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 Status
Claims 1-140 are cancelled. Claims 141-160 are new.
Claims 141-160 are examined on the merits.
Priority
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63241928, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The application fails to provide support for the claims under examination, since there is no disclosure regarding SEQ ID NO 446. Therefore, the effective filling date of claims 150-153, 159-160 is deemed to be September 06, 2022, the filling date of the application PCT/US2022/075992.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). See the nucleotide sequences at Table 2, page 47. See the amino acid sequences at Table 5 at page 51. See the nucleotide sequences at Table 6 at page 53. See the nucleotide sequences at Table 7 at page 54. See the nucleotide sequences at Table 8 at page 55.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Objections
Claim 150 is objected to because of the following informalities: the claim recites, “at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446.” SEQ ID NO: 446 does not contain any degenerate nucleotides. Thus, the recitation of “non-degenerate” is unnecessary. It would be preferrable to amend the claim to recite “at least 80% sequence identity to the sequence of SEQ ID NO: 446.” Appropriate correction is required.
Claim 151 is objected to because of the following informalities: the claim recites, “at least 90% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446.” SEQ ID NO: 446 does not contain any degenerate nucleotides. Thus, the recitation of “non-degenerate” is unnecessary. It would be preferrable to amend the claim to recite “at least 90% sequence identity to the sequence of SEQ ID NO: 446.” Appropriate correction is required.
Claim 158 is objected to because of the following informalities: the claim recites, “at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446.” SEQ ID NO: 446 does not contain any degenerate nucleotides. Thus, the recitation of “non-degenerate” is unnecessary. It would be preferrable to amend the claim to recite “at least 80% sequence identity to the sequence of SEQ ID NO: 446.” Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 141-149 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a product of nature without significantly more. The claims recite a class 2 type V endonuclease and a guide ribonucleic acid.
Claim 141 recites “An engineered nuclease system comprising (a) an endonuclease wherein the endonuclease comprises a RuvCI domain, RuvCII domain, or RuvCIII domain comprising an amino acid sequence having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57; and (b) an engineered guide ribonucleic acid or a nucleic acid encoding the engineered guide ribonucleic acid. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification “Novel families of Type V CRISPR enzymes MG enzymes were identified through a large-scale analysis of metagenomes collected from a variety of complex environments, and representatives of these were developed systems into gene-editing platforms. The majority of these systems come from uncultivated organisms, some of which encode a divergent Type V effector within the same CRlSPR operon (e.g., paragraph 00121). Thus, the claimed endonuclease originates from a naturally occurring organism and represents a naturally occurring biological molecule that has been identified from environmental samples. Additionally, publicly available sequence indicates a substantially homologous transposase sequence, approximately 98% sequence identity present at GenBank database from Alistipes sp. (MBP3473483.1, available 03/24/2021, see alignment). This further demonstrates that the claimed endonuclease is naturally occurring.
The claim further recites an engineered guide RNA. However, the specification does not identify structural modifications that distinguish the claimed guide RNA from naturally occurring CRISPR RNAs present in the source organism. CRISPR guide RNAs naturally occur as components of endogenous CRISPR Cas immune systems in bacteria and archaea.
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Dependent claim 142 encompasses the nuclease system form claim 141, wherein the RuvCI domain, the RuvCII domain, or the RuvCIII domain of the endonuclease comprises an amino acid sequence having at least 90% sequence identity to the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 143 encompasses the endonuclease of claim 142, wherein the RuvCI domain, the RuvCII, or the RuvCIII domain of the endonuclease comprises the amino acid sequence of the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 144 encompasses the endonuclease of claim 141, wherein the endonuclease comprises a WED II domain comprising an amino acid sequence having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 145 encompasses the endonuclease of claim 144, wherein the WED II domain comprises an amino acid sequence having at least 90% sequence identity to the WED II domain of SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 146 encompasses the endonuclease of claim 145, wherein the WED II domain comprises an amino acid sequence having the amino acid sequence of the WED II domain of SEQ ID NO:57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 147 encompasses the endonuclease of claim 141, wherein the endonuclease is configured to be selective for a protospacer adjacent motif sequence comprising 5'-TTR-3'. The additional limitation is configured to be selective for the PAM sequence 5’-TTR-3’, does not render the claim patent eligible because PAM recognition specificity is an inherent biological property of naturally occurring endonuclease. The recited PAM selectivity merely describes a natural functional characteristic of the enzyme.
