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 .
Application Status
Applicant’s response filed March 13, 2026, including remarks, sequence listing, and amendments to the specification, drawings, and claims is acknowledged. Claim 58 was amended, claims 1, 9, 16, 19-22, 25, 30, 33-34, 43, 53, 72-73, 78-79, 81, and 83 were cancelled, and claims 87-105 were introduced. New claims 87-105 are drawn to Group V identified in the restriction requirement mailed January 16, 2026. Claims 58, and 87-105 are pending.
Notice of Non-Compliant Amendment to the Specification
The substitute specification filed March 13, 2026 does not include the amendments to the specification filed September 20, 2023 to the paragraph “CROSS-REFERENCE TO RELATED APPLICATIONS.” In the interest of compact prosecution, the response filed March 13, 2026, including the substitute specification, has been entered. The paragraph below the heading “CROSS-REFERENCE TO RELATED APPLICATIONS” in the substitute specification filed March 13, 2026 should read as follows:
“This application is a 35 U.S.C. § 371 National Stage Application of lnternational Application No. PCT/US2022/021523, filed on March 23, 2022, which claims priority to U.S. Provisional Patent Application Serial No. 63/164,798, filed on March 23, 2021, each of which is incorporated by reference herein in its entirety for all purposes.”
Restriction/Election
Applicant’s election without traverse of Group V (claim 58, and 87-105) in the reply filed on March 13, 2026 is acknowledged. Applicant’s election of the following species is also acknowledged: Species 1: “the Streptococcus constellatus Cas9 of SEQ ID NO: 1,” Species 2: “NLS of SEQ ID NO: 2,” Species 3: “one mutation,” Species 4: “the RuvC domain, and one mutation thereof,” Species 5: “an adenosine deaminase fused with a ScoCas9 D10A nickase of SEQ ID NO: 20,” Species 6: “5'-NGG-3',” Species 7: “a guide RNA of SEQ ID NO: 3,” Species 8: “a nickase,” Species 9: “a lentiviral vector,” Species 10: “editing the target nucleic acid,” Species 11: “a base editor comprising an adenosine deaminase domain, and wherein the one or more guide RNAs target the base editor to effect an A-T to G-C alteration in the polynucleotide.” With respect to the species elections, because Applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the species elections have been treated as elections without traverse (MPEP § 818.01(a)).
All pending claims are drawn to the elected Group and species. No claims are withdrawn from consideration. Claims 58, and 87-105 are under examination hereinafter, accordingly.
Priority
Applicant’s priority claims to Application Nos. 63/164,798 and PCT/US2022/021523 are acknowledged. Claims 58, and 87-105 find support in Application No. 63/164,798. The effective filing date of the claims currently under examination is March 23, 2021, accordingly.
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:
I. Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because the CRF of the “Sequence Listing” is defective. Please refer to the notice dated March 16, 2026, which indicates that an ST.26 format sequence listing was filed March 13, 2026; however, a sequence listing in “ST.25 format is required” in this application.
Required response - Applicant must provide:
A "Sequence Listing" part of the disclosure, as described above in item 1); together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(a)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.825(a)(4); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(a)(3)
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, 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.
If the "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, Applicant must also provide:
A CRF in accordance with 37 CFR 1.821(e)(1) or 1.821(e)(2) as required by 37 CFR 1.825(a)(5); and
A statement according to item 2) a) or b) above.
II. Specific deficiency - This application contains sequence disclosures in accordance with the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR 1.821 - 1.825.
The sequence identifiers associated with several amino acid sequences in Table 2 of the specification (i.e., at least the amino acid sequences associated with “SEQ ID NO: 95” and “SEQ ID NO: 96”) do not correspond to the sequences set forth in SEQ ID NOs: 95-96 in the sequence listing, or in other sections of the disclosure (see pg. 30; Fig. 2F; “FIG. 2D depicts sgRNA comprising SEQ ID NO: 95… SEQ ID NO: 96,” pg. 44). Based on the sequence listing and the specification, SEQ ID NOs: 95-96 correspond to RNA sequences. The correct sequence identifiers corresponding to the amino acid sequences on pgs. 65-66 should be provided. It is noted that other SEQ ID NOs (e.g., SEQ ID NOs: 98-99) disclosed in the specification do not appear to correspond to the sequences set forth in the sequence listing.
Required response – Applicant must provide:
A "Sequence Listing" part of the disclosure, as described above in item 1); as well as
An amendment specifically directing entry of the "Sequence Listing" part of the disclosure into the application in accordance with 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter in accordance with 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, 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;
If the "Sequence Listing" part of the disclosure is submitted according to item 1) b), c), or d) above, Applicant must also provide:
A replacement CRF in accordance with 1.825(b)(6); and
Statement according to item 2) a) or b) above.
Drawings
The drawings are objected to because of the following informalities:
The view numbers for the partial views for Fig. 10B are followed by "CONTINUED" instead of a capital letter such as FIG. 1A, FIG. 1B, etc. 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.”
Appropriate correction is required.
Specification
The specification is objected to because of the following informalities:
The sequence identifier set forth in Fig. 2D (i.e., SEQ ID NOs: 19) do not appear to comprise the sequence identifiers set forth in the brief description of Fig. 2D (i.e, SEQ ID NOs: 95, and 96)(pg. 44). The sequence identifiers set forth in the brief descriptions of Fig. 2D should be amended, accordingly.
The use of terms which are trade names or marks used in commerce, has been noted in this application, e.g., “Lipofectamine” (pg. 126). The terms should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term. Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code, i.e., “www.kazusa.orjp/codon/” (pg. 127). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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.
Claims 87-102 are 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.
