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 .
Election/Restrictions
During a telephone conversation with Yvonne Smith on 08/24/2026 a provisional election was made without traverse to prosecute the invention of the species of a hairpin region of stem-loop 2 nucleic acid sequence that is at least 74% identical of Seq ID NO: 48 and a variant sgRNA nucleic acid sequence that is at least 75% identical of SEQ ID NO:352, encompassing claims 8, 10, and 15. Affirmation of this election must be made by applicant in replying to this Office action. Claims 7, 9, and 11 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected species.
Upon further search and consideration, a hairpin region of stem-loop 2 nucleic acid sequence that is at least 74% identical to Seq ID NO: 48 and a variant sgRNA nucleic acid sequence that is at least 75% identical of SEQ ID NO:352 are free of the art. The search and consideration has been extended to a hairpin region of stem-loop 2 that comprises the nucleic acid sequence of SEQ ID NO: 25 and a variant sgRNA nucleic acid sequence that is at least 75% identical of SEQ ID NO:353 of claim 7 and 11.
Priority
The application claims priority to application 63/484,902 filed 2/14/2023.
Information Disclosure Statement
The information disclosure statement filed 04/16/2024 had been considered.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
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 drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings (see Fig. 1B).
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, 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.
Specification
The use of the term Addgene, Illumina, Thermo Fisher Scientific, Epicentre, Qiagen, Becton Dickinson, Promega, Sigma, Pall, and Life Technologies to name a few, which is a trade name or a mark used in commerce, has been noted in this application. The term 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. A cursory review of the specification has revealed these trademarks or names. It would be remedial to check the specification for additional trademarks or names and amend all upon amendment.
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.
Claim Objections
Claims 1 and 20 is objected to because of the following informalities: Claim 1 recites “impacts increased” This wording is awkward. “Wherein the strengthened interaction impacts increased on-target editing” is grammatically poor. It would be remedial to reword the claim to something such as: “wherein the strengthened interaction results in increased on-target editing and/or increased on-off-target specificity…”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-20 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 1 and 20 recites the limitation "the interaction". There is insufficient antecedent basis for this limitation in the claim.
Claims 2, 16 and 20 recite the limitation "the hairpin". There is insufficient antecedent basis for this limitation in the claim.
Claims 2, 16 and 20 recite the limitation "the stem-loop". There is insufficient antecedent basis for this limitation in the claim.
Claim 5 recites the limitation "the PI domain" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Those claims identified in the statement of rejection but not explicitly referenced in the rejection are also rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1–12 and 14–20 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.
For claims drawn to a genus, MPEP § 2163 states 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 applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 is directed to a variant sgRNA comprising a substitution and/or addition of one or more nucleic-acid residues that strengthens interaction of the sgRNA with “a Cas enzyme,” wherein the strengthened interaction produces increased on-target editing and/or increased on/off-target specificity relative to a wild-type sgRNA lacking the substitution or addition.
Claim 1 does not require that the Cas enzyme be Cas9 or SpCas9; the substitution or addition occur in stem-loop 2; the modification correspond to any of SEQ ID NOS:1–312; the modification interact with SpCas9 His721 or the PI domain; the sgRNA have the scaffold of SEQ ID NO:345; the target-specific guide or spacer have any particular sequence; the sgRNA target any particular gene or class of genes; or the claimed improvement occur at any specified target locus or genomic context. The genus is therefore defined substantially by the desired functional result rather than by structural characteristics demonstrated to reliably produce that result.
