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 the claims
Claims 1-8, 12, 16, 21-23, 25-30 are pending and under exam. Claims 9-11, 13-15, 17-20, and 24 are cancelled.
Drawings
The objections to drawings are withdrawn following amendments to the drawings and specification
MAINTAINED REJECTIONS
Claim Rejections - 35 USC § 112
Claims 1-8, 12, 15-16, 21-23, 25-30 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth 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 1 and 15 require “an area of a cross-section exposed as a result of alteration”. Further it is not clear what is referred as the “index.” The specification did not provide a definition of the index or whether it is a fixed value. Finally, the terms “predetermined value” is unclear. It is unclear how the value is predetermined, what factors or considered or not considered. Additionally, the claim requires the deaminase to “alter the targeted site.” It is not clear from the wording on the claim or the specification as to how the targeted site is altered. It is understood that a deaminase can remove the amino group from a molecule (in this instance, the nucleic acid). The specification does not give an exact definition of the claimed alteration. Appropriate clarifications are required.
Additionally claim 30 requires the deaminase to be smaller in size compared to the wildtype. It is not clear if the 3D size is smaller, or if the primary sequence is smaller or the solvent exposed area is smaller. Appropriate clarification.
Claim 4 requires minimization of hydrophobic residues or ratio of hydrophobic residues. The term “minimized” in claims 4-5 is a relative term which renders the claim indefinite. The term “minimized” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification only gives a general dictionary definition of the term.
Claims 8(3) and 23 (3) requires “the amino acid sequence of (1) or (2) with one or several amino acid” mutations. However, it is not clear how many amino acids make up “several.” As such the claim is unclear.
Claims 2-6, 12, 16, 21 and 30 require that the deaminase is altered. It is unclear from the language of the claim as to how the deaminase altered. It is submitted that it is also unclear if the “alteration” is a functional or structural boundary. It is noted that claim 2-3 and 16 recite that the alteration “comprises a” deletion. It is however, not clear what other mutations are encompassed by the claimed “alteration.”
Claims 2-8, 12, 15-16, 21-23, and 25-30 are rejected for their dependence on the rejected claim.
Response to Arguments: Applicants argued that the "area" or "index" thereof refers to parameters that can be calculated or measured by known methods in the art ( e.g., solvent accessible surface area calculations or molecular modeling). The "predetermined value" is a threshold that a person of ordinary skill in the art would understand as being determinable based on the desired reduction in size and hydrophobic exposure.
Applicants’ arguments have been fully considered but are not persuasive. It is submitted that the cross-sectional area or an index may well be determined by known techniques, however, it is submitted that different techniques yield different results and the specification does not give an objective standard for selecting among such techniques. Accordingly, the scope of the claims cannot be determined with reasonable certainty.
Regarding “alterations of a target site”, Applicants argued that the specification explains that a deaminase alters a targeted site by deamination. Specifically, cytidine deaminases convert cytosine (C) to uracil (U), which is then replicated as thymine (T), as described in specification paragraphs 0038-0040 of the present application. The meaning of "alter the targeted site is well established in the art. Further regarding “minimized” as used in claims 4, 5 and 16, Applicants argued that “[t]he term "minimized" is used in its ordinary meaning, i.e., to reduce to the smallest practicable amount while maintaining the desired function (i.e., deaminase activity). The specification teaches optimization of the deaminase by reducing hydrophobic residues on the exposed surface, as described in paragraphs 0016-0022 of the present application.”
It is submitted that broadest reasonable interpretation of “alterations of a target site” includes deletion, insertion, substitution, domain fusion, rearrangement among others. It is submitted that the claims do not limit the alteration to target and thus does not resolve the indefiniteness. It is pointed out that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” (See MPEP 2111). The claim cannot be interpreted to read solely on truncations.
Further, the claim does not indicate compared to which entity, the claimed parameter is “minimized,” or to what degree as used in claims 4, 5 and 16. Because different artisans could reach different conclusions regarding when the claimed minimization is achieved, the metes and bounds of the claims are unclear. It is submitted that the claims or applicant’s arguments do not resolve indefiniteness
Regarding “several” as used in claim 8, Applicants argued that this is a commonly used term and is understood to mean "more than two but not a large number." This argument is not persuasive. The scope of the claim is unclear. Applicants’ assertion that it encompasses “for example 2-10 amino acids” is not supported by the claim language or the specification.