Dependent claim 148 encompasses the endonuclease of claim 141, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
Dependent claim 149 recites an endonuclease of claim 148, wherein the endonuclease comprises the amino acid sequence of SEQ ID NO: 57. The endonuclease of SEQ ID NO 57 is a natural product as disclosed in the instant specification.
This judicial exception is not integrated into a practical application because no elements in addition of the judicial exception are recited in the claims.
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no elements in addition to the judicial exception are recited in the claims.
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 141-148, 150-157, 159-160 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.
Claim 141 is drawn to a genus of engineered nuclease system. The rejected claims comprise a genus consisted of endonuclease comprising a RuvCI domain, RuvCII domain, or RuvCIII domain comprising an amino acid sequence having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57. The specification does not disclose the amino acid boundaries or locations of the claimed RuvCI, RuvCII or RuvCIII domains within SEQ ID NO 57. Furthermore, the specification does not provide adequate support for the limitation “having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57”, this represent possession of a broader genus of amino acid sequences encompassed by the claimed 75% identity limitation. The specification does not disclose identify of which amino acid substitutions, deletions or insertions are permissible while maintaining the endonuclease activity.
Claim 142 is drawn to a genus of engineered nuclease system. The rejected claims comprise a genus consisted of RuvCI domain, the RuvCII domain, or the RuvCIII domain of the endonuclease comprises an amino acid sequence having at least 90% sequence identity to the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57.
The specification does not disclose the amino acid boundaries or locations of the claimed RuvCI, RuvCII or RuvCIII domains within SEQ ID NO 57. Furthermore, the specification does not provide adequate support for the limitation “having at least 90% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57”, this represent possession of a broader genus of amino acid sequences encompassed by the claimed 90% identity limitation. The specification does not disclose identify of which amino acid substitutions, deletions or insertions are permissible while maintaining the endonuclease activity.
Claim 144 is drawn to a genus of engineered nuclease system. The rejected claims comprise a genus consisted of endonuclease comprises a WED II domain comprising an amino acid sequence having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57. The specification does not disclose the amino acid boundaries or location of the claimed WED II domain within SEQ ID NO 57. Furthermore, the specification does not provide adequate support for the limitation “having at least 75% sequence identity to WED II domain, of SEQ ID NO: 57”, this represent possession of a broader genus of amino acid sequences encompassed by the claimed 75% identity limitation. The specification does not disclose identify of which amino acid substitutions, deletions or insertions are permissible while maintaining the endonuclease activity.
Claim 145 is drawn to a genus of engineered nuclease system. The rejected claims comprise a genus consisted of endonuclease comprises a WED II domain comprising an amino acid sequence having at least 90% sequence identity to the WED II domain of SEQ ID NO: 57. The specification does not disclose the amino acid boundaries or location of the claimed WED II domain within SEQ ID NO 57. Furthermore, the specification does not provide adequate support for the limitation “having at least 90% sequence identity to WED II domain, of SEQ ID NO: 57”, this represent possession of a broader genus of amino acid sequences encompassed by the claimed 90% identity limitation. The specification does not disclose identify of which amino acid substitutions, deletions or insertions are permissible while maintaining the endonuclease activity.
Claim 148 is drawn to a genus of engineered nuclease system. The rejected claims comprise a genus consisted of endonuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. The specification does not provide adequate support for the limitation “having at least 90% sequence identity to SEQ ID NO: 57”, this represent possession of a broader genus of amino acid sequences encompassed by the claimed 90% identity limitation. The specification does not disclose identify of which amino acid substitutions, deletions or insertions are permissible while maintaining the endonuclease activity.
Claim 150 is drawn to a genus of engineered guide ribonucleic acid. The rejected claims comprise a genus consisted of a guide ribonucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 446. The specification does not provide adequate support for the limitation “having at least 80% sequence identity to SEQ ID NO: 446”, this represent possession of a broader genus of nucleotide sequences encompassed by the claimed 80% identity limitation. The specification does not disclose identify of which nucleotide substitutions, deletions or insertions are permissible while maintaining the complementarity to the target locus.
Claim 151 is drawn to a genus of engineered guide ribonucleic acid. The rejected claims comprise a genus consisted of a guide ribonucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 446. The specification does not provide adequate support for the limitation “having at least 90% sequence identity to SEQ ID NO: 446”, this represent possession of a broader genus of nucleotide sequences encompassed by the claimed 90% identity limitation. The specification does not disclose identify of which nucleotide substitutions, deletions or insertions are permissible while maintaining the complementarity to the target locus.
