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 .
Status of Claims
Claims 139-164 are currently pending and under consideration.
Priority
The present application is a continuation of PCT/US2021/049931 filed on 09/10/2021. Acknowledgment is made of applicant’s claim for benefit under 35 U.S.C. 119(e) of Provisional application No. 63/077,060, filed on 09/11/2020. The present application and all claims are being examined with an effective filing date of 09/11/2020. In future actions, the effective filing date may change due to amendments or further review of priority documents.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 04/19/2023, 07/19/2023, 01/19/2024, and 12/09/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
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 143, 147 and 151 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.
Claim 143 is indefinite and unclear due to the recitation of “wherein said endonuclease comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 23…”. However, SEQ ID NO: 23 already contains an alanine residue at position 9. Accordingly, the recited “aspartate to alanine mutation” cannot be determined relative to SEQ ID NO: 23 because there is no aspartate residue present at position 9, from which a mutation to alanine could occur. As a result, it is unclear what structural limitation is required by the claim, and one of ordinary skill in the art cannot reasonably determine the scope of the claim.
Claim 147 recites that the endonuclease comprises an amino acid sequence having “less than 80% sequence identity to a Cas9 endonuclease.” However, the claim fails to identify the specific Cas9 endonuclease against which sequence identity is to be measured. Multiple Cas9 orthologs having substantially different amino acid sequences are known in the art. Because the claim does not specify a reference sequence or otherwise define the comparison standard, one of ordinary skill in the art would be unable to reasonably determine the scope of the claimed limitation. Furthermore, the recitation of “less than 80% sequence identity” establishes no discernible lower boundary for the claimed sequence identity range. Theoretically, the claim encompasses a sequence having zero sequence identity to a Cas9 endonuclease. The limitation therefore encompasses an indeterminate universe of sequences (potentially extending to no sequence identity), while still purporting to define a Cas endonuclease. The claim thus fails to particularly point out and distinctly claim the subject matter regarded as the invention.
Claim 151 is indefinite due to the recitation of “a protospacer adjacent motif (PAM) sequence of nRRnMC”, as it is unclear what the sequence of “nRRnMC” is. The specification provides no definition, description, or example of this motif. Moreover, there is no mention of “nRRnMC” in the disclosure at all. Accordingly, one of ordinary skill in the art cannot determine the scope of the claimed PAM recognition requirement.
Appropriate response and/or amendment for all of the above is requested.
Claim Rejections - 35 USC § 112(a)
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 139-141 and 143-164 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.
It is noted that MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow. Claim 139 has been broadly interpreted as encompassing a genus of engineered nucleic acid editing systems comprising, inter alia, a genus of endonucleases having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. Claims 140-141 encompass a genus of nucleic acid systems comprising, inter alia, a genus of endonucleases having at least 90% or 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, respectively. Claims 149-150 encompass a genus of nucleic acid systems comprising, inter alia, a genus of endonucleases having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a genus of tracer sequences having at least 80% identity to any 60-90 consecutive nucleotides of SEQ ID NO: 29 or the full length sequence set forth in SEQ ID NO: 29, respectively. Claim 152 encompasses a genus of nucleic acid systems comprising, inter alia, a genus of endonucleases having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23 and a genus of base editors comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-17. Claims 154 and 156 encompass a genus of engineered nucleic acid editing systems comprising, inter alia, a genus of endonucleases having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a genus of adenosine deaminases comprising at least 80% sequence identity to SEQ ID NOs: 8 or 164, or a genus of cytosine deaminases comprising at least 80% sequence identity to any one of SEQ ID NOs: 1-7 and 9-17, respectively. Claim 164 encompasses a genus of engineered nucleic acid editing systems comprising, inter alia, a genus of endonucleases having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23 and a genus of deoxyribonucleic acid glycosylase inhibitors comprising at least 70% sequence identity to SEQ ID NO: 18.
MPEP 2163 I. states that to “satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.
MPEP 2163. II.A.3.(a) states that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number' depends on whether one of skill in the art would recognize that the applicant 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. Genome editing systems involving engineered nucleases and base editors constitute an unpredictable biological art in which small sequence changes may substantially alter structure or function.
In the instant case, the recited SEQ ID NOs above define biological macromolecules (e.g., proteins and RNAs) that are intended to have specific functional activity (e.g., nuclease activity, base editing activity, inhibition of DNA repair enzymes, etc.). The specification discloses the specific sequence but fails to describe which regions are essential or necessary to maintain the recited biological function or activity, or how the structure-function relationship is maintained across sequences that share as little as 70% identity with the disclosed sequences, encompassing hundreds of potential amino acid or nucleotide substitutions. Given the substantial size of some of the claimed macromolecules (SEQ ID NO: 23 = 1154 amino acids in length), a sequence identity of even 95% encompasses extensive sequence variation. The breadth of the claimed genera described above encompasses a wide range of possible structural variants throughout the molecule. While the specification does disclose a few variants (e.g., nickases set forth in SEQ ID NOs: 21 and 22 and others disclosed in Table 2), it does not identify any essential or critical residues, nor does it describe which amino acid and/or nucleotide changes (substitutions, insertions, or deletions) can be made, and still result in having the desired activity/function. As such, while the specification defines some species falling within the genus, it fails to define any structural features commonly possessed by members of the genus that distinguish them from others. There is no guidance that would allow a skilled artisan to predict which sequences within the claimed scope would retain the required functional activity.
In the absence of such structural or functional characterization, one of ordinary skill in the art would not be able to visualize or recognize the identity of the members of the genus encompassed by at least 70% or 80% or 90% or 95% sequence identity without undue experimentation. Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the full scope of the claimed invention.
Accordingly, claims 139-141 and 143-164 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.
Claim 151 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 151 recites that the endonuclease is configured to bind a protospacer adjacent motif (PAM) sequence of “nRRnMC”. However, the specification and sequence listing do not disclose the PAM sequence of nRRnMC, nor do they describe a motif corresponding to this sequence. Because the originally filed disclosure does not reasonably convey possession of or support for an endonuclease recognizing the recited PAM sequence of nRRnMC, this is considered new matter. Accordingly, the written description requirement has not been satisfied.