Claims 87-91, 99-100, and 102 recite “the Cas9 protein.” It is not clear which Cas9 protein is referred to by this generic term, because two proteins provided in claim 58 from which the claims depend could be considered a Cas9 protein, i.e., I) the “codon-optimized CRISPR-associated protein,” which is modified from II) the “Streptococcus constellatus Cas9 protein having at least 80% sequence identity to SEQ ID NO: 1.” Neither the specification nor remaining claims provide clarity as to which of the possible Cas9 proteins the generic term “the Cas9 protein” refers; claims 101 and 103, in contrast, explicitly refer to “the codon-optimized Cas9 protein,” and “the Streptococcus constellatus Cas9.”
Because it is not clear to which of the possible Cas9 proteins the generic term “the Cas9 protein” refers, the structures of the systems are unclear. For example, it is not clear whether claim 87 requires that the codon-optimized Cas9 protein have the recited function, or whether the Streptococcus constellatus Cas9 protein from which the codon-optimized Cas9 protein is “modified” must have the recited function. In the latter case, the codon-optimized Cas9 protein, by virtue of being “modified” from the Streptococcus constellatus Cas9 protein, may not have the recited function. Similarly, it is not clear whether the system of claim 88 requires that the codon-optimized Cas9 protein have a D10A mutation in the RuvC domain, or whether the Streptococcus constellatus Cas9 protein from which the codon-optimized Cas9 protein is “modified” must have a D10A mutation in the RuvC domain, such that the codon-optimized Cas9, by virtue of being “modified” from the Streptococcus constellatus Cas9 protein, may not have a D10A mutation in the RuvC domain.
Claims 92-98 are rejected for depending from the aforementioned claims and failing to remedy the indefiniteness. In the interest of compact prosecution, the generic term “the Cas9 protein” will be interpreted hereinafter as referring to the codon-optimized CRISPR-associated protein in claim 58, as this is believed to be Applicant’s preferred embodiment based on the disclosure.
Claim 91 recites that “the [codon-optimized] Cas9 protein comprising the D10A mutation fused to the adenosine deaminase comprises a base editor with an amino acid sequence having at least 90% identity to SEQ ID NO: 20.” Based on the specification, SEQ ID NO: 20 corresponds to the “Amino Acid Sequence of Adenine Deaminase, TadA8.13m-nickase fused to the N-terminal of nickase ScoCas9 (ABE-nScoCas9, D10A mutant)” (Table 15, pg. 143-144). The “base editor” of SEQ ID NO: 20 comprises species of the generic elements of the codon-optimized D10A Cas9-adenosine deaminase fusion protein, which is itself a “base editor” as interpreted herein (pg. 34). The structural implications of the limitations of claim 90 for the codon-optimized D10A Cas9-adenosine deaminase fusion protein are not clear. Specifically, it is not clear whether the claim should be interpreted as requiring I) a codon-optimized D10A Cas9-adenosine deaminase fusion protein comprising a base editor with an amino acid sequence having at least 90% identity to SEQ ID NO: 20, such that claim 91 essentially requires two “base editors,” one generic, and one more specific, or II) a codon-optimized D10A Cas9-adenosine deaminase fusion protein comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 20, such that claim 91 further limits the sequence of the previously recited fusion protein, and the resulting fusion protein is the “base editor.”
Claims 92-98 are rejected for depending from claim 91 and failing to remedy the indefiniteness. In the interest of compact prosecution, claim 91 will be interpreted as in (II) above, i.e., wherein the Cas9 protein comprising the D10A mutation fused to the adenosine deaminase is a base editor comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 20. References to “the base editor” in claims 92-95 are interpreted hereinafter as referring to the structure interpreted as in (II) above.
Claim 100 recites that “the [codon-optimized] Cas9 protein further comprises a nuclear localization sequence (NLS) comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2. Based on the specification, SEQ ID NO: 2 corresponds to the amino acid sequence of “Streptococcus constellatus Cas9 with Nuclear Localization Signal (NLS) and Linker” (Table 1, pg. 49). The phrase “an amino acid sequence that is at least 80% identical to SEQ ID NO: X” would be interpreted as requiring an amino acid sequence that is 80% or more identical to the full-length of the recited SEQ ID NO. The structural implications of claim 100 are confusing, therefore, because the claim requires that “a nuclear localization sequence (NLS)” comprise at least 80% or more identity to a sequence which comprises not only NLS, but also “Streptococcus constellatus Cas9” and “Linker.” It is not clear whether the claim should be interpreted as requiring I) a codon-optimized Cas9 protein which comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, such that the codon-optimized Cas9 protein comprises (i) an NLS sequence set forth in SEQ ID NO: 2, or (ii) an NLS sequence at least 80% identical to SEQ ID NO: 2, or whether the claim should be interpreted as requiring (II) a codon-optimized Cas9 protein further comprising an NLS comprising an amino acid sequence at least 80% identical to an NLS set forth in SEQ ID NO: 2.
In the interest of compact prosecution, claim 100 will be interpreted as in (II) above.
Claim 101 recites that “the codon-optimized Cas9 protein is operably linked to a promoter sequence.” The skilled artisan would interpret “a promoter sequence” as a DNA sequence which is bound by transcriptional machinery and facilitates transcription. The specification does not specifically define the term “promoter sequence.” The specification describes promoters which are a type of “transcriptional control element,” (pg. 96-98), as well as promoters which “include AAV ITR” (pg. 123). The skilled artisan would not have interpreted an “AAV ITR” as within the typical scope of “promoter sequences.” Further, although such linkage is possible, the skilled artisan also would not typically operably link an amino acid sequence (i.e., the codon-optimized Cas9), to a DNA sequence which is bound by transcriptional machinery and facilitates transcription (i.e., a promoter sequence as it would be understood by the skilled artisan). Together, the literal interpretation of the claim, when coupled with the exemplary promoters, raises doubt as to the structures encompassed by claim 101. It is not clear whether the term “promoter sequence” refers to an undisclosed amino acid sequence which “promotes” something, or whether the claim literally requires operable linkage between an amino acid sequence and a DNA sequence, or even, what structures are encompassed by the term “promoter sequence.”