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 envisions engineered nuclease system comprising: an endonuclease having at least 75% sequence identity to SEQ ID NO: 57 or a variant thereof; and an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence (e.g., paragraph 0005). The specification envisions an engineered nuclease system comprising: an engineered guide RNA comprising a sequence with at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NO: 446, and a class 2, type V Cas endonuclease configured to bind to said engineered guide RNA. The specification envisions type 2, class V Cas endonuclease is derived from an uncultivated organism (e.g., paragraph 0009). The specification envisions a method of modifying a target nucleic acid locus. In some embodiments, said target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, said target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, said target nucleic acid locus is in vitro. In some embodiments, said target nucleic acid locus is within a cell. In some embodiments, said cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, a human cell, or a primary cell. In some embodiments, said cell is a primary cell. In some embodiments, said primary cell is a T cell. In some embodiments, said primary cell is a hematopoietic stem cell (HSC). In some embodiments, delivering said engineered nuclease system to said target nucleic acid locus comprises delivering any nucleic acid as disclosed herein or any vector as disclosed herein (e.g., 0017). In some embodiments, the endonuclease comprises a RuvCI, II, or III domain. the RuvCI domain comprises a D catalytic residue. In some embodiments the RuvCII domain comprises an E catalytic residue. In some embodiments
the RuvCIII domain comprises a D catalytic residue. In some embodiments, the RuvC domain does not have nuclease activity. In some embodiments, the endonuclease further comprises a WED II domain having at least about 80%, at least about 90%, identity to a WED II domain of any one of SEQ ID NO: 57 or a variant thereof (e.g., paragraph 0030). The specification envisions that SEQ ID NO: 57 show the full-length peptide sequences of MG119 nuclease (e.g., paragraph 0079).
The examples disclose the characterization of the Class 2 Type V endonuclease MG119-28 Cas. Example 1 discloses metagenomic analysis of samples collected from sediment, soil, and animals. This metagenomic workflow resulted in the delineation of the MG90, MG91A, MG91B, MG91C, MG118, MG119, MG120, MG122, and MG126 families. The corresponding protein and nucleic acid sequences for these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 1-325, 420-431, 476-624, or 629 (e.g., paragraph 00168; example 2).
Example 8 discloses the in vitro activity of MG119-2 in a plasmid library (e.g., paragraph 00174; Fig. 9; example 8).
Example 14 the characterization of Type V nucleases in the MG119 family: discovery of predicted proteins related to nuclease sequences in the MG 119 family of compact type V nucleases based on homology searches . The in vitro characterization of putative tracRNAs and sgRNA design. The in vitro cleavage reaction by different MG119 nucleases to confirm nuclease activity and enable PAM determination (e.g., paragraph bridge [00183-00195]; Table 3; Figs. 11-12). The working example discloses the expression and purification of MG119 candidates. The proteins expressed in this vector have the following sequence architecture: 6xHis-(GS)2-PSP-nucleoplasmin bipartite NLS-(GGS)l-(GS)l-MG119-X-(GGS)3-SV 40 NLS (e.g., paragraph bridge [00197-00205]; Fig. 13; Table 3). The working example disclose in vitro cleavage of intron 1 albumin of mouse Hepa1-6 DNA with ribonucleoproteins (RNPs) comprising the purified MG119-28 (SEQ ID NO 57) nuclease and 119-28 sgRNA3_Mouse_Alb (SEQ ID NO 627) sgRNA (e.g., paragraph 00209; Table 6; Fig. 15A; Table 10). The working examples discloses in vivo cell editing of Hepa 1-6 cells targeting intron 1 of albumin gene using RNP complexes (e.g., paragraph 00212; Table 8; Fig. 15B).
The working examples described in the specification only provides data for the in vitro and in vivo gene editing with a MG119-28 Cas endonuclease (SEQ ID NO 57) (full length) and 119-28 sgRNA3_Mouse_Alb (SEQ ID NO 627) and it is not representative of a very large variations allowed by the claims.
