DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07/28/2026 has been entered.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1, 2, 4, 9, 16, 17, 21, 23, 26, 61, 62, and 65 are pending.
Claim 1 is currently amended.
Claim 15 is currently canceled.
Claims 1, 2, 4, 9, 16, 17, 21, 23, 26, 61, 62, and 65 are examined on the merits.
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.
Claims 1, 2, 4, 9, 16, 17, 21, 23, 26, 61, 62, and 65 are rejected under 35 U.S.C. 103 being unpatentable over Zhang (Feng Zhang, US20200283755A1, Application 2018-05-18, Publication 9-10-2020) in view of Sato (Moritoshi Sato et. al., US 2022/0333089 A1, Application 2019-11-01, Publication 2022-10-20) and Dong (De Dong et. al., Nature (2016) Vol 352, pp522-526).
Under the broadest reasonable interpretation, claim 1 recites an engineered protein comprising: (1) a first polypeptide that is a first portion of a modified protein, where the modified protein: has at least 80%-95% identity to SEQ ID NO: 180, and relative to SEQ ID NO: 180, comprises at least one mutation at one or more of the recited amino acid positions; (2) a second polypeptide that: is heterologous to the first polypeptide and to the third polypeptide, and comprises a first nuclease domain, wherein the first nuclease domain comprises an HNH domain (3) a third polypeptide that is a second portion of the modified protein; wherein: (a) the first portion has a length of about 200 to about 400 amino acids of the modified protein; (b) the second portion has a length of about 800 to about 1,100 amino acids of the modified protein; (c) the first, second, and third polypeptides are different from each other; (d) the second polypeptide is heterologous to the first and third polypeptides; and (e) the first polypeptide and/or the second polypeptide comprise at least one mutation.
Zhang teaches an engineered CRISPR-Cpf1 protein system for targeted nucleic acid editing (Abstract). In particular, Zhang teaches that a CRISPR-Cas protein, including Cpf1, may be engineered by insertion of a heterologous protein sequence into an internal loop or unstructured region of the CRISPR-Cas protein (paragraph 0076, 0077 and 0267), Zhang further teaches “Fusion proteins may without limitation include for instance fusions with heterologous domains or functional domains (e.g. localization signals, catalytic domains, etc.). In certain embodiments, various different modifications may be combined (e.g. a mutated nuclease which is catalytically inactive and which further is fused to a functional domain), such as for instance to induce DNA methylation or another nucleic acid modification, such as including without limitation a break (e.g. by a different nuclease (domain))” (paragraph 0268), Zhang further teaches that, in certain embodiment, a functional domain may be inserted into the CRISPR-Cas protein, such as an adenosine deaminase protein (claim 1), thereby forming a chimeric protein having portions of the original CRISPR-Cas protein separated by the inserted sequence. Zhang further teaches that suitable split positions may be identified in internal loops or unstructured regions where interruption does not disrupt structural elements, and that such sites are preferred for splits and insertions of small protein sequences and can be used to generate chimeric proteins between Cpf1 orthologs (paragraph 0076-0078).
Zhang teaches: “the enzyme is modified by mutation of one or more residues including but not limited positions, including K116, K121, E159, K591, and K601, with reference to amino acid position numbering of LbCpf1- also named as LbCas12a from Lachnospiraceae bacterium ND2006”. The amino-acid sequence alignment of LbCas12a from Lachnospiraceae bacterium ND2006 with SEQ ID NO: 180 establishes the correspondence between the LbCpf1 sequence and SEQ ID NO: 180, such that the mutations taught by Zhang may be mapped to the corresponding positions of SEQ ID NO: 180 (below). The Cpf1 enzymes comprising said one or more mutations have modified, more preferably increased specificity for the target” (paragraph 0387). In some embodiments, the CRISPR-Cas protein is a dead Cpf1, and the dead Cpf1 comprises a mutation in the RuvC domain (paragraph 0069).
Zhang does not teach or identify a specific internal Cpf1 insertion position that would provide a first portion having about 200-400 amino acids and a second portion having about 800-1,000 amino acids, nor does Zhang specifically teach that the inserted nuclease domain comprises an HNH domain.