Claims 1-8, 12, 15-16, 21-23, 25-30 remain 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.
Regarding claim 1: The claim is directed to a composition of a nucleic acid sequence recognition module bound with a deaminase that can alter a target site, where the nucleic acid sequence recognition module binds to a target nucleic acid and the deaminase is smaller than wild-type and an area of cross section exposed as a result of alteration, or an index indicating the area, is less than or equal to a predetermined value.
As such the claim encompasses all deaminases that are smaller than a wild-type that can be fused to a nucleic acid recognition domain (such as a Cas protein for example), and comprises the ability to alter a targeted site (for the sake of compact prosecution, alteration is interpreted to read on deamination) wherein the cross-sectional area is smaller. It is not clear from the specification as to how cross-sectional area of the deaminase is measured. Further, it is not clear how what factors are considered for the predetermination. It is also not understood from the claim as to how the value to determine whether it is determined by molecular weight, surface area or other measurable parameters. It appears that “predetermined value” seems arbitrary and undefined.
As a second matter, the specification only provided support for Cas9 nucleic acid recognition molecules not every single nucleic acid recognition modules such as transcription factors or polymerases among others. (See specification [0135], [0136], [0184]).
It is also noted that the specification did not provide adequate description for all possible deaminases that are smaller than wild-type that have a smaller cross-sectional area than the wild type deaminase. The specification provided support for only the Petromyzon marinus cytosine deaminase 1 or PmCDA1 that have a specific C-terminal and N-terminal deletion. More specifically, the specification provided support for a PmCDA1 deaminase that had residues from 3-161, 6-161, 13-161, 21-161 and 28 to 161 amino acids. (See specification [0159], and [0255]-[0256]) and where 91, 122, 126, 128, and 150 are altered to specific amino acids. Applicant found that deletion of the C and N termini to only encompass 3-161, 6-161, 13-161, 21-161 and 28 to 161 amino acids made the deaminase more spherical, and compact. (See Example 2). As such the specification described only one specific mutation to one specific deaminase.
It is also noted that claims 2-5, 12, 16, 21 and 30 require that the deaminase is altered. The breadth of the claim encompasses all kinds of domain swaps, random mutagenesis among others while the specification only provides description for specific kinds of alterations discussed above.
It is also noted that “fewer amino acids” compared to the wild-type deaminase lacks complete support in the specification. As indicated above, the specification only provided support for specific deletions and insertions. As such the scope of the claim is larger than the teachings and disclosure of the specification.
It is submitted that the specification only provides sufficient disclosure for a one specific compacted deaminase. The claim as drafted covers a genus that extends beyond what the Applicants have demonstrated possession of in the specification.
As such the specification lacks written description.
Regarding claim 7: The claim encompasses mutation of any hydrophobic residue to any hydrophilic amino acid residue. However, the specification only provided mutation of certain hydrophobic residues such as W122E, W133Q, W139R. The specification did not provide support for mutating any hydrophobic residue to any hydrophilic residue as encompassed by the claim.
Regarding claim 15: The claim is directed to a composition of a nucleic acid recognition molecule comprising a deaminase, where the deaminase is inserted between the N and the C-terminus of the nucleic acid recognition molecule, where the protein is properly folded and able to specifically bind a nucleic acid and alter the DNA.
The breadth of the claim encompasses all deaminases that is inserted between any two amino acids within the nucleic acid targeting sequence (for example a Cas9), where the protein is properly folded and can alter the nucleic acid. However, the as indicated above the specification only described a modified PmCDA1 deaminase wild type or one that is modified such that the PmCDA1 deaminase only comprises amino acids 3-161, 6-161, 13-161, 21-161 and 28 to 161 and/or has specific mutations at positions 91, 122, 126, 128, and 150 of the deaminase. (See Figure 8). The specification did not support all deaminases. Further, the specification only provided support for Cas9 nucleic acid recognition molecules not every single nucleic acid recognition modules such as transcription factors or polymerases among others. (See specification [0135], [0136], [0184]).