The specification discloses the full length MG119-28 endonuclease of SEQ ID NO 57. The specification does not disclose representative variants of SEQ ID NO 57; the specification does not disclose the identification of the claimed RuvCI, RuvCII or RuvCIII domains in the SEQ ID NO 57, nor disclose representative variants of the individual RuvC domains; the specification does not disclose the claimed WED II domain in the SEQ ID NO 57, nor variants of the WED II domain. Additionally, the specification fail to provide an adequate structure-function correlation demonstrating which amino acid substitutions within the claimed domains may be made while retaining the recited class 2 Type V endonuclease activity. The disclosure does not identify residues essential for the catalytic activity, residues that may be varies or any predictive principles that would allow one of ordinary skill in the art to recognize members of the claimed genus that retain the claimed function. Thus, further experimentation would be required to determine which variants of MEG119 are functional and which are not.
The examples described in the specification does not meet the limitation of the rejected claims “having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57”. “having at least 90% sequence identity to the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57”. “having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57”. “having at least 90% sequence identity to the WED II domain of SEQ ID NO: 57”. “Having at least 90% sequence identity to SEQ ID NO: 57”. “having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446”. “having at least 90% sequence identity to non- degenerate nucleotides of SEQ ID NO: 446”.
The state of the art with respect to using class 2, type V endonuclease is under developed and unpredictable. Yan et al. (Science, 2019) teaches that Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12 (e.g., abstract). Yan teaches that mutation of the RuvC-I motif of Cas12g1
[Asp513→Ala (D513A)] or omission of the noncoding plasmid substantially decreased interference activity (e.g., paragraph 2nd, middle column, Fig. 2B). Yan teaches that both RNA and ssDNA cleavage by Cas12g1 are metal ion dependent and require an intact RuvC domain that was previously known to cleave only DNA (e.g., paragraph ,2nd, right column, page 1; Fig. 2G, 2H). Zetsche et al. (Cell, 2015) teaches that the putative type V CRISPR-Cas systems contain a large, 1,300 amino acid protein called Cpf1 (CRISPR from Prevotella and Francisella 1). It remains unknown, however, whether Cpf1- containing CRISPR loci indeed represent functional CRISPR systems (e.g., paragraph 1st, left column, page 760). Zetsche teaches that the RuvC-like domain of Cpf1 retains all of the catalytic residues of this family of endonucleases. D917A and E1006A mutations completely inactivated the DNA cleavage activity of FnCpf1, and D1255A significantly reduced nucleolytic activity (e.g., paragraph 2nd, right column, page 762; Fig. 4B).
Thus, the prior art does not overcome the deficiency of the specification with regard to the description of a genus of MEG119 variants. The teachings are consistent with the prior art demonstrating the underdeveloped and unpredictability of the nature of the invention.
The claims encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 141-148, 150-157, 159-160.
Claims 154-157, 159-160 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of modifying a target locus, the method comprising delivering to the target locus: (a) a class 2 type V endonuclease or a nucleic acid encoding the endonuclease, wherein the endonuclease comprises the amino acid sequence of SEQ ID NO: 57; and (b) an engineered guide ribonucleic acid or a nucleic acid encoding the engineered guide ribonucleic acid, wherein the engineered guide ribonucleic acid forms a complex with the endonuclease, and the engineered guide ribonucleic acid comprises a spacer sequence configured to hybridize to a eukaryotic target nucleic acid sequence, wherein the complex modifies the target nucleic acid locus, does not reasonably provide enablement for using endonuclease variants comprising an amino acid sequence having at least 75%, of sequence identity to RuvCI domain, RuvCII domain or RuvCIII domain of SEQ ID NO 57; nor for using the endonuclease comprising a WED II domain comprising an amino acid sequence having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57; nor for using the endonuclease comprising amino acid sequence having at least 90% sequence identity to SEQ ID NO 57; nor for using a guide ribonucleic acid comprising a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988). Wands states, on page 1404: Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex part Forman. These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: The instant claim 154 is drawn to a method of modifying a target nucleic acid locus, the method comprising delivering to the target nucleic acid locus:(a) an endonuclease or a nucleic acid encoding the endonuclease, wherein the endonuclease is a class 2, type V endonuclease, wherein the endonuclease comprises a RuvCI domain, RuvCII domain, or RuvCIII domain, comprising an amino acid sequence having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57. The nature of the claim is complicated, because the claim requires the outcome of modifying a target locus, yet the claim is drawn to delivering an endonuclease where the RuvCI domain, RuvCII domain, or RuvCIII domain, comprising an amino acid sequence having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain of SEQ ID NO: 57.
