivated to form a composition/system being a Type V CRISPR nuclease having recited SEQ ID NO: 1 with several (i.e. 2, 3, 4, or 5) NLS near the N-terminus of the same polypeptide, as well as the remaining features of the rejected claims are stated above, is set forth above. In particular, NLS sequences allows for a nuclease to access the nuclease of a cell of