Sato teaches engineering Cpf1 by dividing the protein at selected internal positions. Sato expressly teaches that the N-terminal and C-terminal Cpf1 fragments are preferably divided outside the RuvC or UK nuclease domains at regions joining secondary-structure elements, such as loop regions oriented toward the outside of the Cpf1 molecular (pa0183). Soto expressly identities positions 265-296 and 309-312 of LbCpf1 among suitable regions at which the protein may be divided (pa0184-0185). Sequence alignment of the LbCpf1 sequence disclosed by Sato with SEQ ID NO: 180 of the instant application shows 100% amino acid sequence identity over the aligned sequence, with the Sato LbCpf1 sequence containing an additional 11 amino acids at the C-terminus (See alignment below). Accordingly, Sato’s amino acid positions 265-296 correspond directly to positions 265-296 of SEQ ID NO: 180. Thus, selecting an internal position within residues 265-296 would provide an N-terminal Cpf1 portion within the claimed about 200-400 amnion acids and remaining C-terminal portion within the claimed about 800-1,100 amino acids, as recited in claim 1.
Sato further teaches that functional domains may be associated with split Cpf1 and expressly identifies nuclease among suitable functional domains (pa0146)
Accordingly, with respect to claim1:
A first polypeptide that is a first portion of a modified protein. Taught by Zhang, which discloses engineering Cpf1 by insertion of a heterologous sequence, thereby generating a modified protein having an N-terminal portion.
A second polypeptide that is heterologous to the first and third polypeptides and comprises a first nuclease domain. Zhang teaches insertion of a heterologous functional domain, including a different nuclease domain, into Cpf1, and Sato expressly identities nuclease domains as suitable functional domains for association with split Cpf1. Thus, the inserted heterologous nuclease-containing polypeptide corresponds to the claimed second polypeptide.
A third polypeptide that is a second portion of the modified protein. Taught by Zhang, which disclosed that insertion separates the Cpf1 protein into portions, including a C-terminal portion.
The first, second, and third polypeptides are different from each other. Taught by Zhang, wherein the inserted heterologous sequence differs from the native Cpf1 portions.
The second polypeptide is heterologous to the first and third polypeptides. Expressly taught by Zhang.
The first polypeptide and/or the second polypeptide comprise at least one mutation. Taught by Zhang, which discloses mutations at multiple amino acid positions in Cpf1.
The first portion has a length of about 200 to about 400 amino acids and the second portion has a length of about 800-1,100 amino acids. Sato expressly identifies LbCpf1 residues 265-296 as a suitable internal division region, resulting in N-terminal and C-terminal Cpf1 portions falling within the claimed size ranges (pa0184-0185)
Zhang and Sato do not specifically teach that the first nuclease domain is an HNH domain.
Dong teaches structure of CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) (Abstract). Dong teaches that Cas9 comprises two nuclease domains, HNH and RuvC, wherein HNH cleaves the target (complementary) DNA strand and RuvC cleaves the non-target strand, while Cpf1 contains only a RuvC nuclease domain and lacks HNH (page 522, left column, paragraph 1 and 3). Thus, Dong teaches that HNH is a distinct catalytic nuclease domain having target-strand cleavage activity. Accordingly, HNH was a known catalytic nuclease domain available to one of ordinary skill in the art.
It would have been obvious to a person of ordinary skill in the art to select an internal position within the 265-296 region taught by Sato for placement of the heterologous nuclease domain taught by Zhang because Sato expressly identifies that region as suitable for dividing LbCpf1 and teaches selecting outwardly oriented loop regions outside the endogenous nuclease domains. Such section would have predictably provided the claimed first and third Cpf1 portions having the recited approximate lengths.
It further would have been obvious to employ the HNH nuclease domain taught by Dong as the heterologous nuclease domain because Zhang teaches insertion of a different nuclease domain into Cpf1, Sato expressly identifies nuclease domains as suitable functional domains for split Cpf1, and Dong identities HNH as a known catalytic nuclease domain having DNA-cleavage activity. Selection of the known HNH nuclease domain as the nuclease domain contemplated by Zhang and Sato would therefore have been a predictable selection of a known nuclease domain for its known DNA-cleavage function. Thus, the combination would have provided the claimed internally positioned heterologous polypeptide comprising an HNH nuclease domain between the first and third Cpf1 portions.
Accordingly, Claim 1 is prima facie obvious over Zhang in view of Sato and further in view of Dong.