In the interest of compact prosecution, claim 101 will be interpreted hereinafter as depending from claim 104, and such that the nucleic acid sequence encoding the codon-optimized Cas9 protein is operably linked to a promoter sequence, which is interpreted as encompassing any nucleic acid sequence which promotes transcription, either directly or indirectly.
Claim 102 recites that “the [codon-optimized] Cas9 protein is expressed in a eukaryotic cell.” It is not clear whether the limitations of claim 102 should be interpreted functionally or structurally. For example, it is not clear whether the claim intends to require that the codon-optimized Cas9 protein is capable of being expressed in a eukaryotic cell, such that, for example, the Cas9 protein is codon-optimized for expression in a eukaryotic cell (pg. 127), or whether the codon-optimized Cas9 in the system must literally be expressed in a cell, such that the system comprises an RNA guide, and a eukaryotic cell comprising the codon-optimized Cas9 protein.
In the interest of compact prosecution, claim 102 will be interpreted as requiring the codon-optimized Cas9 literally expressed in a eukaryotic cell, such that the system comprises an RNA guide, and a eukaryotic cell comprising the codon-optimized Cas9 protein.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 93-98 and 104-105 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 93 recites that “the base editor comprises an amino acid sequence of SEQ ID NO: 20.” The phrase “an amino acid sequence of SEQ ID NO: 20” refers to any two or more consecutive amino acids of SEQ ID NO: 20. Claim 92 requires that the base editor “comprise[] an amino acid sequence having at least 95% identity to SEQ ID NO: 20,” which would be interpreted as a sequence comprising 95% or more identity across the full length of SEQ ID NO: 20. Such a sequence would require a minimum of 1516 identical amino acids to SEQ ID NO: 20. Thus, claim 93 does not further limit the subject matter of the claim upon which it depends, and does not include all the limitations of the claim upon which it depends, because it only requires identity to only two or more consecutive amino acids of SEQ ID NO: 20.
Claims 94-98 are rejected for depending from claim 93 and failing to resolve the improper dependency above. Claim 93 will be interpreted hereinafter as requiring the limitations of the claim upon which it depends, i.e., as having equivalent scope to claim 92.
Claim 97 recites that “the RNA guide comprises a sequence of SEQ ID NO: 3.” The phrase “a sequence of SEQ ID NO: 3” refers to any two or more consecutive nucleotides of SEQ ID NO: 3. Claim 96 requires that the RNA guide “comprise a sequence having at least 90% identity to SEQ ID NO: 3,” which would be interpreted as a sequence comprising 90% or more identity across the full length of SEQ ID NO: 3. Such a sequence would require a minimum of 83 identical nucleotides to SEQ ID NO: 3. Thus, claim 97 does not further limit the subject matter of the claim upon which it depends, and does not include all the limitations of the claim upon which it depends, because it requires identity to only two or more consecutive nucleotides of SEQ ID NO: 3.
Claim 98 is rejected for depending from claim 97 and failing to resolve the improper dependency above. Claim 97 will be interpreted hereinafter as requiring the limitations of the claim upon which it depends, i.e., as having equivalent scope to claim 96.
Claim 104 recites a “nucleic acid encoding the system of claim 58,” and claim 105 recites a “vector comprising the system of claim 58, wherein the vector is a plasmid or viral vector… a lentiviral vector.” Claims 104-105 are drawn to nucleotide sequences encoding the system of claim 58, including DNA sequences. The system of claim 58 requires “a codon-optimized CRISPR-associated protein” composed of amino acids, and “an RNA guide” composed of RNA nucleotides. Claims 104-105 encompass nucleic acids and vectors which substitute the amino acids and RNA nucleotides of claim 58, for nucleotide sequences, including DNA sequences. The claims do not include all the limitations of the claim upon which they depend.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 58, and 87-105 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.
MPEP 2163.II.A3.(a).(i) states the following:
“The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the inventor was in possession of the claimed genus.”
“Satisfactory disclosure of a "representative number" depends on whether one of skill in the art would recognize that the inventor was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are "representative of the full variety or scope of the genus," or by the establishment of "a reasonable structure-function correlation." Such correlations may be established "by the inventor as described in the specification," or they may be "known in the art at the time of the filing date.” See AbbVie, 759 F.3d at 1300-01, 111 USPQ2d 1780, 1790-91 (Fed. Cir. 2014).”
Species Encompassed – Claims 58, 87-95, 99-100, and 102-103
The claims recite systems comprising “a codon-optimized CRISPR-associated protein modified from Streptococcus constellatus Cas9 protein having at least 80% sequence identity to SEQ ID NO: 1.” The phrase “codon-optimized” is interpreted as a “type of optimization known in the art [which] entails the mutation of foreign-derived DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein” (pg. 66). The specification states “the codons are changed, but the encoded protein remains unchanged” (pg. 66). The codon-optimized Cas protein must also be “modified” from a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1. Thus, the recited Cas protein is a product-by-process. The structure conferred by the process (i.e., expression from a codon-optimized transcript, and modification relative to the Streptococcus constellatus Cas9) is a Cas protein comprising at least one modification (e.g., substitution, deletion, insertion, etc.) relative to a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1, because, as evidenced by the specification, codon-optimization does not impact the encoded protein.