Claims 139-141 and 143-164 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 an engineered nucleic acid editing system comprising an endonuclease having the amino acid sequence set forth in SEQ ID NOs: 21-23, an engineered tracr sequence comprising SEQ ID NO: 27-29, a base editor comprising a sequence corresponding to one of SEQ ID NOS: 1–17 and 19-20, an adenosine deaminase comprising SEQ ID NO: 164, and a UGI comprising SEQ ID NO: 18, does not reasonably provide enablement for the full scope of the claimed genus of engineered nucleic acid editing systems, including a genus of endonucleases with as little as 80% sequence identity to SEQ ID NO: 23, a genus of tracr sequences comprising as little as 80% sequence identity to any 60 consecutive nucleotides of SEQ ID NO: 29, a genus of base editors comprising as little as 70% identity to any of SEQ ID NOs: 1-17, a genus of adenosine deaminases comprising as little as 80% sequence identity to SEQ ID NO: 164, and a genus of UGIs comprising as little as 70% identity to SEQ ID NO: 18. 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.
The specification fails to enable the claim (or the full scope of the claim) if a person of ordinary skill in the art would be faced with an undue burden of experimentation when trying to implement the invention based on the disclosure. In re Wands (858 F.2d 731 at 737, 8 USPQ2d 1400 at 1404 (Fed. Cir. 1988)) sets forth a non-exclusive list of factors by which this burden of experimentation may be judged to be due or undue; factors that are germane to the instant case include the breadth of the claims, nature of the invention, the amount of direction provided by the inventor, the existence of working examples, the state of the prior art, and quantity of experimentation required.
Breadth of the Claims and Nature of the Invention
As indicated above, claim 139 encompasses a genus of systems, which comprise in part, endonucleases having as little as 80% sequence identity to SEQ ID NO: 23. Because SEQ ID NO: 23 encodes a protein exceeding one thousand amino acids in length, an 80% identity limitation permits extensive amino acid substitutions distributed throughout the molecule. Similar breadth arises from the claimed identity ranges for tracr RNA sequences and base editor enzymes.
This scope includes, without limitation, a large genera of biological macromolecules potentially including hundreds or thousands of structurally distinct protein and RNA variants. Thus, the claims extend far beyond the specifically disclosed sequences to encompass vast numbers of uncharacterized variants.
State of the Prior Art and Level of Predictability
While CRISPR technology for gene editing is known in the art, engineered CRISPR nucleases, guide RNAs, and base editors constitute an unpredictable biological art, particularly because even small sequence variations may substantially alter nuclease activity, RNA binding, protein folding, target recognition, editing efficiency, etc. The unpredictability of biological systems weighs against enablement across the full scope of the claims.
Amount of Direction and Presence of Working Examples
The only detailed working example described is construction and testing of metagenomic base editor plasmids comprising specific nickase variants (iMG4-2 (D28A), SEQ ID NO: 21; iMG7-1 (D10A), SEQ ID NO: 22; and iMG16-1 (D9A), SEQ ID NO: 23) fused to defined deaminase components, including TadA (ABE8.17m; SEQ ID NO: 164) or APOBEC1 cytidine deaminase, together with a uracil glycosylase inhibitor (UGI; SEQ ID NO: 18) and sgRNA constructs assembled using disclosed scaffold sequences. The example demonstrates plasmid construction, protein expression and purification, in vitro nickase activity assays, and evaluation of base-editing efficiency in E. coli using these specifically disclosed sequence combinations. However, the example is limited to these particular identified sequences and experimental constructs and does not provide guidance for designing or validating variants across the broad sequence-identity ranges recited in the claims, including endonucleases having as little as 80% sequence identity to SEQ ID NO: 23, tracer RNA variants defined only by partial sequence identity to SEQ ID NO: 29, or base editor, deaminase, and UGI variants defined solely by percentage identity.
The specification therefore provides no teaching enabling a person of ordinary skill in the art to determine which amino acid substitutions across these claimed genera would preserve nuclease deficiency, target DNA binding, or base-editing activity without extensive experimentation.
Undue Experimentation
Absent additional guidance, a person of ordinary skill in the art would be required to engage in extensive experimentation, including generating numerous candidate variants meeting identity thresholds, empirically testing nuclease deficiency, validating RNA–protein complex formation, evaluating base editing activity, and confirming inhibition of DNA repair mechanisms. In view of the breadth of the claims relative to the narrow disclosure, the limited number of working examples, the complexity and relative unpredictability of the art, and the amount of experimentation required to extend the disclosed example to the full range of systems encompassed by the claims, one of ordinary skill in the art would not be able to practice the claimed invention without undue experimentation.
Thus, claims 139-141 and 143-164 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for lack of enablement.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 139-141, 144-146, 148, 151-152, 155-164 are rejected under 35 U.S.C. 103 as being obvious over Thomas et al. (WO2020168234, cited in the IDS) and Liu and Gaudelli (WO2018176009, herein “Liu”, cited in the IDS).