The disclosure acknowledges a broad CRISPR/Cas universe. The specification identifies numerous CRISPR-Cas effectors as examples of “Cas” proteins and thereafter describes Cas9 merely as one embodiment. It additionally contemplates Cas9 orthologs, mutants, truncated proteins, and chimeric Cas9 proteins [pg. 25-28]. The specification states that the inventors sought to increase activity by engineering the interaction between an enzyme and its sgRNA scaffold and then focuses on variant sgRNAs having substitutions or additions, particularly in stem-loop 2 [pg. 2-3]. The working examples use SpCas9 and systematically engineer stem-loop 2 [Example 1]. The library was generated by altering upper stem-loop-2 sequences, extending stem length, introducing selected base-pair mutations, and combining these modifications [pg. 34-36]. The most successful experimentally characterized variants, including SV48 and SV240, concern the particular stem-loop-2 structures and particular modeled interactions with SpCas9. The specification states that SV48 and SV240 increased SpCas9 editing and models interactions of the engineered stem-loop-2 structures with His721 and the PI domain [pg. 47; 58-59]. Thus, although the disclosure reasonably demonstrates possession of particular SpCas9-compatible stem-loop-2 engineering strategies and particular successful scaffold species, that disclosure is not representative of the full structural diversity embraced by claim 1.
The breadth of claim 1 is further increased because the claim places no meaningful limitation on the target-specific guide or spacer sequence. The specification expressly explains that an sgRNA includes a guide sequence that specifies the target site and that the guide is generally complementary to the target DNA sequence [pg. 17]. Thus, claim 1 embraces sgRNA variants containing target-specific guide sequences directed to essentially any target sequence or target gene. This is important because claim 1 requires a particular functional result: the variant must exhibit strengthened Cas interaction resulting in increased on-target editing and/or increased on/off-target specificity relative to the corresponding wild-type sgRNA. The disclosure does not establish that these functional improvements are independent of the identity of the target-specific spacer, the corresponding target sequence, or the genomic context. The working examples instead evaluate a limited number of guides and target loci. The constructs reported in Tables 1 and 3 include particular guides directed to RFP, FANCF, EMX1, PD1, DNMT1, HPRT, CXCR4 and HBG. The specification states that SV48 and SV240 increased editing at the five endogenous loci tested and discusses CXCR4 and HBG as particular examples [Example 2]; thereby demonstrating that selected scaffold modifications can function with several particular target-specific guides. They do not establish that the claimed functional effect is guide-independent or identify a structural characteristic by which a skilled artisan could recognize, throughout the substantially larger universe of scaffold-modification/guide-sequence combinations, those members possessing the required functional properties.
Accordingly, claim 1 encompasses diversity along at least the following independent dimensions: Cas protein, sgRNA scaffold modification, target-specific guide/spacer, target sequence/genomic context. The specification does not disclose representative species reflecting the full diversity produced by those variables and does not establish a reasonable structure-function relationship permitting the successful members of that genus to be identified from structure.
The state of the art further supports this conclusion. Briner (Briner et al., Molecular Cell 56:333–339 (2014)) teaches that Cas9 activity depends on guide-RNA interactions, that evolutionarily divergent Cas9 nucleases can operate orthogonally, and that the guide-RNA nexus and hairpins contribute to defining that orthogonality [abstract]. This supports the conclusion that results obtained with one Cas9/sgRNA architecture do not establish a universally applicable guide-scaffold/Cas relationship. The disclosure itself likewise recognizes that the engineered effect is associated with particular SpCas9/scaffold interactions and identifies His721 and PI-domain contacts as mechanistic features of the preferred stem-loop-2 embodiments pg. 59]. The art therefore does not supply the missing reasonable correlation by which arbitrary sgRNA modifications across arbitrary Cas proteins and arbitrary target-specific guides can be recognized as possessing the claim-1 function.
Dependent claims 2–12 and 14–20 are rejected for the same reasons because, although they further limit particular aspects of the Cas enzyme, sgRNA scaffold, stem-loop-2 sequence, sequence identity, vector, ribonucleoprotein complex, cell, method, or kit, they do not cure the lack of representative species or a disclosed structure-function correlation sufficient to demonstrate possession of the full claimed genera, including the remaining breadth in Cas/Cas9 identity, sgRNA scaffold variation, target-specific guide/spacer sequence, and target-sequence context.