The instant claim 155 is drawn to a method wherein the RuvCI domain, the RuvCII domain, or the RuvCIII domain comprises an amino acid sequence having at least 90% sequence identity to the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57. The nature of the claim is complicated, because the claim requires the outcome of modifying a target locus, yet the claim is drawn to delivering an endonuclease wherein the RuvCI domain, RuvCII domain, or RuvCIII domain, comprising an amino acid sequence having at least 90% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain of SEQ ID NO: 57.
The instant claim 156 is drawn to a method wherein the endonuclease comprises a WED II domain comprising an amino acid sequence having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57. The nature of the claim is complicated, because the claim requires the outcome of modifying a target locus, yet the claim is drawn to delivering an endonuclease wherein the WED II domain, comprising an amino acid sequence having at least 75% sequence identity to WED II domain of SEQ ID NO: 57.
The instant claim 157 is drawn to a method wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. The nature of the claim is complicated, because the claim requires the outcome of modifying a target locus, yet the claim is drawn to delivering an endonuclease, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57.
The instant claim 159 is drawn to a method wherein the engineered guide ribonucleic acid comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446. The nature of the claim is complicated, because the claim requires the outcome of modifying a target locus, yet the claim is drawn to delivering an engineered guide ribonucleic acid comprising a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446.
Breadth of the claim: The claims encompass a method of modifying a target nucleic acid locus by delivering a endonuclease and an engineered guide RNA. The claims are broad with respect to the RuvCI, RuvCII, RuvCIII and WED II domains of the endonuclease and to the guide RNA. The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: The specification envisions engineered nuclease system comprising: an endonuclease having at least 75% sequence identity to SEQ ID NO: 57 or a variant thereof; and an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence (e.g., paragraph 0005). The specification envisions an engineered nuclease system comprising: an engineered guide RNA comprising a sequence with at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NO: 446, and a class 2, type V Cas endonuclease configured to bind to said engineered guide RNA. The specification envisions type 2, class V Cas endonuclease is derived from an uncultivated organism (e.g., paragraph 0009). The specification envisions a method of modifying a target nucleic acid locus. In some embodiments, said target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, said target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, said target nucleic acid locus is in vitro. In some embodiments, said target nucleic acid locus is within a cell. In some embodiments, said cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, a human cell, or a primary cell. In some embodiments, said cell is a primary cell. In some embodiments, said primary cell is a T cell. In some embodiments, said primary cell is a hematopoietic stem cell (HSC). In some embodiments, delivering said engineered nuclease system to said target nucleic acid locus comprises delivering any nucleic acid as disclosed herein or any vector as disclosed herein (e.g., 0017). In some embodiments, the endonuclease comprises a RuvCI, II, or III domain. the RuvCI domain comprises a D catalytic residue. In some embodiments the RuvCII domain comprises an E catalytic residue. In some embodiments
the RuvCIII domain comprises a D catalytic residue. In some embodiments, the RuvC domain does not have nuclease activity. In some embodiments, the endonuclease further comprises a WED II domain having at least about 80%, at least about 90%, identity to a WED II domain of any one of SEQ ID NO: 57 or a variant thereof (e.g., paragraph 0030). The specification envisions that SEQ ID NO: 57 show the full-length peptide sequences of MG119 nuclease (e.g., paragraph 0079).
The examples disclose the characterization of the Class 2 Type V endonuclease MG119-28 Cas. Example 1 discloses metagenomic analysis of samples collected from sediment, soil, and animals. This metagenomic workflow resulted in the delineation of the MG90, MG91A, MG91B, MG91C, MG118, MG119, MG120, MG122, and MG126 families. The corresponding protein and nucleic acid sequences for these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 1-325, 420-431, 476-624, or 629 (e.g., paragraph 00168; example 2).
Example 8 discloses the in vitro activity of MG119-2 in a plasmid library (e.g., paragraph 00174; Fig. 9; example 8).