The applied reference has a common applicant with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Thomas et al. discloses an engineered nuclease system comprising: (a) an endonuclease comprising a RuvC III domain and an HNH domain, wherein said endonuclease is derived from an uncultivated microorganism, and wherein said endonuclease is a class 2, type II Cas endonuclease; and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence (correlating to a “non-guide ribonucleic acid sequence”) configured to bind to said endonuclease (claim 1); wherein the endonuclease is configured to bind to a protospacer adjacent motif (claim 10); wherein the guide ribonucleic acid sequence is 15-23 nucleotides in length (claim 14) and is complementary to a prokaryotic, bacterial, eukaryotic, fungal, plant, mammalian, or human genomic sequence (claim 13); wherein the endonuclease may comprise one or more nuclear localization sequences (claim 15). Specifically, Thomas et al. discloses an endonuclease (i.e., MG16) set forth in SEQ ID NO: 43, having 99.9% sequence identity to instant SEQ ID NO: 23 (see sequence alignment below), and further teaches that “in some cases the endonuclease may comprise a variant having at least about 30%...or at least about 99% identity to any one of SEQ ID NOs: 43-44” (para 0176). Thomas et al. further teaches wherein the endonuclease can comprise variants of an HNH and/or RuvC III domain (para 0174- 0175) – indicating that the nuclease is amenable to engineering that can include activity alteration. Furthermore, Thomas et al. teaches wherein said sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT, or Smith -Waterman homology search algorithm, and more specifically, wherein said sequence identity is determined by said BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment. Thomas et al. does not teach wherein the engineered nuclease system comprises a base editor coupled directly or via a linker to the endonuclease (claims 139 and 160), wherein the base editor is an adenosine deaminase comprising at least 80% sequence identity to SEQ ID NOs: 8 or 164 (claims 153-154), wherein the base editor is a cytosine deaminase comprising at least 80% sequence identity to SEQ ID NOs:1-7 or 9-17 (claims 155-156), wherein said endonuclease comprises a nickase mutation (claim 144), wherein the RuvC domain lacks nuclease activity (claim 145), wherein the endonuclease is configured to cleave one strand of a double stranded target deoxyribonucleic acid (claim 146), and wherein said system further comprises a uracil deoxyribonucleic acid glycosylase inhibitor comprising at least 70% sequence identity to SEQ ID NO: 18 (claims 163-164).
Liu teaches that targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and has the potential to provide new therapies for human genetic diseases by effecting a specific nucleotide change at a specific location in the genome. Liu further teaches that programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR) (Specification, para 0001). However, “both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration”. Accordingly, Liu teaches nucleic acid programmable DNA binding proteins (napDNAbp), including dCas9 (catalytically inactive) and Cas9 nickase (cleaving one strand of double stranded DNA), fused to a cytidine deaminase, via a linker for deaminating target cytidine residues, and a uracil glycosylase inhibitor (UGI) domain (Specification, para 0003-0008). Liu teaches that base editing (e.g., deaminating target cytidine residues) can be reversed by endogenous DNA repair mechanisms – “Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity” (para 0023). Liu teaches that these fusion proteins “generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain” (para 0006). Therefore, the inclusion of an UGI would improve efficiency of deaminating target nucleotides. Liu discloses a UGI set forth in SEQ ID NO: 178 having 100% sequence identity to instant SEQ ID NO: 18, and a cytidine deaminase set forth in SEQ ID NO: 168 having 100% sequence identity to instant SEQ ID NO: 9 (see sequence alignments below). It is noted that Liu teaches point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in dCas9 (para 0004). More specifically, Liu teaches that mutations within the HNH or RuVC subdomains “can silence the nuclease activity of Cas9”, such as D10A and H840A, which “completely inactivate the nuclease activity of S. pyogenes Cas9” (para 00214 and para 0008).
It is noted that the term “engineered nucleic acid editing system”, as recited in claim 139, has been interpreted as a composition comprising the recited structural components. Accordingly, the claimed “system” encompasses a composition including the endonuclease, the base editor coupled thereto, and the engineered guide ribonucleic acid structure.
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, Thomas et al. discloses an engineered nucleic acid editing system comprising a Cas9 endonuclease, such as MG16, together with an engineered guide ribonucleic acid structure to direct the endonuclease to target a DNA sequence via protospacer adjacent motif recognition, and a tracr sequence to bind said endonuclease. In view of Liu’s disclosure of nucleobase editor fusion proteins comprising programmable DNA binding proteins (including dCas9 and Cas9 nickase) linked to cytidine deaminase (SEQ ID NO: 168), for more precision editing compared to programmable cleavage that may result in unwanted gene alterations, a person of ordinary skill in the art would have been motivated to incorporate a base editor, such as SEQ ID NO: 168 into the Thomas et al. system in order to enable direct conversion of target nucleotides at specific genomic loci using sequence specific targeting, because Liu teaches the potential for treating diseases by effecting a specific nucleotide change at a specific location in the genome. Said practitioner would further be motivated to modify the endonuclease of Thomas et al. in view of Liu’s disclosure of catalytically inactive Cas9 proteins, obtained by mutations in the RuvC domain of Cas9, because reduction or elimination of nuclease cleavage activity avoids formation of double strand DNA breaks and thereby reduces undesired insertions and deletions associated with cleavage based genome editing while maintaining programmable DNA binding capability. Said practitioner would also be motivated to further modify the system of Thomas et al. by incorporating a uracil glycosylase inhibitor (UGI), including SEQ ID NO: 178, as disclosed by Liu, in order to inhibit base excision repair mechanisms that would otherwise reverse cytidine deamination events, thereby improving base editing efficiency. Because Liu expressly demonstrates programmable DNA-binding proteins operating in combination with deaminase editors, nickase or catalytically inactive variants, and UGI domains to perform targeted nucleobase editing, a person of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings with the engineered CRISPR system of Thomas et al. to arrive at the claimed engineered nucleic acid editing system. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Thus, claims 139-141, 144-146, 148, 151-152, 155-164 are rejected under 35 U.S.C. 103 as being obvious over Thomas et al. (WO2020168234, cited in the IDS) and Liu and Gaudelli (WO2018176009, herein “Liu”, cited in the IDS).
Claims 153-154 are rejected under 35 U.S.C. 103 as being unpatentable over Thomas et al. and Liu, as applied to claim 139 above, and further in view of Liu and Gaudelli (WO2018027078, herein “Liu2”, cited in PTO-892).