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Claim 2 is interpreted as requiring that the engineered protein of claim 1 comprises at least one amino acid substitution selected from a specifically enumerated list of substitutions at defined residue positions “…G532R….. K538V…. Y542R..” relative to SEQ ID NO:180.
Zhang teaches, “the Cpf1 protein is modified…. or at position 532, 538, 542, and/or 595 of LbCpf1, ….. or preferably mutated amino acid residues at positions 532 and 538 (and optionally 542), wherein said mutations preferably are 532R and 538V (and optionally 542R), such as G532R and K538V (and optionally Y542R) (paragraph 0392).
Accordingly, claim 2 is obvious over Zhang, Sato and Dong.
Claim 4 is drawn to claim 1 and further comprising a mutation in a nuclease active site.
Zhang teaches, “In some embodiments, the CRISPR-Cas protein is a dead Cpf1 which comprises a mutation in the RuvC domain”(paragraph 0069).
Sato similarly teaches nuclease-inactive Lbcpf1 obtained by mutation of the nuclease active center, including D832A, E925A, or D1180A (pa0069).
Claim 9 is drawn to claim 1, wherein the third polypeptide comprises a second nuclease domain that is active or inactive.
For the same reason set forth above with respect to claim 1, Zhang teaches that CRISPR-associated proteins may be engineered to include heterologous domains, including nuclease domains, and further teaches that nuclease domains in CRISPR effector proteins may be rendered catalytically inactive by mutation, while such domains may also remain intact and active (paragraph 0069). Accordingly, Zhang explicitly teaches the limitation of a nuclease domain that is either active or inactive.
Sato further teaches both nuclease-active and nuclease-inactive Cpf1 proteins and expressly teaches dividing Cpf1 into N-terminal and C-terminal portions.
The claimed invention in claims 4 and 9 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Claim 16 is drawn to claim 1, wherein the second polypeptide is between two amino acids that are two consecutive amino acids of the modified protein.
For the same reasons set forth above with respect to claim 1, it would have been obvious to place the second polypeptide at an internal insertion site between two consecutive amino acids as a position within the LbCpf1 265-296 region expressly identified by Sato as suitable for division (pa0184-0185).
Claim 17 is drawn to claim 1, wherein the second polypeptide is positioned in a location that corresponds to an interdomain linker region of the modified protein.
For the same reasons set forth above with respect to claim 1, the claimed invention in claims 16 and 17 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Claim 23 is drawn to claim 1, further comprising a first linker between the first polypeptide and the second polypeptide and/or a second linker between the second polypeptide and the third polypeptide.
For the same reasons set forth above with respect to claim 9, Zhang teaches that fusion proteins formed from two separate proteins typically involve the use of spacers or linkers. The Cpf1 protein can be fused to the catalytic domain thereof on either the N- or C-terminal end thereof. Suitable linkers for use in the methods are well known to those of skill in the art (paragraph 0058-0060).
Sato additionally teaches that a functional domain may be bound to a Cpf1 fragment though a linker and specifically teaches a flexible linker comprising glycine and serine residues (pa0154-0155).
Accordingly, claim 23 is obvious over Zhang Sato and Dong.
Claim 26 is drawn to claim 1, wherein the engineered protein comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence of a wild-type CRISPR-Cas effector protein. Claim 61 is drawn to claim 1, wherein the engineered protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any one of SEQ ID NOs:181-192.
Claim 26 expands scope by allowing the engineered protein to have 70-99% identity to any wild-type CRISPR-Cas effector protein, not limited to SEQ ID NO: 180. Claim 61 further expends scope to Cpf1 mutant lines.
Zhang teaches engineered Type V CRISPR-Cas effector proteins (e. g., AsCpf1, LbCpf1, and FnCpf1) with amino acid substitutions, insertions or deletions, where the resulting modified protein retains at least 60%, 70%, 80%, 85%, 90%, 95% , or higher sequence identity to the wild-type Cas effector protein while improving activity, specificity, or functional attributes (paragraph 0368-0370). SEQ ID NOs: 181-192 correspond to engineered Cpf1 mutant variants differing from the parent Cpf1 protein by only 1-3 amino acid substitutions. One of ordinary skill in the art would have understood that, in a protein of over 1000 amino acids, 1–3-point mutations present minor sequence variations that do not affect the overall sutural scaffold.