The claims encompass systems comprising Cas9 proteins comprising mutations, e.g., D10A, D1117M, S118Q, etc. (pg. 67), and additional sequences (e.g., NLS, linkers, tags, etc.), as well as “base editor” fusion proteins comprising a D10A mutated Cas9 fused to an adenosine deaminase. For example, a base editor comprising a D10A mutated Cas9 fused to an adenosine deaminase, wherein the base editor comprises at least 90% or 95% identity to SEQ ID NO: 2, sequences which further comprise various nuclear localization sequences (NLSs) and linkers as evidenced by the specification (Table 1, pg. 49). The claims encompass Cas9 proteins and Cas9 fusion proteins with various functional properties, e.g., capability of binding to the RNA guide and modifying a target nucleic acid sequence complementary thereto, functioning as a nickase (pg. 31), a base editor (pg. 34), and recognizing various PAM sequences, e.g., PAM sequences comprising 5'-NGG- 3' as in claim 99, or comprising 5’-NGC-3’ (pg. 67).
Together, the claims encompass systems comprising a genus of Cas9 proteins modified from a Streptococcus constellatus Cas9 protein comprising 80% identity to SEQ ID NO: 1, and fusion proteins comprising the Cas9 proteins, and having the functional limitations described in the claims. The specification has not sufficiently described the genus of Cas9 proteins or fusion proteins comprising the Cas9 proteins, which function as a base editor, or which recognize various PAM sequences for the reasons that follow.
Species Disclosed in the Specification
The specification describes a Streptococcus constellatus Cas9 protein consisting of SEQ ID NO: 1. The specification describes various mutations in the RuvC and/or HNH domains which may be made with respect to the sequence of SpCas9, and confer nickase or catalytically inactive Cas9 proteins (pg. 58). The specification also describes various mutations which may be made with respect to the sequence of SpCas9 that result in enhanced specificity or reduced off-target activity (pg. 59). The specification also describes working examples of such proteins, including a modified Streptococcus constellatus Cas9 protein comprising D10A (Table 15, SEQ ID NO: 20), and two modified Streptococcus constellatus Cas9 proteins comprising mutations which confer alternative PAM recognition (i.e., 5’-NGC-3’, SEQ ID NOs: 98-99, pg. 161-162).
Based on the specification, the functionality of modified Streptococcus constellatus Cas9 proteins comprising comprising various known mutations in, for example, the RuvC or HNH domains relative to known Cas9 proteins, e.g., D10A relative to a known SpCas9 sequence, would be predictable. The functionality of a Streptococcus constellatus Cas9 protein prepared with the mutations set forth in SEQ ID NOs: 98-98 on pg. 161-162 relative to the Streptococcus constellatus Cas9 protein sequence 100% identical to SEQ ID NO: 1 would also be predictable based on the specification. The specification does not, however, appear to describe any other modified Streptococcus constellatus Cas9 proteins comprising mutation(s) which confer alternative PAM recognition. The specification also does not appear to provide guidance to determine which other mutation(s) in a Streptococcus constellatus Cas9 protein comprising 80% or more identity to SEQ ID NO: 1 would promote alternative PAM sequence recognition.
The specification also describes various adenosine deaminase domains, e.g., adenosine deaminase domains comprising various mutations relative to ecTadA (pg. 88-93). The specification also provides three specific working examples of base editor proteins: (I) a base editor comprising a D10A Cas9 modified from SEQ ID NO: 1, fused to a specific adenosine deaminase (“TadA8.13m”)(Table 15, SEQ ID NO: 20), and (II)-(III) two base editors comprising a Cas9 modified from SEQ ID NO: 1 to comprise mutations resulting in alternative PAM recognition, fused to a specific adenosine deaminase which comprises 98.7% identity to “TadA8.13m” based on alignment between the adenosine deaminase sequences set forth in SEQ ID NOs: 98-99 on pg. 161-162, and SEQ ID NO: 20.
Taken together, the specification discloses three specific base editor fusion proteins comprising a Cas9 proteins modified from a Streptococcus constellatus Cas9 protein sequence 100% identical to SEQ ID NO: 1, i.e., SEQ ID NO: 20, and SEQ ID NOs: 98-99 disclosed on pgs. 161-162. The specification does not provide any other working examples of base editor fusion proteins, and does not provide any guidance as to which other adenosine deaminase proteins or variants thereof encompassed by the claims, may be fused to the many modified Cas9 proteins encompassed by the claims and retain “base editor” function.
Guidance in the Prior Art
The prior art describes a Streptococcus constellatus Cas9 protein comprising 100% identity to SEQ ID NO: 1. See alignment in Appendix II with SEQ ID NO: 98 of Liu (Liu and Hu, WO 2019/168953 A1, published 06 September 2019). The prior art also describes various mutations in a Streptococcus constellatus Cas9 protein comprising 100% identity to SEQ ID NO: 1, including D10A, as well as guidance to predict the consequences of the various mutations in the RuvC and HNH domains therein based on domain information obtained from sequence alignment with other well-characterized Streptococcus Cas9 proteins (see Liu, Example 1, [00347]-[00356]).