The teachings of Thomas et al. and Liu, as they apply to claim 139 have already been discussed above. Briefly, Thomas et al. discloses an engineered nucleic acid editing system comprising a Cas9 endonuclease, such as MG16, together with an engineered guide ribonucleic acid structure to direct the endonuclease to target a DNA sequence via protospacer adjacent motif recognition, and a tracr sequence to bind said endonuclease. Liu discloses fusion proteins comprising Cas9, a base editor such as cytidine deaminase and at least one UGI for improved efficiency in targeted base editing in DNA. Neither Thomas et al. nor Liu expressly teach wherein the base editor is an adenosine deaminase, specifically having at least 70% sequence identity to SEQ ID NOs : 1-17 and 80% sequence identity to SEQ ID NOs: 8 or 164.
Liu2 teaches that many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, an A to G or a T to C change in a specific codon of a gene associated with a disease). Accordingly, Liu2 discloses compositions, kits, and methods of modifying a polynucleotide (e.g., DNA) using an adenosine deaminase (base editor) and a nucleic acid programmable DNA binding protein (e.g., Cas9) (Specification, para 0001-0002). Liu2 further teaches said fusion proteins, further comprising a nuclear localization sequence (NLS), and/or an inhibitor of base repair (e.g., UGI) (Abstract). Specifically, Liu2 discloses an adenosine deaminase set forth in SEQ ID NO: 1, having 100% sequence identity to instant SEQ ID NO: 8 (see sequence alignment below).
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, the teachings of Liu2, which discloses fusion proteins comprising Cas9 and adenosine deaminase (base editor) for targeted base editing of DNA for the potential benefit of treating disease, would have motivated a person of ordinary skill in the art to modify the engineered nucleic acid editing system made obvious by Thomas et al. and Liu, and substitute the cytidine deaminase with an adenosine deaminase, such as SEQ ID NO: 8. Given that both cytidine deaminase and adenosine deaminase are known alternative nucleobase editors enabling different base conversions, as part of an engineered nucleic acid editing system comprising Cas9, it would have been obvious for a person of ordinary skill in the art, to make these substitutions and expect predictable results (see MPEP 2144.06, “Substituting equivalents known for the same purpose”). Selection of either editor would have represented routine optimization depending on the desired nucleotide modification. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Thus, claims 152-154 are rejected under 35 U.S.C. 103 as being unpatentable over Thomas et al. and Liu, as applied to claim 139 above, and further in view of Liu and Gaudelli (WO2018027078, herein “Liu2”, cited in PTO-892).
Sequence alignment of SEQ ID NO: 43 of Thomas et al. and instant SEQ ID NO: 23
BIF06675
(NOTE: this sequence has 2 duplicates in the database searched.
See complete list at the end of this report)
ID BIF06675 standard; protein; 1154 AA.
XX
AC BIF06675;
XX
DT 01-OCT-2020 (first entry)
XX
DE Bacterial derived endonuclease MG16-1 effector, SEQ 43.
XX
KW CRISPR-Cas9 system; dna cleavage; endonuclease; enzyme production;
KW genome editing.
XX
OS Bacteria.
XX
CC PN WO2020168234-A1.
XX
CC PD 20-AUG-2020.
XX
CC PF 14-FEB-2020; 2020WO-US018353.
XX
PR 14-FEB-2019; 2019US-0805893P.
XX
CC PA (META-) METAGENOMI IP TECHNOLOGIES LLC.
XX
CC PI Thomas B, Brown C, Kantor R, Devoto A, Butterfield C;
CC PI Alexander L, Goltsman DSA, Liu J;
XX
DR WPI; 2020-79946U/073.
XX
CC PT Engineered nuclease system for modifying target nucleic acid locus,
CC PT comprises endonuclease comprising RuvC III domain and HNH domain, and
CC PT engineered guide ribonucleic acid structure configured to form complex
CC PT with endonuclease.
XX
CC PS Claim 24; SEQ ID NO 43; 109pp; English.
XX
CC The present invention relates to a novel engineered nuclease system
CC (CRISPR-Cas9 system) for modifying a target nucleic acid locus. The
CC engineered nuclease system comprises: (a) an endonuclease comprising RuvC
CC III domain of SEQ ID NO:45-88 and 246-262 (see BIF06677-BIF06720 and
CC BIF06878-BIF06894) and and HNH domain, where the endonuclease is derived
CC from an uncultivated microorganism, and where the endonuclease is a class
CC 2, type II Cas endonuclease; and (b) an engineered guide ribonucleic acid
CC structure configured to form a complex with the endonuclease comprising:
CC (i) a guide ribonucleic acid sequence configured to hybridize to a target
CC deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid
CC sequence configured to bind to the endonuclease. The invention also
CC provides: an engineered guide ribonucleic acid polynucleotide; a
CC deoxyribonucleic acid polynucleotide encoding the engineered guide
CC ribonucleic acid polynucleotide; a nucleic acid comprising an engineered
CC nucleic acid sequence optimized for expression in an organism; a
CC deoxyribonucleic acid polynucleotide encoding the engineered guide
CC ribonucleic acid polynucleotide; a nucleic acid comprising an engineered
CC nucleic acid sequence optimized for expression in an organism; a vector
CC comprising a nucleic acid sequence encoding a class 2, type II Cas
CC endonuclease comprising a RuvC III domain and an HNH domain; a cell
CC comprising the vector; a method for manufacturing an endonuclease; a
CC method for binding, cleaving, marking, or modifying a double-stranded
CC deoxyribonucleic acid polynucleotide; and a method for modifying a target
CC nucleic acid locus. The discovery of new Cas enzymes with unique
CC functionality and structure may offer the potential to further disrupt
CC DNA editing technologies, improving speed, specificity, functionality,
CC and ease of use.