In addition to the above the specification did not provide support for insertion of the deaminase into any position within the nucleic acid targeting sequence. For example FIG. 21 showed insertion of deaminases between positions 204-205, 535-536, 1023-1024, 1023-1055 or 1054-1055 of nCas9. Thus, Applicants were not in possession of the full scope of the claimed invention at the time of filing of the instant invention.
Regarding claim 23: The claim requires an amino acid having 90% or greater sequence identity to SEQ ID NO: 1.
Applicant’s claims do not provide support for all nucleic acids comprising 90% identity to SEQ ID NO: 1. The specification as filed does not provide sufficient evidence that Applicants were in possession of the full scope of the claimed invention at the time of filing of the instant invention. As such, >90% identity to a 208 amino acid polypeptide, encompasses
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polypeptide molecules. This amounts to enormous number of polypeptides which do not have support in the specification. The specification also does not provide any guidance on how or where the nucleotides need to be changed except for 91, 122, 126, 128, and 150 of amino acid sequence between 30-150 of SEQ ID NO: 1. It is clear that the polypeptide of the invention was restricted to SEQ ID NO: 1. For example, see instant specification at [0085], [0160].
Thus, Applicants were not in possession of the full scope of the claimed invention at the time of filing of the instant invention.
Claims 2-8, 12, 16, 21-23, 25-30 are rejected for their dependence on the rejected claim.
Response to Arguments: Applicants argued that the application describes the general concept of miniaturizing deaminases by truncation and identifies structural features that are common to functional miniaturized deaminases (reduced hydrophobic exposure on the surface created by truncation) (see paragraphs 0016-0022 of the present application). While the specification provides detailed examples using PmCDA1, the disclosed principles are applicable to other AID/ APOBEC family deaminases that share similar structural characteristics. Applicants further argued that a person of ordinary skill in the art would recognize that the specification adequately describes the full scope of the combination of a nucleic acid sequence recognition module with a miniaturized deaminase. The specification teaches how to create such combinations using known components (e.g., CRISPR-Cas systems) with modified deaminases (see paragraphs 0039-0053 of the present application).
Applicant’s arguments have been fully considered but are not persuasive. Written description requirement is only satisfied if the specification reasonably conveyed to a person of ordinary skill in the art that the inventor had possession of the claimed invention at the time of filing.
It is first noted that specification primarily described PmCDA1 and specific truncation variants thereof. The specification does not disclose other deaminases meeting the claimed structural parameters nor provide a representative number of species across full scope of the claimed genus. It is pointed out that written description is not satisfied by described by general concept or research plan, rather through demonstration of possession of the claimed genus, for example, through disclosure of representative species or structural features common to the genus.
It is pointed out that neither claim amendments nor arguments address the limited number of hydrophilic residues disclosed as required by claim 7.
Claims 1-8, 12, 16, 21-23, 25-30 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement.
The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Regarding claims 1-5: Applicant's specification is found enabling for a Cas9-deaminase complex comprising SEQ ID NO: 1.
Applicant's specification is not found to be enabling for a composition of any nucleic acid sequence recognition module bound with any deaminase that can alter a target site, where the nucleic acid sequence recognition module binds to a target nucleic acid wherein the deaminase is smaller than wild-type and relative to a predetermined value with respect to its cross-sectional area.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to carry out the method of the invention commensurate in scope with the current claims.
Analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention without undue or unreasonable experimentation. See Mineral Separation v. Hyde, 242 U.S. 261, 270 (1916). The key word is 'undue,' not experimentation.' " (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all these factors are considered, a sufficient number are discussed below so as to create a prima facie case.
Applicants' claims are directed to:
any nucleic acid sequence recognition module
any deaminase
determination of a cross sectional area
arrival at a predetermined value or an index determining the cross sectional area.
The breadth of the claims includes any nucleic acid targeting sequence such as polymerases, transcription factors as well as all CRISPR enzymes such as Talens, ZFNs, and all Cas proteins and all deaminases such as APOBEC3, ADARs, Activation induced deaminases as well as other deaminases which are not yet identified or synthesized.
The specification provides support for compaction of Pm cytidine deaminase 1 by deleting N-terminal region and substitution of specific hydrophobic residues to specific hydrophilic residues (W122E; W139R; W139Q, for example) based on the 3D structure of the PmCDA1 deaminase. (Examples 1 and 2). The altered deaminase was fused to the N-terminus of Cas9 protein using a linker (Example 3) and insertion of deaminase in the Cas9 region between 1-201, 1-197, 1-190, 1-183, 1-179, 1-176, and 1-161 (Example 7).