Claims 1-20 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 variant sgRNA that comprises a nucleic acid sequence of SEQ ID NO 352 or 353 and a ribonucleoprotein comprising such variant sgRNA and a spCAS9 where the variant gRNA is targeted to a particular target gene, does not reasonably provide enablement for the full scope of the claimed invention as written without undue experimentation. 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.
Nature of the Invention
The claims are directed to a variant sgRNA comprising a substitution and/or addition of one or more nucleic-acid residues that strengthens interaction of the sgRNA with “a Cas enzyme,” wherein the strengthened interaction produces increased on-target editing and/or increased on/off-target specificity relative to a wild-type sgRNA lacking the substitution or addition.
Breadth of the claims
The claims embraces substantially any sgRNA having an unspecified substitution and/or addition that strengthens interaction with a Cas enzyme; and produces increased on-target editing and/or increased on/off-target specificity relative to the wild-type sgRNA. The claims are not limited as to: type or species of Cas enzyme; sgRNA scaffold; position of modification; number or identity of modified nucleotides beyond “one or more”; guide/spacer sequence; target sequence; target gene; genomic context; or cell type. Accordingly, the functional genus encompasses combinations arising from multiple sources of structural and biological variation. A particularly important aspect of this breadth is the unrestricted gene-specific guide/spacer sequence. An sgRNA includes a guide sequence specifying its target sequence, as the specification itself explains. The claims therefore encompass sgRNAs directed to essentially any target gene or sequence. The claims require the resulting sgRNA variant to exhibit the claimed enhancement in editing and/or specificity.
Predictability
The claimed functions result from interactions among the Cas protein, sgRNA scaffold, guide sequence, target DNA sequence, mismatched potential off-target sequences, and genomic environment. The specification's own experimental strategy reflects this complexity. Rather than deriving all successful scaffolds from a general predictive rule, applicant generated and screened a 312-member scaffold library [pg. 7]. The library incorporated different upper stem-loop-2 sequences, stem extensions, selected base-pair mutations, and combinations thereof [pg. 34-36]. A substantial number of entries in Table 4 are identified as “NA”, while experimentally evaluated variants differ appreciably in measured SpCas9 activity. Thus, even within the applicant's much narrower SpCas9/stem-loop-2 library, empirical screening was used to determine which variants had desirable activity.
Guidance from the Specification
The specification supplies substantial guidance concerning a narrower SpCas9/stem-loop-2 invention. The specification describes the WT scaffold; a 312-member stem-loop-2 library; particular stem extensions; selected tetraloop alterations; selected base-pair substitutions; the preferred SV48 and SV240 variants; GUIDE-seq; several endogenous target loci; and measurements of SpCas9 editing. This guidance is meaningful, but it does not provide a general rule that permits a skilled artisan to predict, throughout the claimed genus: which sgRNA modification will strengthen a selected Cas interaction; whether that modification will increase rather than decrease editing; whether any increase will persist when the spacer sequence is changed; whether specificity will improve for the new target/off-target sequence landscape; whether the effect transfers to another Cas or Cas9 protein; or whether the same effect occurs in another biological environment. The disclosure therefore provides assays and screening methods by which additional successful embodiments can be discovered, rather than a teaching enabling their predictable production throughout the scope. The working examples are limited substantially to SpCas9 and engineered stem-loop-2 scaffolds (see examples). Applicants initially perform screening in reporter systems and thereafter tests selected constructs against a limited group of endogenous targets [Example 3]. The application reports that SV48 and SV240 increased editing at five endogenous loci and specifically discusses CXCR4 and HBG [Example 3]. Thus, the specification establishes that selected scaffold modifications can provide improvements for several target-specific guides. The specification not establish that: all claimed scaffold modifications work; every SEQ ID NO:1–312 scaffold exhibits the functional improvement with arbitrary guides; the disclosed successful scaffold effects are invariant across spacer sequences; arbitrary percentage-identity variants retain the effect; the effect is transferable across Cas proteins or Cas9 orthologs; or all target genes encompassed by the method claims can be edited with the required comparative enhancement.