Example 14 the characterization of Type V nucleases in the MG119 family: discovery of predicted proteins related to nuclease sequences in the MG 119 family of compact type V nucleases based on homology searches . The in vitro characterization of putative tracRNAs and sgRNA design. The in vitro cleavage reaction by different MG119 nucleases to confirm nuclease activity and enable PAM determination (e.g., paragraph bridge [00183-00195]; Table 3; Figs. 11-12). The working example discloses the expression and purification of MG119 candidates. The proteins expressed in this vector have the following sequence architecture: 6xHis-(GS)2-PSP-nucleoplasmin bipartite NLS-(GGS)l-(GS)l-MG119-X-(GGS)3-SV 40 NLS (e.g., paragraph bridge [00197-00205]; Fig. 13; Table 3). The working example disclose in vitro cleavage of intron 1 albumin of mouse Hepa1-6 DNA with ribonucleoproteins (RNPs) comprising the purified MG119-28 (SEQ ID NO 57) nuclease and 119-28 sgRNA3_Mouse_Alb (SEQ ID NO 627) sgRNA (e.g., paragraph 00209; Table 6; Fig. 15A; Table 10). The working examples discloses in vivo cell editing of Hepa 1-6 cells targeting intron 1 of albumin gene using RNP complexes (e.g., paragraph 00212; Table 8; Fig. 15B).
The working examples described in the specification only provides data for the in vitro and in vivo gene editing with a MG119-28 Cas endonuclease (SEQ ID NO 57) (full length) and 119-28 sgRNA3_Mouse_Alb (SEQ ID NO 627) and it is not representative of a very large variations allowed by the claims.
The specification discloses the full length MG119-28 endonuclease of SEQ ID NO 57. The specification does not disclose representative variants of SEQ ID NO 57; the specification does not disclose the identification of the claimed RuvCI, RuvCII or RuvCIII domains in the SEQ ID NO 57, nor disclose representative variants of the individual RuvC domains; the specification does not disclose the claimed WED II domain in the SEQ ID NO 57, nor variants of the WED II domain. Additionally, the specification fail to provide an adequate structure-function correlation demonstrating which amino acid substitutions within the claimed domains may be made while retaining the recited class 2 Type V endonuclease activity. The disclosure does not identify residues essential for the catalytic activity, residues that may be varies or any predictive principles that would allow one of ordinary skill in the art to recognize members of the claimed genus that retain the claimed function. Thus, further experimentation would be required to determine which variants of MEG119 are functional and which are not.
The examples described in the specification does not meet the limitation of the rejected claims “having at least 75% sequence identity to RuvCI domain, RuvCII domain, or RuvCIII domain, respectively, of SEQ ID NO: 57”. “having at least 90% sequence identity to the RuvCI domain, the RuvCII domain, or the RuvCIII domain, respectively, of SEQ ID NO: 57”. “having at least 75% sequence identity to the WED II domain of SEQ ID NO: 57”. “having at least 90% sequence identity to the WED II domain of SEQ ID NO: 57”. “Having at least 90% sequence identity to SEQ ID NO: 57”. “having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 446”. “having at least 90% sequence identity to non- degenerate nucleotides of SEQ ID NO: 446”.
Predictability and state of the art: The state of the art with respect to using class 2, type V endonuclease is under developed and unpredictable. Yan et al. (Science, 2019) teaches that Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12 (e.g., abstract). Yan teaches that mutation of the RuvC-I motif of Cas12g1 [Asp513→Ala (D513A)] or omission of the noncoding plasmid substantially decreased interference activity (e.g., paragraph 2nd, middle column, Fig. 2B). Yan teaches that both RNA and ssDNA cleavage by Cas12g1 are metal ion dependent and require an intact RuvC domain that was previously known to cleave only DNA (e.g., paragraph ,2nd, right column, page 1; Fig. 2G, 2H). Zetsche et al. (Cell, 2025) teaches that the putative type V CRISPR-Cas systems contain a large, 1,300 amino acid protein called Cpf1 (CRISPR from Prevotella and Francisella 1). It remains unknown, however, whether Cpf1- containing CRISPR loci indeed represent functional CRISPR systems (e.g., paragraph 1st, left column, page 760). Zetsche teaches that the RuvC-like domain of Cpf1 retains all of the catalytic residues of this family of endonucleases. D917A and E1006A mutations completely inactivated the DNA cleavage activity of FnCpf1, and D1255A significantly reduced nucleolytic activity (e.g., paragraph 2nd, right column, page 762; Fig. 4B). Taylor et a. (Nat. Struct. & Mol. Biol., 2026) teaches characterization of miniature CRISPR–Cas12f nucleases (Type V) a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells (e.g., abstract). Taylor teaches that to understand the importance of intermediate structures,
the RuvC lid in OsCas12f and Al3Cas12f were examined. States I and II
in OsCas12f (8-bp R-loop formed) show the RuvC lid in a ‘closed’ conformation. OsCas12f State III structure is flexible and not modeled (D335–L342). This lid is essential for cleavage by substituting E334–R343 to alanine, preventing the lid from undergoing the rearrangements necessary for cleavage and resulting in significantly reduced OsCas12f activity in GFP depletion assays. In addition to the RuvC loop, W345 in OsCas12f positions DNA in the active site of the RuvC domain, like what has been observed in UnCas12f. A W345A substitution significantly hindered its activity in E. coli, indicating that this residue is essential for activity in OsCas12f. Moreover, we also observed a smaller loop region (residues 229–235, denoted as lid 2) that undergoes similar conformational changes. Alanine substitutions of the residues 229–231 abolished OsCas12f activity, indicating an essential role in nuclease activity (e.g., paragraph 1st, left column, page 763; Fig. 5).