The prior art also describes base editor fusion proteins comprising a D10A Cas9 protein modified from a Streptococcus constellatus Cas9 protein comprising 100% identity to SEQ ID NO: 1 (see Liu, [0060]; [0072]; [0074]). The prior art provides working examples of base editor fusion proteins comprising a D10A Cas9 protein linked to an ecTadA*7.10 adenosine deaminase by an XTEN linker, and comprising an NLS (see Liu, [00230]-[00231]). Based on the prior art, it would be predictable to prepare a base editor fusion protein comprising a D10A Cas9 protein modified from the Streptococcus constellatus Cas9 protein disclosed by Liu in SEQ ID NO: 98 (which comprises 100% identity to SEQ ID NO: 1), linked by an XTEN linker to the ecTadA*7.10 adenosine deaminase used in the working examples of Liu.
The prior art does not appear to provide any guidance to determine the mutation(s) in Streptococcus constellatus Cas9 protein comprising 100% identity to SEQ ID NO: 1 required to promote non-natural PAM sequence recognition. The prior art also provides evidence that the function of a base editor fusion protein comprising a modified Cas protein fused to an adenosine deaminase may be unpredictable for some Cas proteins; while many Cas homologs, including several Cas9 homologs are compatible with adenosine deaminase domains, not every Cas protein is capable of functioning with an adenosine deaminase, or may have substantially low activity (see Richter et al., 16 March 2020, Nature Biotechnology, 38, pg. 883-891, pg. 883). While more generally compatible adenosine deaminases have been described in the prior art, e.g., ABE8 variants described by Richter et al., the prior art still does not provide guidance to predict which of the many Cas9 proteins modified from Streptococcus constellatus Cas9 protein would be functional in a base editor fusion protein. The prior art, while describing various mutations in adenosine deaminases which affect their function (see, for example, Richter et al., with respect to TadA), also does not describe which of the many adenosine deaminase variants, including adenosine deaminases from other species, would be functional in the Cas9 base editor fusion protein.
Conclusion
Considering the large variation in the genus, the small percentage of species described in the specification, and the lack of predictability provided by the art for the full scope of the genus of systems, and more specifically, the lack of predictability for (I) the genus of Cas9 proteins which comprise alternative PAM sequence recognition, and (II) the genus of base editor fusion proteins comprising any member of the genus of Cas9 proteins modified from a Streptococcus constellatus Cas9 protein comprising 80% identity to SEQ ID NO: 1, fused to any member of the genus of adenosine deaminase proteins encompassed by the claims, it is reasonable to conclude that Applicant did not possess the invention as claimed at the time of filing.
Dependent Claims
Claims 96-98 are rejected for depending from claim 94 and failing to remedy the indefiniteness. Under the interpretation applied herein in view of the improper dependency above, the claims are directed to systems comprising the base editor of claim 92, wherein the base editor comprises at least 95% identity to SEQ ID NO: 20. SEQ ID NO: 3 is a RNA guide scaffold sequence (pg. 29). Means to prepare sequences with 90% or more sequence identity to SEQ ID NO: 3 would be well known to the skilled artisan. However, the specification only describes an RNA guide which functions with ScoCas9 comprising 100% identity to SEQ ID NO: 3 (pg. 44). The specification does not provide any guidance to determine which nucleotides of SEQ ID NO: 3 may be modified and still retain the scaffold sequence function. The prior art does not teach any sequences with 90% or more identity to SEQ ID NO: 3, or provide any guidance to determine which nucleotides of SEQ ID NO: 3 may be modified and still retain the scaffold sequence function. The claims also do not sufficiently limit the modified Cas9 protein element or base editor comprising the Cas9 protein in the system so as to resolve the written description issues described above.
Claims 101, and 104-105 are rejected for depending from claim 58 and failing to remedy the written description issues described above. The claims are directed to nucleic acids and vectors, i.e., plasmid vectors and lentiviral vectors, comprising the system. Means to prepare codon-optimized sequences encoding Cas9 proteins are well known, and means to prepare nucleic acids, plasmid vectors, and lentiviral vectors comprising such sequences and RNA guides are also well known. However, the claims do not sufficiently limit the modified Cas9 protein element or base editor comprising the Cas9 protein in the system so as to resolve the written description issues described above.
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 58, 87, 99, 101, and 104 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a law of nature or natural phenomena without significantly more. The claims recite an “engineered, non-naturally occurring CRISPR-Cas system comprising: an RNA guide comprising a crRNA and a tracrRNA; and a codon-optimized CRISPR-associated protein modified from Streptococcus constellatus Cas9 protein having at least 80% sequence identity to SEQ ID NO: 1.” The phrase “codon-optimized” is interpreted as a “type of optimization known in the art [which] entails the mutation of foreign-derived DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein” (pg. 66). The specification states “the codons are changed, but the encoded protein remains unchanged” (pg. 66). The codon-optimized Cas protein must also be “modified” from a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1.
The recited Cas protein is a product-by-process. The structure conferred by the process (i.e., expression from a codon-optimized transcript, and modification relative to the Streptococcus constellatus Cas9) is a Cas protein comprising at least one modification (e.g., substitution, deletion, insertion, etc.) relative to a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1, because, as evidenced by the specification codon-optimization does not impact the encoded protein. Accordingly, the claims are directed to an engineered, non-naturally occurring system comprising an I) RNA guide, which is interpreted as an RNA sequence comprising (i) a crRNA, i.e., an RNA sequence comprising at least a sequence complementary to a target sequence (a “spacer”) (pg. 38, 69-70), and (ii) a tracrRNA, i.e., an RNA sequence capable of forming a complex with the Cas9 (pg. 43, 71), and II) a Cas protein comprising at least one modification (e.g., substitution, deletion, insertion, etc.) relative to a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1.