State of the Art
The state of the art reinforces the lack of universal predictability. Briner teaches that guide-RNA structural modules control Cas9 activity and that the nexus and hairpins contribute to orthogonality between different Cas9 systems [abstract]. Kim (Kim et al., Nature Biotechnology 38:1328–1336 (2020)), teaches that selecting the optimal SpCas9 variant for a given target sequence remained difficult and experimentally assessed 26,891 target sequences across 13 SpCas9 variants before developing predictive models [abstract]. Kim further reports materially different activity and specificity among the tested SpCas9 variants [entire paper]. Boyle (Boyle et al., Science Advances 7:eabe5496 (2021)) also investigated Cas9 binding and cleavage over diverse guide/target sequence combinations [abstract]. These references demonstrate that guide/target sequence is a material variable affecting the same Cas9 activity and specificity properties recited by the present claims.
Experimentation Required
To identify a new member across the full claim-1 genus, a skilled artisan would need to select or generate a candidate sgRNA modification and then, depending on the embodiment, determine whether the resulting RNA adopts a compatible scaffold structure; determine whether it interacts appropriately with the selected Cas enzyme; determine whether interaction is strengthened relative to WT; pair that scaffold with the desired gene-specific spacer; introduce or express the resulting components in the relevant cellular system; quantify on-target editing; compare editing to the corresponding WT scaffold; identify potential off-target sequences; determine off-target editing/specificity; repeat such testing for different guide sequences and target contexts; and where different Cas proteins are encompassed, repeat the analysis for those proteins. The successful members therefore cannot simply be selected from disclosed structural rules; they must be experimentally identified by determining whether they possess the function that defines the claim. The amount of experimentation becomes particularly substantial when the independent dimensions of the claim are considered together: scaffold modification, Cas protein, gene-specific spacer, and target/off-target sequence landscape. Kim's need to assay 26,891 target sequences merely to characterize the sequence-specific behavior of 13 SpCas9 variants illustrates the degree to which activity remained target-sequence dependent even within SpCas9 technology. The amount of experimentation is not merely limited to verification of predictable embodiments but amounts to a screening program necessary to identify which members of the claimed genus actually work as required. Accordingly, the disclosure does not enable the full scope of claims 1–20 without undue or unreasonable experimentation.
Dependent claims 2–20 are rejected for the same reasons because their additional limitations do not eliminate the need for undue experimentation to identify operative combinations across the remaining claimed variables, including Cas or Cas9 identity, sgRNA scaffold modification, sequence variants, target-specific guide/spacer sequence, target sequence and genomic context, and, where applicable, cellular or in-vivo conditions, that actually provide the recited increase in on-target editing and/or on/off-target specificity. Claim 19 is additionally not enabled because the specification provides no in-vivo working example and does not provide guidance sufficient to practice the claimed method across the unrestricted range of organisms, tissues, target cells, delivery routes, formulations, doses, and physiological environments encompassed by “in vivo” administration.
Claim Rejections - 35 USC § 101
Claim 17 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 17 is drawn to a “cell.” The specification teaches that the cell can be in vivo and can be found in a subject from any organism. Thus, the term “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 that the cell be an isolated cell, an in vitro human cell, or a non-human cell.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claims 1-4, 6, 7, 12–14, and 16–20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US 2018/0312824 A1; hereinafter “Zhang”) in view of Chen et al., Cell 155:1479–1491 (2013) (“Chen”), and Dang et al., Genome Biology 16:280 (2015) (“Dang”).