At the time the invention was made several deaminases and nucleic acid targeting sequences were known to a person of ordinary skill in the art. The specification fails to provide specific methods to identify any and all nucleic acid targeting domains and deaminases and indicate how they can be modified to be used together. As such the specification fails to provide a generalizable composition as currently claimed.
It is submitted that the specification fails to provide a method of determining the cross section and how it is indexed and how it is reduced with respect to a predetermined value. It is known in the art that the cross section can be determined by crystallographic studies, molecular modelling, Small angle X-ray scattering or hydrodynamic radius. In all cases the protein environment and the structure has to be considered and leads to highly divergent results. As such it is understood by a person of ordinary skill in the art that cross section of proteins are not routinely determined by straightforward methods and require series of approximations assumptions and parameters, none of which have any support in the specification. (See Durham Abstract; Published 2009; PTO-892). Further there is no straightforward method or parameters to predetermine a value or index the cross sectional area in the specification. As such one of ordinary skill in the art would not be able to make and use the claimed composition without undue experimentation.
Regarding claim 7 and 22: The claim encompasses any and all mutation of any hydrophobic residue with any hydrophilic residue. The specification as indicated above provided only certain specific mutations in PmCDA1 that enabled this specific protein to be globular, which were chosen from the 3D structure of the deaminase. It is known in the art that hydrophobic residues form the core part of the protein and are important for the structure of a protein. Modifying these core, structure conferring hydrophobic residues will result in misfolding, or aggregation or loss of protein activity of a protein. As such one of ordinary skill in the art would have to mutate each hydrophobic residue into a hydrophilic residue in any deaminase to arrive at the composition which alters a target site, which requires undue experimentation by the person.
Regarding claim 8 and 23: The claim requires a protein that is an ortholog of the deaminase. The specification lists a list of possible PmCDA1 orthologs, but provides no structural analysis or experimental data showing that the described orthologs function in the claimed complex. As such the full genus of the “orthologs” lacks enablement.
Regarding claim 15: The claim requires any nucleic acid recognition sequence that had any inlaid deaminase domain. The breadth of the claims includes any nucleic acid targeting sequence such as polymerases, transcription factors as well as all CRISPR enzymes such as Talens, ZFNs, and all Cas proteins and all deaminases such as APOBEC3, ADARs, Activation induced deaminases as well as other deaminases which are not yet identified or synthesized. Additionally, the specification specifically taught insertion of deaminases between positions 204-205, 535-536, 1023-1024, 1023-1055 or 1054-1055 of nCas9. [0265] of the specification taught that “[s]tructurally, the position of 1054 amino acid in the RuvC domain of Cas9 is on a flexible protein surface, and is close to a non-target DNA strand subjected to deamination.“ As such the specification clearly taught that the insertion of a foreign domain should be carefully planned according to the 3D structure of the nCas9. To operate the claimed invention, one of ordinary skill in the art would have to analyze the 3D structure of all nucleic acid recognition sequence, find an appropriate position to insert any deaminase, and analyze its ability to target and alter a site in dsDNA. As such the breadth of the claim encompasses incorporating any deaminase into any position in any nucleic acid recognition domain. This which requires undue experimentation by a person of ordinary skill in the art.
Therefore, there was a recognized level of unpredictability with regards to all the aspects of the claims noted above.
Due to the lack of teachings in the art regarding the aspects noted above, and the recognized unpredictability in the area of protein engineering and function, a large amount of guidance and teachings would be necessary in order to be enabling for methods of such.
Guidance and teachings provided by Applicants in the instant specification is limited to disclosure SEQ ID NO: 1. The Examiner acknowledges that the Office does not require the presence of working examples to be present in the disclosure of the invention (see MPEP §2164.02). However, in light of the state of the art, discussed above, which recognizes a high level of unpredictability in the field of protein engineering for deaminases, and limited teachings with regards to the claimed composition, the Office would require appropriate disclosure to support the claimed composition. The amount of guidance or direction needed to enable the invention is inversely related to the amount of knowledge in the state of the art as well as the predictability in the art. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970). Thus, due to the high level of unpredictability in the art, the current specification would have to provide greater amounts of teachings and guidance directed to methods of carrying out the claimed invention.