Laws of nature and natural phenomena, as identified by the courts, include naturally occurring principles/relations and nature-based products that are naturally occurring or that do not have markedly different characteristics compared to what occurs in nature. MPEP 2106.04(b)(II) also provides that “product of nature exceptions include both naturally occurring products and non-naturally occurring products that lack markedly different characteristics from any naturally occurring counterpart.” In other words, the phrase “engineered, non-naturally occurring” does not render the product eligible, if the product is structurally identical to a naturally-occurring counterpart.
SEQ ID NO: 1 is 100% identical to a known Streptococcus constellatus Cas9 protein sequence, i.e., WP_006269658.1. Several naturally-occurring Cas9 proteins comprise modifications relative to the known Streptococcus constellatus Cas9 protein sequence 100% identical to SEQ ID NO: 1. For example, Streptococcus anginosus Cas9 protein corresponding to WP_003030002.1, and Streptococcus viridians Cas9 protein corresponding to WP_115233568.1. See ABSS search results file “us-18-283-148-1.rup” in Appendix I. As evidenced by the UniProtKB and UniParc entries associated with the Cas9 proteins corresponding to WP_003030002.1 and WP_115233568.1, the Cas9 proteins are part of a naturally-occurring system comprising a crRNA and a tracrRNA (see “Function” section of each entry). The system of claims 58 and 103 are not markedly different from the naturally occurring systems, and therefore, recite product of nature judicial exceptions.
The UniProtKB and UniParc entries also teach that the aforementioned Cas9 proteins meet the functional limitations of claims 87 (see “Function” section of each entry). The system of claim 87 is not markedly different from the naturally occurring systems, and therefore, recites a product of nature judicial exception.
Haider (Haider et al., 2025, Virulence, Vol. 16, No. 1, pg. 1-15) teaches that S. anginosus Cas9 protein is capable of recognizing a PAM sequence comprising 5’-NGG-3’ (“This study demonstrated, that the type II-A system of S. anginosus is a functional adaptive immune system through a plasmid interference assay… Cas9 could efficiently cleave the target DNA in the presence of a TGG sequence thereby preventing or highly reducing plasmid transformation… this confirmed that PAMs of streptococcal type II-A systems work with different types of Cas9 nucleases,” pg. ). The system of claim 99 is not markedly different from a naturally occurring system, and therefore, recites a product of nature judicial exception.
The systems are expressed in nature in bacterial cells, which must necessarily comprise nucleic acid encoding the systems as recited in claim 104. Because the Cas9 proteins are expressed in nature in bacterial cells, the naturally-occurring nucleic acid encoding the Cas9 must necessarily be operably linked to a promoter sequence. The systems of claims 101 (which is interpreted herein as depending from claim 104 in view of the indefiniteness described above) and 104 are not markedly different from the naturally occurring systems, and therefore, recite product of nature judicial exceptions.
The claims do not recite any additional elements, each element of the systems is present in the naturally-occurring counterparts. Therefore, the judicial exceptions are not integrated into a practical application and do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
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.
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claim 102 is rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101).
Claim 102 is interpreted herein as a system comprising an RNA guide, and a eukaryotic cell comprising the codon-optimized Cas9 protein. Neither the specification nor claim expressly exclude cells within a human organism comprising the system elements. The specification describes human cells and cells in a subject comprising the system elements (at least pg. 32-33). Thus, the term “eukaryotic cell” could reasonably be interpreted as encompassing cells within a human organism, which is non-statutory subject matter. The rejection may be obviated by requiring an in vitro eukaryotic cell, for example, as described in Example 4 of the specification.
Claim Rejections - 35 USC § 102 – Liu
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.
Claims 58, 87-90, 99-100, 102, and 103 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Liu (Liu and Hu, WO 2019/168953 A1, published 06 September 2019).
As described above, the term “codon-optimized” while describing the structure from which the protein is produced, does not impact the encoded protein based on the specification (pg. 66). Accordingly, claim 58 is interpreted as an engineered, non-naturally occurring system comprising an I) RNA guide, which is interpreted as an RNA sequence comprising (i) a crRNA, i.e., an RNA sequence comprising at least a sequence complementary to a target sequence (a “spacer”) (pg. 38, 69-70), and (ii) a tracrRNA, i.e., an RNA sequence capable of forming a complex with the Cas9 (pg. 43, 71), and II) a Cas protein comprising at least one modification (e.g., substitution, deletion, insertion, etc.) relative to a Streptococcus constellatus Cas9 protein comprising at least 80% identity to SEQ ID NO: 1.
Regarding claims 58, 87, and 103, Liu teaches a Cas9 protein modified from a Streptococcus constellatus Cas9 protein comprising 100% identity to instant SEQ ID NO: 1 (“This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NO: 9 are mutated as described herein… mutations in Cas9 sequences other than SEQ ID NO: 9 that correspond to mutations identified in SEQ ID NO: 9 herein… for example, the mutations corresponding to the D10A mutation… A total of 250 Cas9 sequences (SEQ ID NOs: 10-262) were aligned using the same algorithm and alignment parameters outlined above…,” Example 1, [00347]-[00356]; SEQ ID NO: 98, [00355], pg. 149). See attached alignment in Appendix II. Liu also teaches an RNA guide comprising a crRNA and a tracrRNA, to which the Cas9 protein is capable of binding and modifying a target sequence complementary to the RNA guide ([0064]). Thus, the elements disclosed by Liu are considered a “system,” i.e., a set of elements which work together, wherein the system is considered “engineered, non-naturally occurring,” by virtue of modification relative to the Streptococcus constellatus Cas9 protein sequence disclosed by Liu.