Regarding claim 1, Zhang teaches CRISPR-Cas systems comprising a guide RNA and Cas enzyme for sequence-specific modification of target nucleic acids [0038-0043]. Zhang teaches that Cas proteins include Cas9 as well as numerous other Cas proteins, and expressly identifies SpCas9 and other Cas9 orthologs [0028, 0174]. Zhang further teaches that SpCas9 enzymatic action is defined by the guide sequence and tracr sequence and produces sequence-specific cleavage at genomic target sites [0247, 0464]. Zhang further teaches modification of sgRNA architecture, including replacement and insertion of nucleotides In particular, Zhang states that where a nucleotide or stretch of nucleotides is modified by replacement, corresponding nucleotides are inserted, and that sgRNA stem loops may be engineered by introducing or removing nucleotides to obtain the desired secondary structure [0388, 0444, 0272]. Zhang further recommends guides modified according to known guide-engineering approaches. Zhang experimentally performed mutational analysis of sgRNA scaffolds and determined the effects of nucleotide changes upon the ability of the resulting sgRNAs to induce indels at the EMX target locus [0186].
Zhang does not expressly teach that a particular substitution or addition to the sgRNA strengthens the interaction between the sgRNA and Cas enzyme such that the strengthened interaction results in increased on-target editing and/or increased on/off-target specificity relative to the corresponding wild-type sgRNA.
Chen teaches modifying an sgRNA “to increase its stability and to enhance its assembly with the dCas9 protein” [pg. 1481, col.2, para 2]. Chen removed a four-U Pol III termination sequence by an A-U base-pair flip and, separately, extended the dCas9-binding hairpin specifically to improve sgRNA-dCas9 assembly [pg. 1481, col.2, para 2]. Both modified sgRNAs increased functional performance, while the combined F+E sgRNA increased observable target puncta approximately two-fold and the signal-to-background ratio approximately five-fold [pg. 1481, col.2, para 2]. Chen characterizes the resulting system as comprising a “structurally optimized sgRNA that improves its interaction with the dCas9 protein” [pg. 1481, col.1, para 2] Chen therefore directly establishes that nucleotide substitution/addition in an sgRNA could be used to improve the sgRNA-Cas9 interaction. However, Chen's Cas9 was catalytically inactive and the measured improvement concerned Cas9 binding/imaging and gene regulation rather than increased nuclease-mediated on-target genome editing.
Dang, however, investigated essentially the same type of sgRNA structural engineering in nuclease-active Cas9 genome editing. Dang extended the sgRNA duplex by 1, 3, 5, 8, or 10 base pairs and found that each extension significantly increased knockout efficiency for the tested sgRNAs, with approximately a five-base-pair extension providing the highest efficiency [pg. 2; Fig. 1]. The increased target-site modification was independently confirmed by deep sequencing [pg. 2]. Dang additionally introduced nucleotide substitutions into the sgRNA, finding that the mutations increased knockout efficiency and that T→C or T→G substitutions, particularly at position 4, provided the highest efficiencies [pg. 3, col. 2]. Dang demonstrated that the improvement was not peculiar to a single target-specific guide. A typical optimized sgRNA containing a T→G substitution and five-base-pair extension significantly increased knockout efficiency for 15 of 16 different CCR5-targeting sgRNAs, and the same optimized architecture significantly increased knockout efficiency for eight sgRNAs targeting CD4 in a different cell line [pg. 4; Fig. 4]. Dang therefore concluded that the optimized sgRNA structure generally increased knockout efficiency.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the sgRNAs taught by Zhang using the sgRNA substitutions/additions taught by Chen and Dang. Zhang itself expressly invites modification and optimization of sgRNA architecture and experimentally evaluates sgRNA mutations for genome-editing activity. Chen provides an express reason to make such structural modifications—to improve sgRNA-Cas9 assembly/interactions—while Dang establishes that substantially the same sgRNA structural optimization produces significantly increased Cas9-mediated target editing. A person of ordinary skill therefore would have had reason to modify Zhang's sgRNAs by nucleotide substitution and/or addition according to Chen and Dang to strengthen sgRNA-Cas9 association and thereby improve the functional efficiency of the CRISPR-Cas9 complex, with a reasonable expectation of obtaining increased on-target editing relative to the corresponding unmodified sgRNA. This motivation is reinforced by Dang's demonstration of increased editing across numerous distinct gene-specific sgRNAs and two different target genes.