Therefore, due to the sum of all the aforementioned factors, one of ordinary skill in the art, at the time the invention was made, would not expect success carrying out the claimed composition. It is submitted that the skilled artisan would be faced with the impermissible burden of undue experimentation in order to practice the claimed invention using any species of the claimed composition. Accordingly, claims 1-8, 12, 16, 21-23, 25-30 are deemed properly rejected.
Claims 6, 8, 12, 16, 21, 25-30 are rejected for their dependency on the rejected claims.
Response to Arguments: Applicants argued that the specification provides detailed guidance on how to create miniaturized deaminases, including specific examples of truncation sites and amino acid substitutions (see paragraphs 0016-0022 of the present application) and so a person of ordinary skill in the art can apply these teachings to create functional variants other than those variants specifically disclosed in the specification. Applicants further argued that methods for fusing deaminases to nucleic acid recognition modules (such as CRISPR-Cas systems) were well known in the art as of the filing date for the present application.
Applicants’ arguments are not persuasive. It is submitted that pending claims recite a broad genus encompassing essentially any nucleic acid recognition module combine with a truncated deaminase satisfying structural limitations relating to cross-sectional area or indexed values, as well as a wide-ranging substitutions, insertion configurations, orthologs and sequence identity variants. The specification however, primarily discloses specific embodiments involving Cas-based recognition modules and particular PmCDA1 truncations and mutations. It does not provide a sufficient guidance to reliably predict which combinations across full scope of the claim retain proper protein folding, functional activity and stability.
In order to practice the full scope of the claims, a skilled artisan would be required to experimentally determine suitable recognition modules, identify compatible deaminases, optimize truncation boundaries, evaluate cross-sectional parameters relative to a pre-determined value and assess numerous substitution and insertion permutations. Such extensive trial-and-error experimentation across the full breadth of the claim constitutes undue experimentation. Presence of limited examples and general design principles does not eliminate the need for substantial empirical optimization to achieve the full claimed scope.
As such Applicants arguments do no overcome the enablement rejection and the rejection is maintained.
Claim Rejections - 35 USC § 102
Claims 1-6, 8, 12 and 26-27 and 29-30 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tan et al (Nat Commun 10, 439 (2019); See IDS filed 3/3/2023; hereinafter "Tan").
Regarding claims 1-3, 8, and 12: Tan was directed to high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. (See Tan Abstract). Tan used “a nickase form of SpCas9 (nSpCas9, to stimulate cellular DNA mismatch repair) fused to a nucleobase deaminase enzyme as well as an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI).” (See Tan p. 2, col. 1, para 1). Tan “generated a series of 22 base editors with C-terminally truncated CDA1 versions fused to nCas9” to improve efficiency and specificity. (See Tan p. 4, col. 2, para 2; Figure 3). Tan indicated that large “deletions made editing more precise and substantially narrowed the activity window of the base editors” Tan taught that “[t]he enzyme tolerated truncations up to amino acid residue 158 without a significant loss in editing efficiency.” (See Tan p. 4, col. 2, para 2). While Tan did not explicitly teach or disclose a deaminase with a cross sectional area exposed as a result of alteration less than a predetermined value, it is expected by a person of ordinary skill in the art that a deaminase that is smaller as disclosed in Tan is expected to have a smaller cross sectional area as required by the claim. As such Tan disclosed all elements of the instant claims and thus anticipates it. Tan teaches a nSpCas9 as required by claim 12 (it is known that nickases have one nuclease domain inactivated) and a CDA deaminase as required by claim 8.
Regarding claims 4-5: Tan did not specifically disclose minimization of hydrophobic residues. However as described above Tan “generated a series of 22 base editors with C-terminally truncated CDA1 versions fused to nCas9” to improve efficiency and specificity. (Ref; Figure 3). It is noted that the C-terminus of CDA1 comprises hydrophobic restudies and it is submitted that Tan teaches reducing the number of hydrophobic residues.
Regarding claim 6: Tan disclosed that they “tested the impact of N- and C-terminal truncations (removing potential linker-like fragments) on base editing.” (See Tan, p. 4, col. 2 para 1).