Regarding claims 88-89, Liu teaches the Cas9 protein comprises a D10A mutation in the RuvC domain. Liu teaches that the resulting Cas9 protein is a nickase that cleaves one strand of a target nucleic acid (“The term “Cas9 nickase” or “Cas9n,” as used herein, refers to a Cas9 domain that is capable of cleaving one strand of the duplexed nucleic acid molecule… a Cas9 nickase comprises a D10A mutation… of SEQ ID NO: 9, or a corresponding mutation in any of SEQ ID NOs: 10-262… such a Cas9 nickase has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing is desired,” [0074]; This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NO: 9 are mutated as described herein… mutations in Cas9 sequences other than SEQ ID NO: 9 that correspond to mutations identified in SEQ ID NO: 9 herein… for example, the mutations corresponding to the D10A mutation… A total of 250 Cas9 sequences (SEQ ID NOs: 10-262) were aligned using the same algorithm and alignment parameters outlined above…,” Example 1, [00347]-[00356]).
Regarding claim 90, Liu teaches a base editor comprising the Cas9 protein comprising the D10A mutation in the RuvC domain, fused to an adenosine deaminase (“the base editor comprises a Cas9 nickase (Cas9n) fused to an adenosine deaminase domain,” [0060]; “In some embodiments, the Cas9 domain comprises a D10A mutation… as provided in SEQ ID NO: 9, or at corresponding positions in any of the amino acid sequences provided in SEQ ID NOs: 10-262,” [0072]; “The term “Cas9 nickase” or “Cas9n,” as used herein, refers to a Cas9 domain that is capable of cleaving one strand of the duplexed nucleic acid molecule… a Cas9 nickase comprises a D10A mutation… of SEQ ID NO: 9, or a corresponding mutation in any of SEQ ID NOs: 10-262, [0074]).
Regarding claim 99, Liu’s Cas9 protein comprises a substantially identical structure to the Cas9 protein in the specification which comprises the function recited in the claim (“The consensus PAM sequence recognized by Streptococcus constellatus Cas9 was identified as 5’-NGG-3’,” pg. 135). The functional limitations of claim 99, therefore, are understood to be inherent properties of Liu’s Cas9 protein of substantially identical structure to the claimed Cas9 protein, and the Cas9 protein disclosed in the specification as having the function. See MPEP 2112.01.
Regarding claim 100, Liu teaches the Cas9 protein further comprises an NLS comprising an amino acid sequence at least 80% identical to an NLS set forth in SEQ ID NO: 2 ([0089]; “The fusion proteins of the present disclosure may comprise one or more additional features… the fusion protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the fusion protein is localized between the nucleic acid editing domain and the Cas9 domain… the NLS of the fusion protein is localized C-terminal to the Cas9 domain… the NLS of the fusion protein is localized N-terminal to the Cas9 domain… the NLS comprises the amino acid sequence of SEQ ID NO: 520,” [00186]; [00220]). See alignment below in Fig. A between Liu, SEQ ID NO: 520, and an NLS of SEQ ID NO: 2 as evidenced by the specification (bolded, Table 1, pg. 49).
FIG. A
MPKKKRKV NLS of instant SEQ ID NO: 2
PKKKRKV Liu, SEQ ID NO: 520
Regarding claim 102, Liu teaches eukaryotic cells comprising the Cas9 protein ([00317]).
Notice to Joint Inventors
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim Rejections - 35 USC § 103 – Liu
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 91-95 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu and Hu, WO 2019/168953 A1, published 06 September 2019) as applied to claims 58, 87-90, 99-100, 102, and 103 above.
The teachings of Liu are described above and applied as to claims 87-90, 99-100, 102, and 103 therein.
Regarding claims 91-93, as described above, Liu teaches a base editor comprising a Cas9 protein modified from a Streptococcus constellatus Cas9 protein comprising 100% identity to instant SEQ ID NO: 1 (i.e., SEQ ID NO: 98), wherein the Cas9 protein comprises a D10A mutation. Liu also teaches the architecture for a base editor – “NH2-[NLS]-[adenosine deaminase]-[Cas9]-COOH” ([0221]). Liu teaches the NLS may comprise any disclosed NLS, e.g., PKKKRKV (SEQ ID NO: 520) ([0221]). Liu also teaches the sequences of adenosine deaminases ([00219]), and teaches working examples of base editor fusion proteins comprising the adenosine deaminase ecTadA*7.10 ([00230]-[00231]). Liu teaches that each “-“ in the aforementioned architecture may correspond to a linker sequence, e.g., an XTEN linker corresponding to “SGGSSGGSSGSETPGTSESATPESSGGSSGGS,” which is disposed between the adenosine deaminase and D10A Cas9 protein in the working examples of base editor fusion proteins Liu ([00219]; [00230]-[00231]).
Liu does not teach a base editor comprising at least 90%, or at least 95% identity to SEQ ID NO: 20.
However, using the aforementioned architecture of Liu, one of the two NLS sequences disclosed by Liu (i.e., SEQ ID NO: 520), the exemplary adenosine deaminase ecTadA*7.10 used in the working examples of Liu, a known XTEN linker which is disposed between the adenosine deaminase and D10A Cas9 in the working examples of Liu, and the modified Cas9 protein comprising a D10A mutation relative to Liu SEQ ID NO: 98, which Liu states is compatible in the base editor fusion proteins ([0060]; [0072]; [00074]), the skilled artisan would arrive at the following sequence in Fig. B, which as shown in the alignment in Appendix III is 95.1% identical to instant SEQ ID NO: 20.