XX
SQ Sequence 1154 AA;
Query Match 99.9%; Score 6041; Length 1154;
Best Local Similarity 99.9%;
Matches 1153; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 MIKNILGLALGVGSIGWALIQTEDDQPKQIIGMGSRIVPLTKDDSDQFTKGQAISKNAER 60
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MIKNILGLDLGVGSIGWALIQTEDDQPKQIIGMGSRIVPLTKDDSDQFTKGQAISKNAER 60
Qy 61 TARRTTRKGYDRYQLRRALLTQVLRQNGMLPECMDENMIDLWKLRSDAATEGKQLTLQQI 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TARRTTRKGYDRYQLRRALLTQVLRQNGMLPECMDENMIDLWKLRSDAATEGKQLTLQQI 120
Qy 121 GRVLYHINQKRGYKHAKSDDNGDSKQTKYVEAVNLRYKEIQEKNVTVGQHFYAELLNSKV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GRVLYHINQKRGYKHAKSDDNGDSKQTKYVEAVNLRYKEIQEKNVTVGQHFYAELLNSKV 180
Qy 181 ESGNGPYYTFRIKDKVFPRAAYIAEFDQIMGVQKEYYPNVLTDELIETLRNRIIFYQRPL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ESGNGPYYTFRIKDKVFPRAAYIAEFDQIMGVQKEYYPNVLTDELIETLRNRIIFYQRPL 240
Qy 241 KSCKHLVGLCEFEMRPYKKDGKIVYGGPKCAPRTSPLAQLCAMWETVNNITLTNRNNERL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 KSCKHLVGLCEFEMRPYKKDGKIVYGGPKCAPRTSPLAQLCAMWETVNNITLTNRNNERL 300
Qy 301 EISNEQRRQLVQFLCTHETLKLTDLYKILGITKKDGWYGGKAIGKGIKGNVTLNQLRKAL 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 EISNEQRRQLVQFLCTHETLKLTDLYKILGITKKDGWYGGKAIGKGIKGNVTLNQLRKAL 360
Qy 361 DGKYSQWLEMPIERIDVVDRNTAEAFWAVSPKVEETPLFQLWHAVYSLQNVEELTKTLQN 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 DGKYSQWLEMPIERIDVVDRNTAEAFWAVSPKVEETPLFQLWHAVYSLQNVEELTKTLQN 420
Qy 421 RFSITDPQVIDALCKIDFVKPGYANKSHKFIRRLLPYLMEGMMYSEACACIQINHSNSMT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 RFSITDPQVIDALCKIDFVKPGYANKSHKFIRRLLPYLMEGMMYSEACACIQINHSNSMT 480
Qy 481 KAEREARPLAERIELLQKNALRQPVIEKILNQMINLVNRLQQEYGPIDEARVELARELKQ 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 KAEREARPLAERIELLQKNALRQPVIEKILNQMINLVNRLQQEYGPIDEARVELARELKQ 540
Qy 541 SREERKDAFDRNNKNEKRNKEISALISEQGIRPSRSRIQKYKMWEESEHRCMYCGKVVNL 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 SREERKDAFDRNNKNEKRNKEISALISEQGIRPSRSRIQKYKMWEESEHRCMYCGKVVNL 600
Qy 601 SEFLNGADVEIEHIIPRSILFDDSFSNKVCACRDCNREKDNMTAMDYMASKPEGEFEAYK 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 SEFLNGADVEIEHIIPRSILFDDSFSNKVCACRDCNREKDNMTAMDYMASKPEGEFEAYK 660
Qy 661 QRVDEAFNAHRISKTKRDHLLWRRADIPQDFIDRQLRLSQYIATKAVEILQQGIRQVWTS 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 QRVDEAFNAHRISKTKRDHLLWRRADIPQDFIDRQLRLSQYIATKAVEILQQGIRQVWTS 720
Qy 721 GGGVTDFLRHQWGYDEILHTLNLPRYRQVEDLTEMVHYEHAGQEHDEERIKNWSKRIDHR 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 GGGVTDFLRHQWGYDEILHTLNLPRYRQVEDLTEMVHYEHAGQEHDEERIKNWSKRIDHR 780
Qy 781 HHAIDALTVALTRQSYIQRLNTLEASHEHMEKLVKEANTPYKEKKSLLEKWVALQPHFSV 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 HHAIDALTVALTRQSYIQRLNTLEASHEHMEKLVKEANTPYKEKKSLLEKWVALQPHFSV 840
Qy 841 EEVTTQVDGILVSFRAGKRVTTPARRAVYHGGKRTIVQRGIQVPRGALTEDTIYGKLGDK 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 EEVTTQVDGILVSFRAGKRVTTPARRAVYHGGKRTIVQRGIQVPRGALTEDTIYGKLGDK 900
Qy 901 FVVKYALDHPSMKPENIVDPTIRLLVENRITALGKKDAFKTPLYSAEGMEIKSVRCYTSL 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FVVKYALDHPSMKPENIVDPTIRLLVENRITALGKKDAFKTPLYSAEGMEIKSVRCYTSL 960
Qy 961 SEKGVVPIKYNEKGNAIGFAKKGNNHHVAIYKDQSGQYQEMVVSFWDAVERKLYGVPTVI 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 SEKGVVPIKYNEKGNAIGFAKKGNNHHVAIYKDQSGQYQEMVVSFWDAVERKLYGVPTVI 1020
Qy 1021 TNPKTVWDELLEKELPQDFLEKLPKDNWQYVLSMQENEMFVLGMEEDEFNDAIDTQDYNT 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 TNPKTVWDELLEKELPQDFLEKLPKDNWQYVLSMQENEMFVLGMEEDEFNDAIDTQDYNT 1080
Qy 1081 LNKHLYRVQKLSHADYTFRFHTETKVDDKYDGVENGRNTSMSLKALVRIRSFNGLFTQFP 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 LNKHLYRVQKLSHADYTFRFHTETKVDDKYDGVENGRNTSMSLKALVRIRSFNGLFTQFP 1140
Qy 1141 HKVKIDIMGRITKA 1154
||||||||||||||
Db 1141 HKVKIDIMGRITKA 1154
Sequence alignment of SEQ ID NO: 168 of Liu and instant SEQ ID NO: 9
BFR63263
(NOTE: this sequence has 5 duplicates in the database searched.
See complete list at the end of this report)
ID BFR63263 standard; protein; 1544 AA.