Regarding claim 26-27: Tan discloses a nucleic acid and vector comprising nucleic acid according to claim 1. (See Tan p. 8, col. 2, para 3).
Regarding claims 29-30: Tan discloses canavanine reversion mutations using the nucleic acid of claim 1. (See Tan Figure 3; p. 9, col. 1, para 1). As such Tan disclosed a method of altering a dsDNA using the nucleic acid of claim 1.
Claims 15 and 23 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang et al (Signal Transduct Target Ther. 2019 Sep 20; hereinafter "Wang;" See PTO-892).
Regarding claims 15 and 23: Wang disclosed a “novel editing system based on the position information of the Cas9/DNA/ sgRNA complex.” (See Wang, p. 1, col. 1, 2nd para). Wang disclosed a Cas9 construct where APOBEC1, a cytidine deaminase (as required by claim 23) was inserted inside the coding region of a SpyCas9 protein. (See Wang Fig. 1b BE-PIGS and BE-RuvCGE). Wang further demonstrated that these constructs were active, (See Fig. 1d). As such Wang disclosed all elements of the claim and thus anticipates it.
Response to Arguments: Applicants argued that Tan does not anticipate the subject claims as it does not expressly disclose “an area of a cross-section exposed as a result of alteration as compared to the wild-type deaminase or an index indicating the area is less than or equal to a predetermined value.” Applicants further argued that Tan did not teach truncation based on cross sectional parameter or predetermined threshold, and therefore does not disclose this structural limitation.
Applicants’ arguments are not persuasive. As set forth in the rejection, Tan expressly discloses CRISPR-based base editors comprising a nickase spCas9 fused to cytidine deaminase variants, including C-terminal truncations of CDA-1 and demonstrated progressive truncation narrows the editing window and improves precision. Tan therefore discloses the claimed nucleic acid recognition module bound to deaminase and capable of altering a targeted site in double-stranded DNA.
With respect to the cross-sectional area limitation, Applicant’s argument focusses on the absence of an explicit numerical predetermined value in Tan. However, anticipation does not require verbatim disclosure of claim terminology. “The elements must be arranged as required by the claim, but this is not an ipsissimis verbis test, i.e., identity of terminology is not required. In re Bond, 910 F.2d 831, 15 USPQ2d 1566 (Fed. Cir. 1990). “ (See MPEP 2131). Tan disclosed that truncated deaminase variants relative to wild-type CDA1. A truncated protein necessarily has reduced amino acid length and altered structural dimensions compared to a full-length enzyme. To the extent that the claim is construed broadly, such that the “predetermined value” encompasses a threshold reflecting reduced structural cross-sectional characteristics relative to wild-type, Tan’s truncated CDA1 variants inherently satisfy this limitation. The reference does not need to recognize or describe the cross-sectional parameter for inherency to apply; it is sufficient that the structural consequence is necessarily present.
Applicant’s assertion that Tan does not design truncations according to a cross sectional metric does not overcome the rejection, because anticipation does not require the reference disclose the same design rationale as the Applicant. The question is whether the structural limitations of the claim are met by prior art disclosure. Here, Tan’s truncated deaminase embodiments necessarily possess reduced structural dimensions relative to wildtype CDA1 and therefore fall within the scope of the claimed limitation under a reasonable interpretation.
Further regarding Wang, Applicant argued that Wang does not anticipate the claim as Wang did not disclose specific structural arrangement wherein the N-terminal and C-terminal fragments of the nucleic acid sequence recognition module are "refolded" with the deaminase inserted therebetween. Applicants’ arguments are not persuasive. Wang disclosed an inlaid Cas-9-deaminase fusion within the coding sequence of SpyCas9, and the resulting protein is functional in targeted base editing. For such an internally modified Cas9 protein to retain DNA-binding and editing activity, it must necessarily adopt a functional 3D conformation. This even if Wang does not explicitly use the term “refolded,” the structural and functional requirements of the disclosed construct inherently require that the N-and C-terminal regions form a properly folded, active configuration. As indicated above, anticipation does not require verbatim disclosure of claim terminology. Further inherency does not require express recognition or discussion in the reference. It is sufficient that the limitation is necessarily present in the disclosed structure. Because Wang disclosed a functional inlaid Cas9-deaminase complex that necessarily achieves the claimed structural relationship, Applicant’s arguments do not overcome the anticipation rejection.