FIGURE B - “NH2-[NLS]-[adenosine deaminase]-[Cas9]-COOH,” wherein “-“ between [adenosine deaminase] and [Cas9] is a linker
MPKKKRKVSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGSMGKPYSIGLAIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSFLVDEDKRGEHHPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKFRGHFLYEGDLKAENTDVQALFKDFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNTLFGNLIALSLDLHPNFKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYDAILLSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYASYIESGVKQDEFYKYLKGILLKINGSGDFLDKIDREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNFDDILDKEKSAEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFDTNMKQEIFDHVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFLDDKANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLSYKLINGIRNKENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSPAIKKGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNPVNIKNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVLTRSDKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKFDNLTKAERGGLTEEDKAGFIKRQLVETRQITKHVAQILDERFNTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKEDIQTNEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTKNSYWDPKKYGGFDSPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQRGYRNVRLEKIIKLPKYSLFELENKRRRLLASAKELQKGNELVIPQRFTTLLYHSYRIEKDYEPEHREYVEKHKDEFKELLEYISVFSRKYVLADNNLTKIEMLFSKNKDAEVSSLAKSFISLLTFTAFGAPAAFNFFGENIDRKRYTSVTECLNATLIHQSITGLYETRIDLSKLGED
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have prepared a base editor comprising at least 90% or 95% identity to instant SEQ ID NO: 20 in view of Liu. It would have amounted to preparing a base editor using a known base editor architecture, and known elements compatible with the base editor architecture, by known means to yield predictable results. Liu provides multiple examples of functional base editors comprising a Cas9 protein sequence comprising a D10A mutation, fused to an ecTadA*7.10 sequence, wherein the Cas9 sequence and ecTadA*7.10 sequence are linked by an XTEN linker having the sequence underlined above in Fig. B. The base editor fusion proteins also comprise an NLS corresponding to SEQ ID NO: 520. Based on Liu, it was well within the purview of the skilled artisan to prepare fusion proteins comprising the aforementioned base editor architecture of Liu, and the skilled artisan would have had a reasonable expectation that the base editor protein of Fig. B would be functional, owing to the shared architecture and sequences with the base editor working examples of Liu (i.e., ecTadA*7.10, XTEN linker, and Cas9 comprising D10A). The skilled artisan would have been motivated to prepare base editors with one of the architectures of Liu, using a successful adenosine deaminase domain and XTEN linker disclosed by Liu, and a D10A Cas9 modified from the Streptococcus constellatus Cas9 sequence disclosed by Liu, in view of Liu’s working examples, and Liu’s disclosure that a D10A Cas9 modified from the Streptococcus constellatus Cas9 sequence of SEQ ID NO: 98 is compatible as a base editor.
Regarding claim 94, Liu teaches the base editor is capable of binding to the RNA guide and editing a target nucleic acid sequence complementary to the RNA guide ([00428]).
Regarding claim 95, Liu teaches the base editor effects an A-T to G-C alteration in a target nucleic acid ([00428]).
Claim Rejections - 35 USC § 103 – Liu in view of Kabadi
Claims 101, and 104-105 are rejected under 35 U.S.C. 103 as being unpatentable over Liu (Liu and Hu, WO 2019/168953 A1, published 06 September 2019) as applied to claims 58, 87-90, 99-100, 102, and 103 above, in view of Kabadi (Kabadi et al., 2014, Nucleic Acids Research, Vol. 42, No. 19, e147, pg. 1-11).
The teachings of Liu are described above and applied as to claims 87-90, 99-100, 102, and 103 therein.
Based on the specification (pg. 66), the codon-optimized Cas9 protein of claim 58 is a product-by-process, which is produced from a codon-optimized transcript. Thus, the nucleic acid and vectors encoding the system must, necessarily, comprise codon-optimized sequences encoding the Cas9 protein.
Liu teaches nucleic acids encoding the system, e.g., plasmid vectors ([00434]-[00436]; Table 6). Liu teaches nucleic acids encoding Cas9 operably linked to a promoter sequence (Table 6). Liu teaches that the system is expressed in a eukaryotic cell, e.g., a human cell ([00426]-[00428]).
Liu does not teach that the nucleic acid, or plasmid and lentiviral vectors encoding the system comprise a codon-optimized sequence encoding the Cas9 protein.
However, Kabadi also teaches a nucleic acid encoding a system similar to that of Liu, wherein the nucleic acid comprises a codon-optimized sequence encoding a Cas9 protein (“The gene encoding human codon optimized Cas9 (hCas9) nuclease was obtained from Addgene,” “Plasmid constructs,” pg. 2). Kabadi teaches plasmid and lentiviral vectors comprising the codon-optimized sequence encoding the Cas9 protein (“Plasmid constructs,” pg. 2; “Protocol for assembly of custom lentiviral vectors,” pg. 2-3; Fig. 2). Kabadi demonstrates that the sequences are expressed in human cells (at least pg. 6, right col. to pg. 7, left col.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have prepared a nucleic acid, or plasmid or lentiviral vector comprising a codon-optimized sequence encoding the Cas9 protein. It would have amounted to preparing a known nucleic acid or vector encoding a known Cas9 protein sequence, using a codon-optimization technique known to one of ordinary skill, by known means to yield predictable results. As evidenced by Kabadi, it was well within purview of one skilled in the art to prepare a codon-optimized sequence encoding a known Cas9 protein sequence, and a nucleic acid, plasmid or lentiviral vector comprising the codon-optimized sequence and an RNA guide. Kabadi teaches the codon-optimized sequence is expressed in human cells. The skilled artisan would have been motivated to apply the known technique to sequences encoding the Cas9 protein in the nucleic acid and vectors of the prior art, such that the Cas9 protein may be delivered to and expressed in human cells as taught in Liu and Kabadi.
Conclusion
No claims are allowed.
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/JENNA L PERSONS/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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