XX
AC BFR63263;
XX
DT 15-NOV-2018 (first entry)
XX
DE APOBEC1-linker-SaCas9d-UGI-UGI-NLS protein construct, SEQ 168.
XX
KW CRISPR-Cas9 system; antiinflammatory; antimicrobial-gen.;
KW autoimmune disease; cardiovascular disease; cardiovascular-gen.;
KW cytostatic; dermatological; dermatological disease; endocrine disease;
KW endocrine-gen.; gastrointestinal disease; gastrointestinal-gen.;
KW genome editing; hematological disease; hematological-gen.;
KW immunomodulator; immunosuppressive; infectious disease;
KW inflammatory disease; lymphatic system disease; metabolic disorder;
KW metabolic-gen.; musculoskeletal disease; musculoskeletal-gen.; neoplasm;
KW neurological disease; neuroprotective; nutrition-disorder-gen.;
KW nutritional disorder; protein production; protein therapy;
KW psychiatric disorder; psychiatric-gen.; recombinant protein;
KW respiratory disorder; respiratory-gen.; therapeutic;
KW urinary tract disease; uropathic.
XX
OS Synthetic.
OS Unidentified.
XX
CC PN WO2018176009-A1.
XX
CC PD 27-SEP-2018.
XX
CC PF 23-MAR-2018; 2018WO-US024208.
XX
PR 23-MAR-2017; 2017US-0475830P.
PR 26-APR-2017; 2017US-0490587P.
PR 26-MAY-2017; 2017US-0511934P.
PR 30-AUG-2017; 2017US-0551951P.
XX
CC PA (HARD ) HARVARD COLLEGE.
XX
CC PI Liu DR, Komor AC, Chen L, Rees HA;
XX
DR WPI; 2018-754796/71.
XX
CC PT New fusion protein comprises a nucleic acid programmable DNA binding
CC PT protein (napDNAbp), a cytidine deaminase domain, and a uracil glycosylase
CC PT inhibitor (UGI) domain, for preparing pharmaceutical composition for
CC PT treating cancer.
XX
CC PS Example 18; SEQ ID NO 168; 552pp; English.
XX
CC The present invention relates to a novel fusion protein, useful for
CC preparing a pharmaceutical composition for treating cancer. The fusion
CC protein comprises: (a) a nucleic acid programmable DNA binding protein
CC (napDNAbp); (b) a cytidine deaminase domain; and (iii) a uracil
CC glycosylase inhibitor (UGI) domain. The napDNAbp is a CasX, CasY, Cpf1,
CC C2c1, C2c2, C2c3, or argonaute protein. The invention also provides: a
CC method for producing a ribonucleoprotein (RNP) complex; a method for
CC purifying a base editor protein; and a method for editing a nucleobase
CC pair of a double-stranded DNA sequence. The fusion protein is useful for
CC treating autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis,
CC psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis,
CC pelvic inflammatory disease); infectious diseases (e.g. viral infections,
CC bacterial infections, fungal infections, and sepsis); neurological
CC disorders (e.g. Alzheimer's disease, Huntington's disease; autism;
CC Duchenne muscular dystrophy); cardiovascular disorders (e.g.
CC atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders,
CC angiogenic disorders such as macular degeneration); proliferative
CC disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g.
CC chronic obstructive pulmonary disease); digestive disorders (e.g.
CC inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g.
CC fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders
CC (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease);
CC psychological disorders (e.g. depression, schizophrenia); skin disorders
CC (e.g. wounds, eczema); and blood and lymphatic disorders (e.g. anemia,
CC hemophilia). The present sequence is an APOBEC1-linker-SaCas9d-UGI-UGI-
CC NLS protein construct, which is used for constructing the fusion protein
CC of the invention.
XX
SQ Sequence 1544 AA;
Query Match 100.0%; Score 1280; Length 1544;
Best Local Similarity 100.0%;
Matches 229; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK 60
Qy 61 HVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 HVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY 120
Qy 121 HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL 180
Qy 181 ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 229
|||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 229
Sequence alignment of SEQ ID NO: 178 of Liu and instant SEQ ID NO: 18
BFR63273
ID BFR63273 standard; protein; 1709 AA.
XX
AC BFR63273;
XX
DT 15-NOV-2018 (first entry)
XX
DE BE4-N protein construct, SEQ 178.
XX
KW CRISPR-Cas9 system; antiinflammatory; antimicrobial-gen.;
KW autoimmune disease; cardiovascular disease; cardiovascular-gen.;
KW cytostatic; dermatological; dermatological disease; endocrine disease;
KW endocrine-gen.; gastrointestinal disease; gastrointestinal-gen.;
KW genome editing; hematological disease; hematological-gen.;
KW immunomodulator; immunosuppressive; infectious disease;
KW inflammatory disease; lymphatic system disease; metabolic disorder;
KW metabolic-gen.; musculoskeletal disease; musculoskeletal-gen.; neoplasm;
KW neurological disease; neuroprotective; nutrition-disorder-gen.;
KW nutritional disorder; protein production; protein therapy;
KW psychiatric disorder; psychiatric-gen.; recombinant protein;
KW respiratory disorder; respiratory-gen.; therapeutic;
KW urinary tract disease; uropathic.
XX
OS Synthetic.
OS Unidentified.
XX
CC PN WO2018176009-A1.
XX
CC PD 27-SEP-2018.
XX
CC PF 23-MAR-2018; 2018WO-US024208.
XX
PR 23-MAR-2017; 2017US-0475830P.
PR 26-APR-2017; 2017US-0490587P.
PR 26-MAY-2017; 2017US-0511934P.
PR 30-AUG-2017; 2017US-0551951P.
XX
CC PA (HARD ) HARVARD COLLEGE.
XX
CC PI Liu DR, Komor AC, Chen L, Rees HA;
XX
DR WPI; 2018-754796/71.