Claim Rejections - 35 USC § 103
Claim 7 remains rejected under 35 U.S.C. 103 as being unpatentable over Tan et al (Nat Commun 10, 439 (2019); See IDS filed 3/3/2023; hereinafter "Tan") in view of Harjes et al (J Mol Biol. 2009 Jun 26; hereinafter "Harjes;" See PTO-892).
Regarding claim 7: The teachings of Tan are set forth above. Tan did not teach a truncated deaminase and a nucleic acid recognition module with hydrophobic amino acid substitution in the truncated deaminase. Harjes taught an APOBEC deaminase “[d]erivatives with five amino acid substitutions, L234K, C243A, F310K, C321A and C356A (2K3A), were also made to enable concentrated protein preparations for NMR studies.” (ref). Harjes clearly taught that the substitutions enabled improved solubility and stability of the deaminase protein. A person of ordinary skill in the art would have found it obvious to apply the substitutions to eh inlaid deaminase of Wang to improve the protein stability and solubility. Given that the references demonstrate predictable functional results there would have been reasonable expectation of success in combining the teachings of cited art.
Claim 16 remains rejected under 35 U.S.C. 103 as being unpatentable over Wang et al (Signal Transduct Target Ther. 2019 Sep 20; hereinafter "Wang;" See PTO-892) in view of Jin et al (Mol Cell. 2020 Sep 3; hereinafter "Jin;" See PTO-892).
Regarding claim 16: The teachings of Wang are set forth above. Wang did not teach a truncated or modified deaminase for insertion within a Cas9 protein. Jin optimized and validated the orthogonal R-loop assay for application in plants and used it to evaluate the sgRNA-independent off-target activities of a series of A3Bctd deaminase (a truncated human APOBEC3B cytidine deaminase) variants. It would have been obvious to a person of ordinary skill in the art to use a truncated or modified deaminase as taught by Jin in the inlaid Cas9architecture taught by Wang. It is noted that both Wang and Jin are directed to base editing by deamination. The person would be motivated to use the modified deaminase of Jin in the Cas9-deaminase architecture of Jin as Jin taught higher activity of the modified/truncated APOBEC3B compared to wildtype. The person would have a reasonable expectation that the modified deaminase would yield higher performance in view of teachings of Jin.
Claim 21 and 25 remain rejected under 35 U.S.C. 103 as being unpatentable over Wang et al (Signal Transduct Target Ther. 2019 Sep 20; hereinafter "Wang;" See PTO-892) in view of Tan et al (Nat Commun 10, 439 (2019); See IDS filed 3/3/2023; hereinafter "Tan").
Regarding claim 21: The teachings of Wang are set forth above. Wang did not teach a N and C termini truncated or modified deaminase for insertion within a Cas9 protein. Tan taught a CDA1 that was truncated at the N and C termini as indicated above. A person of ordinary skill in the art would be motivated to substitute the deaminase domain of Tan in the inlaid Cas9-deaminase architecture taught by Wang. Wang taught higher activity with the inlaid Cas-deaminase architecture (See Fig. 1d). Wang taught that the inlaid architecture (BE-PIGS) has a “wider editing window than that of BE3 or BE4.” (ref). Tan achieved a compact and more efficient deaminase by their modifications. Tan indicated that large “deletions made editing more precise and substantially narrowed the activity window of the base editors” As such a person of ordinary skill in the art would have combined the teachings of Tan and Wang to generate a deaminase that was truncated at both ends (such as the deaminase taught by Tan) in the inlaid architecture taught by Wang to narrow the editing window or to achieve an efficient editing machinery as taught by Tan. The person would have a reasonable expectation of success in view of the teachings of Tan that the truncated deaminase retains function.
Regarding claim 25: The teachings of Wang are set forth above. Wang did not teach a CRISPR-Cas system wherein at least one DNA cleaving capability of a Cas protein is inactivated as required by the claim. Tan taught truncated CDA1 fused to a Cas9 nickase and shows that to inactivating one nuclease domain improved editing precision and reduced double stranded breaks. A person of ordinary skill in the art would have been motivated to inactivate one nuclease domain in the Wang inlaid deaminase architecture to achieve combined benefits of Wang’s improved efficiency and Tan’s improved precision. Given that Tan demonstrated preserved deaminase activity with a truncated deaminase and inactivated Cas9, there would have been a reasonable expectation of success in producing a functional inlaid Cas9-deaminase with at least one nuclease domain inactivated.