Regarding claim 2, Zhang teaches that at least one loop of the sgRNA that is preferably modified by the insertion of distinct RNA sequence is either one or both of the tetraloop or the stem loop 2 [0447].
Regarding claims 3 and 4, Zhang expressly teaches Cas9 and particularly teaches use of Cas9 derived from S. pyogenes (SpCas9) [0010, 0028].
Regarding claims 6-7, Zhang teaches SEQ ID NO: 136, an SaCas9 sgRNA that comprises the architecture upon which the claimed SEQ ID NO:355 framework is based and a hairpin region of stem-loop 2 that comprises the sequence of ACUUGAAAAAGU which is 12 nucleotides long and corresponds to the instant application’s SEQ ID NO:25 [0196, sequence listing]. Zhang also teaches that a guide sequence can be selected to target any target sequence, including genomic target sequences, and expressly describes comparing test and control guide sequences for target cleavage [0457].
Regarding claim 12, Zhang expressly teaches forming Cas9-sgRNA complexes (RiboNucleoProtein) [0008, 0169, 0185].
Regarding claim 13, the teachings of Zhang are discussed above as applied to claim 4.
Regarding claim 14, the teachings of Zhang are discussed above as applied to claims 6-7.
Regarding claim 16, Zhang teaches vectors comprising the sgRNAs [0032-0033].
Regarding claim 17, Zhang teaches use of its engineered CRISPR-Cas systems in eukaryotic cells and vectors operable in eukaryotic cells [0008, 0031, 0036].
Regarding claim 18, Zhang teaches that a guide may be selected to target any target sequence, including genomic sequences, and that Cas9-guide complexes may be introduced into cells followed by assessment of preferential target cleavage [0154, 0157, 0457].
Regarding claim 19, Zhang expressly contemplates in-vivo delivery and instructs the skilled artisan to select CRISPR concentrations that provide the highest level of on-target modification while minimizing off-target modification for in-vivo delivery [0658, 0669].
Regarding claim 20, Zhang teaches kits containing the methods and composition of the invention [0663-0664].
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US 2018/0312824 A1; hereinafter “Zhang”) in view of Chen et al., Cell 155:1479–1491 (2013) (“Chen”), and Dang et al., Genome Biology 16:280 (2015) (“Dang”) as discussed above as applied to claim 1, and further in view of Weissman et al. (US 2022/0259593 A1; “Weissman”).
The teachings of Zhang, Chen and Dang are discussed above as applied to claim 1 and similarly apply to claim 2.
Zhang teaches that at least one loop of the sgRNA that is preferably modified by the insertion of distinct RNA sequence is either one or both of the tetraloop or the stem loop 2 [0447].