XX
CC PT New fusion protein comprises a nucleic acid programmable DNA binding
CC PT protein (napDNAbp), a cytidine deaminase domain, and a uracil glycosylase
CC PT inhibitor (UGI) domain, for preparing pharmaceutical composition for
CC PT treating cancer.
XX
CC PS Example 14; SEQ ID NO 178; 552pp; English.
XX
CC The present invention relates to a novel fusion protein, useful for
CC preparing a pharmaceutical composition for treating cancer. The fusion
CC protein comprises: (a) a nucleic acid programmable DNA binding protein
CC (napDNAbp); (b) a cytidine deaminase domain; and (iii) a uracil
CC glycosylase inhibitor (UGI) domain. The napDNAbp is a CasX, CasY, Cpf1,
CC C2c1, C2c2, C2c3, or argonaute protein. The invention also provides: a
CC method for producing a ribonucleoprotein (RNP) complex; a method for
CC purifying a base editor protein; and a method for editing a nucleobase
CC pair of a double-stranded DNA sequence. The fusion protein is useful for
CC treating autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis,
CC psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis,
CC pelvic inflammatory disease); infectious diseases (e.g. viral infections,
CC bacterial infections, fungal infections, and sepsis); neurological
CC disorders (e.g. Alzheimer's disease, Huntington's disease; autism;
CC Duchenne muscular dystrophy); cardiovascular disorders (e.g.
CC atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders,
CC angiogenic disorders such as macular degeneration); proliferative
CC disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g.
CC chronic obstructive pulmonary disease); digestive disorders (e.g.
CC inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g.
CC fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders
CC (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease);
CC psychological disorders (e.g. depression, schizophrenia); skin disorders
CC (e.g. wounds, eczema); and blood and lymphatic disorders (e.g. anemia,
CC hemophilia). The present sequence is a BE4-N protein construct, which is
CC used for constructing the fusion protein of the invention.
XX
SQ Sequence 1709 AA;
Query Match 100.0%; Score 423; Length 1709;
Best Local Similarity 100.0%;
Matches 84; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS 60
Qy 61 DAPEYKPWALVIQDSNGENKIKML 84
||||||||||||||||||||||||
Db 61 DAPEYKPWALVIQDSNGENKIKML 84
Sequence alignment between SEQ ID NO: 1 of Liu2 and instant SEQ ID NO: 8
BFB10528
(NOTE: this sequence has 135 duplicates in the database searched.
See complete list at the end of this report)
ID BFB10528 standard; protein; 167 AA.
XX
AC BFB10528;
XX
DT 08-SEP-2022 (revised)
DT 05-APR-2018 (first entry)
XX
DE E. coli tRNA adenosine deaminase A (TadA) protein SEQ 1.
XX
KW CRISPR-Cas9 system; TadA; cytostatic; genetic disorder;
KW genetic-disease-gen.; genome editing; lysosome storage disease;
KW metabolic disorder; metabolic-gen.; neoplasm; neuroprotective;
KW recombinant protein; tRNA adenosine deaminase A; therapeutic.
XX
OS Escherichia coli.
XX
CC PN WO2018027078-A1.
XX
CC PD 08-FEB-2018.
XX
CC PF 03-AUG-2017; 2017WO-US045381.
XX
PR 03-AUG-2016; 2016US-0370684P.
PR 02-FEB-2017; 2017US-0454035P.
PR 20-MAR-2017; 2017US-0473714P.
XX
CC PA (HARD ) HARVARD COLLEGE.
XX
CC PI Gaudelli N, Liu DR;
XX
DR WPI; 2018-11292C/13.
XX
CC PT New adenosine deaminase capable of deaminating adenine of deoxyadenosine
CC PT in DNA useful for editing nucleobase pair of double-stranded DNA
CC PT sequence.
XX
CC PS Claim 6; SEQ ID NO 1; 1450pp; English.
XX
CC The invention relates to a novel adenosine deaminase capable of
CC deaminating adenine of deoxyadenosine in DNA useful for editing
CC nucleobase pair of double-stranded DNA sequence. The invention claims: 1)
CC a fusion protein; 2) a complex comprising the fusion protein and a guide
CC RNA (gRNA) bound to the napDNAbp of the fusion protein, where the
CC napDNAbp is a clustered regularly interspaced short palindromic repeat
CC (CRISPR) associated protein 9 (Cas9) domain, a Cpfl, a CasX, a CasY, a
CC C2c1, a C2c2, or a C2c3.; 3) a method involving (a) contacting a nucleic
CC acid molecule with the fusion protein and a gRNA, where the gRNA is 15-
CC 100 nucleotides long and comprises a sequence of at least 10 contiguous
CC nucleotides that is complementary to a target sequence, or (b) contacting
CC a nucleic acid molecule with the complex; 4) a method for editing a
CC nucleobase pair of a double-stranded DNA (dsDNA) sequence; 5) a nucleic
CC acid-guided adenosine deaminase coupled to an inhibitor of base excision
CC repair; 6) a kit comprising a nucleic acid construct; 7) a polynucleotide
CC encoding the adenosine deaminase, or the fusion protein; 8) a vector
CC comprising the polynucleotide; and 9) a cell comprising (a) the adenosine
CC deaminase, or the fusion protein, (b) the complex, or (c) the nucleic
CC acid molecule encoding the adenosine deaminase, or the fusion protein.
CC The adenosine deaminase is used in pharmaceutical composition for editing
CC a nucleobase pair of a dsDNA sequence; for modifying a polynucleotide,
CC and treating a proliferative disease, genetic disease, neoplastic
CC disease, metabolic disease, and lysosomal storage disease. The adenosine
CC deaminase has excellent stability. The present sequence represents an
CC Escherichia coli tRNA adenosine deaminase A (TadA) protein used for
CC editing nucleobase pair of double-stranded DNA sequence. Note: The
CC present sequence is used as the parent sequence for the creation of
CC variants (see BFB10592-BFB10610, BFB10947-BFB10959, BFB11199-BFB11211,
CC BLM40733-BLM40803, BLM40806-BLM40834) based on the information given in
CC example 1 of the specification.