Thus, the teachings of the post-filing art are consistent with the prior art demonstrating the underdeveloped and unpredictable nature of the invention.
Amount of experimentation necessary: It would require large amount of experimentation to make use of any Class 2 Type V CRISPR Cas variants for targeted genome modification in a cell.
In view as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 154-157, 159-160 are not considered to be fully enabled by the instant disclosure.
In view of the breadth of the claims, the lack of guidance provided by the specification, the lack of the predictability of the art to which the invention pertains, undue amount of experimentation would be required to make and use the claimed invention for gene editing in a cell, with a reasonable expectation of success. Because the specification does not contain a detailed description of how to make and use the method based on the delivery of the claimed Class 2 Type V endonuclease variants, or representative variants of the individual RuvC domains of the class 2 Type V endonuclease according to the invention, or variants of the WED II domain of the class 2 Type V endonuclease according to the invention, and absent working examples that provide evidence that is reasonably predictive of the ability of gene editing of cells, the claims are not enabled commensurate in scope with the claimed invention.
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.
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Claims 141-146, 148-160 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 5, 17, 32, of copending Application No. 19162390 (hereinafter ‘390 application).
Although the claims at issue are not identical, they are not patentably distinct from each other. Claim 1 of the ‘390 application is drawn to “An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-3251-325, 420-431, 476-624, 629, 1065-1090, 1114- 1118, 1746-1752, and 1842-1872; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence”. Claim 2 of the ‘390 application that depends of claim 1, is drawn to the engineered nuclease system of claim 1, wherein the endonuclease comprises a sequence having at least 90% or 100% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-3251-325, 420-431, 476-624, 629, 1065-1090, 1114-1118, 1746-1752, and 1842-1872. Instant claims 141-146, 149 encompasses embodiments where the endonuclease is SEQ ID NO 57 (see alignment below). Accordingly, instant claims 141-146, 149 are anticipated by claims 1-2 of ‘390 application.
SEQ ID NO 57 vs SEQ ID NO 57:
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Claim 5 of ‘390 application depends of claim 1, is drawn to “The engineered nuclease system of claim 1, wherein the engineered guide polynucleotide comprises a sequence having at least 90% or 100% sequence identity to any one of SEQ ID NOs: 333-335, 355-357, 410-411, 346-347, 368-369, 412- 413, 326-332, 336-345, 348-354, 358-367, 414-419, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 647-766, 1091-1113, 1119-1120, 1697-1731, 1807-1836, 1876, and 1887-1894”. Instant claims 150-152, 159-160 encompass embodiments where the engineered guide polynucleotide is SEQ ID NO 446 (see alignment below). Accordingly, instant claims 150-152, 159-160 are anticipated by claim 5 of ‘390 application.
SEQ ID NO 446 vs SEQ ID NO 446:
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294
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Claim 17 of the ‘390 is drawn to “An engineered nuclease system comprising: (iv) a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-150, 420-431, 476-624, and 629; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 326-332, 336-345, 348-354, 358-367, 414-419, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 647-766, and 1697-1731. Instant claim 153 encompasses embodiments where the endonuclease is SEQ ID NO 57 and the engineered guide polynucleotide is SEQ ID NO 446 (see alignment below). Accordingly, instant claim 153 is anticipated by claim 17 of ‘390 application.
Claim 32 of ‘390 application depends of claim 1, is drawn to “A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of claim 1 (an endonuclease of SEQ ID NO 57 and an engineered guide ribonucleic acid)”. Instant claims 154-158 encompass embodiments where the endonuclease is SEQ ID NO 57. Accordingly, instant claims 154-158 are anticipated by claim 32 of ‘390 application.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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