Accordingly, the claimed invention in claims 26 and 61 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Claim 62 is drawn to a complex comprising: the engineered protein of claim 1; a guide nucleic acid; and a deaminase. Claim 65 recites a method of modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with: the engineered protein of the engineered protein of a guide nucleic acid, wherein the engineered protein and the guide nucleic acid form a complex or are comprised in a complex, thereby modifying the target nucleic acid.
For the same reasons set forth above with respect to claim 1, Zhang teaches “Preassembled recombinant CRISPR-Cpf1 complexes comprising Cpf1 and crRNA” (paragraph 0347), Zhang further teaches a CRISPR-Cpf1 gene-editing system and method comprising a Cpf1 nickase protein (claim 1), a guide molecule (guide nucleic acid) (claim 47), and a n adenosine deaminase protein (claim 13), wherein the guide molecule forms a complex with the Cpf1 protein and directs the complex to a target nucleic acid for modification (claim 1 and claim 27).
Sato additionally identities deaminases as suitable functional domains for Cpf1 (pa0146).
The claimed invention in claims 62 and 65 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Claim 21 is rejected under 35 U.S.C. 103 being unpatentable over Zhang (2020) in view of Sato (2022), Dong as applied to claim 1, and further in view of Naqvi (Mohsin M. Naqvi et. al., Nature Chemical Biology (2022) vol 18, pp1014–1022).
Claim 1 as the teachings of Zhang, Sato and Dong are discussed above.
Claim 21 is interpreted as dependent of claim 1.
Claim 21 is drawn to claim 1, wherein the engineered protein comprises a Rec domain and a Rec2 domain and the second polypeptide is between the Rec1 domain and the Rec2 domain.
As discussed above with respect to claim 1. Zhang teaches engineering Cpf1 by inserting a heterologous functional polypeptide, including a catalytic or nuclease domain, into an internal loop or unstructured region of the Cpf1 protein. Sato further teaches selecting internal Cpf1 division sites at outwardly oriented loop regions outside the endogenous nuclease domains and expressly identifies LbCpf1 positions 265-296 as a suitable division region. Dong teaches the HNH domain as a catalytic nuclease domain, as discussed above with respect to claim 1.
Naqvi teaches Lachnospiraceae bacterium ND2006 Cas12a (Cpf1) protein includes a “linker” region, described as a loop connecting the reC1 and reC2 lobes, and teaches the linker. (See e.g., Extended Data Fig. 1 “Domains, ternary structure, and key mechanistic elements of Lachnospiraceae bacterium ND2006 Cas12a”) (also see below). Naqvi further identifies this linker region as spanning approximately amino acid position 276-288. This linker region effectively divides the LbCpf1 protein into Two portions. Thus, the REC1/REC2 linker identified by Naqvi fails within the 265-295 Cpf1 division region expressly taught by Sato.
Accordingly, it would have been obvious to one of ordinary skill in the art to position the heterologous HNH-containing polypeptide of Zhang and Dong at an internal position within the 265-296 region taught by Sato, and particularly within the REC1/REC2 linker identified by Naqvi, because Sato expressly identifies that region as suitable for division of LbCpf1 and Naqvi identifies the corresponding region as the linker between the REC1 and REC domains. The resulting engineered protein would therefore comprise the second polypeptide positioned between the REC1 and REC2 domains, as recited in claim 21.
Accordingly, claim 21 is prima facie obvious over Zhang in view of Sato and Dong, and further in view of Naqvi.
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Response to Applicant’s Remarks:
Applicant argues that Zhang does not identify an internal Cpf1 position within the claimed 200-400 amino acid range as suitable for insertion, that Naqvi merely identifies a functional REC1/REC2 linker, and that Dong does not provide a reason to insert an HNH domain into Cpf1.
These arguments have been considered but are not persuasive in view of Sato. Sato expressly teaches that LbCpf1 may be divided at outwardly oriented loop regions and specifically identities residues 265-296 as a suitable division region (pa0183-0185). Sato further teaches that functional domains associated with split Cpf1 may include nuclease (pa0146). Thus, Sato provides both the claimed positional guidance and a reason to employ an additional nuclease domain, while Dong is relied upon for teaching HNH as a known CRISPR catalytic nuclease domain.
Accordingly, the present rejection does not rely on Naqvi merely to infer that the REC1/REC2 linker is a suitable insertion site. Sato directly addresses that deficiency, and Applicant’s arguments therefore do not overcome the rejection.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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