Claim 22 remains rejected under 35 U.S.C. 103 as being unpatentable over Wang et al (Signal Transduct Target Ther. 2019 Sep 20; hereinafter "Wang;" See PTO-892) in view of Harjes et al (J Mol Biol. 2009 Jun 26; hereinafter "Harjes;" See PTO-892).
Regarding claim 22: The teachings of Wang are set forth above. Wang did not teach an in laid deaminase with hydrophobic amino acid substitution. Harjes taught an APOBEC deaminase “[d]derivatives with five amino acid substitutions, L234K, C243A, F310K, C321A and C356A (2K3A), were also made to enable concentrated protein preparations for NMR studies.” (ref). Harjes clearly taught that the substitutions enabled improved solubility and stability of the deaminase protein. A person of ordinary skill in the art would have found it obvious to apply the substitutions to eh inlaid deaminase of Wang to improve the protein stability and solubility. Given that the references demonstrate predictable functional results there would have been reasonable expectation of success in combining the teachings of cited art.
Claim 28 remains rejected under 35 U.S.C. 103 as being unpatentable over Tan et al (Nat Commun 10, 439 (2019); See IDS filed 3/3/2023; hereinafter "Tan") in view of Lau et al (F1000Res. 2017 Dec 20; See PTO-892; hereinafter “Lau”).
As indicated above, Tan discloses a nucleic acid and vector comprising nucleic acid according to claim 1. (See Tan p. 8, col. 2, para 3). However, Tan did not teach that the vector is an adenoviral vector. It is submitted as taught by Lau “With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing.“ (See Lau Abstract). As such one of ordinary skill in the art would have been motivated to combine the teachings of Lau and Tan to generate an AAV vector in order to package, deliver, and express CRISPR components for targeted gene editing. As such the claim is obvious.
Response to Arguments: Applicants argued that the cited references address different problems, do not teach the specific design principle of controlling exposed cross-sectional area and would not have been combined with a reasonable expectation of success. These arguments are not persuasive.
The references are directed to the same field of endeavor: CRISPR-based base editing systems and collectively teach truncation of deaminases, internal and external insertion into Cas architectures, hydrophobic residue substitutions, nuclease inactivation and vector-based delivery. A reference need not address the identical problem articulated in the specification for its teachings to be combined; rather the relevant inquiry is whether a person of ordinary skill in the art would have been motivated to combine the references and prior art elements according to known methods to yield predictable results. The structural modifications relied upon in the rejections: truncations, substitutions, inactivation of nuclease activity are routine protein engineering strategies expressly taught in the prior art, and the prior art demonstrated that such modifications preserve or improve base editing base editing functions. Accordingly, the proposed combinations represent predictable use of prior art elements to achieve expected improvements in stability, precision or performance and therefore a reasonable expectation of success. As such obviousness is maintained.
WITHDRAWN REJECTIONS
Further, the term “smaller” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 16 recites that a deaminase that has a smaller size. The term “smaller” in claim 16 is a relative term which renders the claim indefinite. The term “smaller” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
This rejection is withdrawn form claims 1 and 16, due to cancellation of the term “smaller.”
Claims 1-6, 12, 16, 21 and 30 require that the deaminase is altered. It is unclear from the language of the claim as to how the deaminase altered. It is submitted that it is also unclear if the “alteration” is a functional or structural boundary.
This rejection is withdrawn form claims 1 and 16, due to cancellation of the term “deaminase is altered.”
NEW REJECTIONS NECESSITATED BY CLAIM AMENDMENTS
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 1-8, 12, 15-16, 21-23, 25-30 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth 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.
The term “fewer amino acids” in claims 1 and 16 is a relative term which renders the claim indefinite. The term “fewer” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claims 2-8, 12, 15-16, 21-23, and 25-30 are rejected for their dependency.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JAGAMYA NMN VIJAYARAGHAVAN/ Examiner, Art Unit 1633
/EVELYN Y PYLA/Primary Examiner, Art Unit 1633