CC
CC Revised record issued on 05-SEP-2022 : Correction to PS line and
CC comments.
XX
SQ Sequence 167 AA;
Query Match 100.0%; Score 877; Length 167;
Best Local Similarity 100.0%;
Matches 167; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI 60
Qy 61 MALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 MALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDV 120
Qy 121 LHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 167
|||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 167
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 139-142, 144-146, 148, 155, 157-164 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claima 112-114, 116, and 118 of copending Application No. 17431135 in view of Liu (WO2018176009, cited in the IDS).
Claim 116 of application ‘135 recites an engineered nuclease system comprising:(a) an endonuclease comprising a RuvC_III domain and an HNH domain, wherein said endonuclease is derived from an uncultivated microorganism, wherein said HNH domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 131, and wherein said endonuclease is a class 2, type II Cas endonuclease; and(b) an engineered guide ribonucleic acid structure that forms a complex with said endonuclease, wherein said engineered guide ribonucleic acid structure comprises: i) a guide ribonucleic acid sequence that hybridizes to a target deoxyribonucleic acid sequence; and ii) a tracr ribonucleic acid sequence that binds to said endonuclease; wherein said endonuclease comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least 80% sequence identity thereto. It is noted that the endonuclease set forth in instant SEQ ID NO: 23, shares 99.9% sequence identity with SEQ ID NO: 43 of application ‘135 (see application file for sequence alignment), and therefore is a variant of SEQ ID NO: 43 having at least 80% sequence identity thereto. Claim 112 recites wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, fungal, plant, mammalian, or human genomic sequence.
Claim 113 recites wherein said guide ribonucleic acid sequence is 15-23 nucleotides in length.
Claim 114 recites wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
Claim 118 recites wherein said sequence identity is determined by a BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
As described above, Liu teaches that targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and has the potential to provide new therapies for human genetic diseases by effecting a specific nucleotide change at a specific location in the genome. Liu further teaches that programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR) (Specification, para 0001). However, “both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration”. Accordingly, Liu teaches nucleic acid programmable DNA binding proteins (napDNAbp), including dCas9 (catalytically inactive) and Cas9 nickase (cleaving one strand of double stranded DNA), fused to a cytidine deaminase, via a linker for deaminating target cytidine residues., and a uracil glycosylase inhibitor (UGI) domain (Specification, para 0003-0008). Liu teaches that base editing (e.g., deaminating target cytidine residues) can be reversed by endogenous DNA repair mechanisms – “Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity” (para 0023). Liu teaches that these fusion proteins “generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain” (para 0006). Therefore, the inclusion of an UGI would improve efficiency of deaminating target nucleotides. Liu discloses a UGI set forth in SEQ ID NO: 178 having 100% sequence identity to instant SEQ ID NO: 18, and a cytidine deaminase set forth in SEQ ID NO: 168 having 100% sequence identity to instant SEQ ID NO: 9 (see sequence alignments below). It is noted that Liu teaches point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in dCas9 (para 0004). More specifically, Liu teaches that mutations within the HNH or RuVC subdomains “can silence the nuclease activity of Cas9”, such as D10A and H840A, which “completely inactivate the nuclease activity of S. pyogenes Cas9” (para 00214 and para 0008).
In view of claim 116 of application ‘135 which discloses an engineered nucleic acid editing system comprising a type II Cas endonuclease together with an engineered guide ribonucleic acid structure to direct the endonuclease to target a DNA sequence and a tracr sequence to bind said endonuclease, and Liu’s disclosure of nucleobase editor fusion proteins comprising programmable DNA binding proteins (including dCas9 and Cas9 nickase) linked to cytidine deaminase (SEQ ID NO: 168), for more precision editing compared to programmable cleavage that may result in unwanted gene alterations, a person of ordinary skill in the art would have been motivated to incorporate a base editor, such as SEQ ID NO: 168 into the system disclosed by application ‘135 in order to enable direct conversion of target nucleotides at specific genomic loci using sequence specific targeting, because Liu teaches the potential for treating diseases by effecting a specific nucleotide change at a specific location in the genome. Said practitioner would further be motivated to modify the endonuclease of application ‘135 in view of Liu’s disclosure of catalytically inactive Cas9 proteins, obtained by mutations in the RuvC domain of Cas9, because reduction or elimination of nuclease cleavage activity avoids formation of double strand DNA breaks and thereby reduces undesired insertions and deletions associated with cleavage based genome editing while maintaining programmable DNA binding capability. Because Liu expressly demonstrates programmable DNA-binding proteins operating in combination with deaminase editors, nickase or catalytically inactive variants, to perform targeted nucleobase editing, a person of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings with the engineered CRISPR system of application ‘135 to arrive at the claimed engineered nucleic acid editing system. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
This is a provisional nonstatutory double patenting rejection.
Pertinent Art
Zheng et al. (Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion, Commun Biol 1, 32 (2018), cited in PTO-892) teaches CRISPR/Cas9-guided base editing systems comprising a Cas9 nickase fused to a cytidine deaminase for programmable conversion of specific nucleobases in genomic DNA without introducing double-strand breaks. Zheng et al. further teaches engineered guide RNAs directing the Cas9–deaminase fusion to target loci to achieve efficient and precise single-base substitutions in bacterial cells. Zheng et al. demonstrates that base-editing fusion proteins provide an alternative to cleavage-based genome editing by enabling targeted nucleotide modification with reduced undesired mutations. Zheng et al. does not teach an endonuclease derived from an uncultivated microorganism; and
an endonuclease comprising at least 80% sequence identity to SEQ ID NO:23.
Conclusion
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NAGHMEH NINA MOAZZAMI whose telephone number is (703)756-4770. The examiner can normally be reached Monday-Friday, 9:00-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached at 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NAGHMEH NINA MOAZZAMI/Examiner, Art Unit 1652
/RICHARD G HUTSON/Primary Examiner, Art Unit 1652