Weissman explicitly demonstrates mutations within the hairpin region of stem-loop 2. Weissman teaches modified sgRNAs containing nucleotide changes within the sgRNA constant region—the portion outside the targeting sequence that is required for binding to Cas9—and expressly teaches constant-region nucleotide and base-pair substitutions [0030-0031; 0044]. Weissman's Table 6 provides 995 constant-region variants comprising all possible single-nucleotide substitutions, base-pair substitutions, and combinations thereof, including variants whose activities exceeded the unmodified constant region [0047, 0087]. Weissman specifically maps its mutations onto the sgRNA secondary structure and teaches that mutations within the hairpin region of stem-loop 2 were well tolerated [0030, 0164]. More significantly, Weissman reports that several variants carrying mutations in stem-loop 2 had consistently increased activities [0164]. It would have been obvious to one of ordinary skill, seeking additional sgRNA substitutions capable of increasing the activity of the Zhang/Chen/Dang CRISPR-Cas9 system, to investigate and employ the stem-loop-2 substitutions expressly identified by Weissman or Zhang as producing consistently increased activity. One of ordinary skill would be motivated to make the modification because the references address the same recognized variable—the sgRNA constant-region/scaffold architecture—and the same objective of modifying sgRNA structure to alter or improve Cas9-dependent functional activity.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US 2018/0312824 A1; hereinafter “Zhang”) in view of Chen et al., Cell 155:1479–1491 (2013) (“Chen”), Dang et al., Genome Biology 16:280 (2015) (“Dang”), and Weissman et al. (US 2022/0259593 A1; “Weissman”) as discussed above as applied to claims 1-2, and further in view of Nishimasu et al., Cell 156:935–949 (2014) (“Nishimasu”).
The teachings of Zhang, Chen and Dang are discussed above as applied to claims 1-2 and similarly apply to claim 5.
Zhang, Chen and Dang do not teach wherein the substitution and/or addition of one or more nucleic acid residues strengthens the sgRNAs interaction with residue His721 and/or the PI domain of SpCas9.
Nishimasu determined the 2.5-Å crystal structure of SpCas9 in complex with an sgRNA and target DNA, expressly identifying the molecular interactions between the sgRNA and Cas9 [abstract]. Nishimasu specifically teaches that the single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe [pg. 945; col. 2; para 2-4]. Nishimasu teaches the sgRNA backbone interacts with the RuvC and PI domains, and the 2′-hydroxyl group of sgRNA nucleotide A65 forms a hydrogen bond with His721 [pg. 945; col. 2; para 2]. Nishimasu further identifies direct Cas9 recognition of stem loop 2 through the A68:G81 pair and Cas9 residues Ser1351, Tyr1356, and Thr1358 [pg. 945; col. 2; para 3]. Nishimasu expressly explains that its high-resolution structure and functional analyses revealed the molecular mechanism of RNA-guided DNA targeting and provided a basis for rational design of new genome-editing technologies [abstract]. Accordingly, one of ordinary skill seeking to implement the activity-enhancing sgRNA modifications taught by Zhang, Chen, Dang, and Weissman would have had reason to use Nishimasu's known SpCas9-sgRNA structure to select and evaluate sgRNA modifications capable of increasing stabilizing contacts with SpCas9, including contacts involving His721 and/or the PI-domain region, because Nishimasu expressly identifies these as sgRNA-contacting structural features and presents the structure as enabling rational engineering of Cas9 systems.
Allowable Subject Matter
The following is a statement of reasons for the indication of allowable subject matter: The prior art of record teaches modification and optimization of single-guide RNA (sgRNA) scaffold structures for use with CRISPR/Cas systems, including nucleotide substitutions and/or additions within sgRNA constant regions and stem-loop structures. Thus, modification of an sgRNA scaffold, including modification of stem-loop 2 to alter or improve CRISPR/Cas activity, was known in the art. However, the prior art of record does not teach or suggest the particular sgRNA sequences and sequence-identity genera recited in claims 8–11 and 15. Specifically, the prior art does not suggest a variant sgRNA comprising a stem-loop-2 hairpin having the sequence GCGGGGUGCCGC (SEQ ID NO:48), or a sequence having the recited degree of sequence identity thereto, as required by claim 8. Nor does the prior art teach or suggest a variant sgRNA comprising a stem-loop-2 hairpin having the sequence GGGCCGGGGUGCCGGCCC (SEQ ID NO:240), or a sequence having the recited degree of sequence identity thereto, as required by claim 9. Therefore, the prior art does not teach or suggest a variant sgRNA comprising a nucleic acid sequence of SEQ ID NOs: 352 and 353, or a sequence having the recited degree of sequence identity thereto.
Conclusion
No claims allowed.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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