DETAILED ACTION
Status of the Application
Claims 1-5, 7-8, 10-11, 13, 15, 17-20, 22, 25-30, 32-33, 35, 37-40, 42-44, 46-50, 52 and 54 are pending.
A preliminary amendment cancelling claims 6, 9, 12, 14, 16, 21, 23-24, 31, 34, 36, 41, 45, 51, and 53 and amending claims 4-5, 7-8, 11, 13, 15, 17-20, 22, 25, 29-30, 32-33, 35, 37-40, 42-44, 46, 48, 50, and 52 as submitted in a communication filed on 01/13/2025 is acknowledged.
Priority
This application is a CON of PCT/US2023/066501 filed on 05/02/2023 and acknowledgement is made of a claim for domestic priority under U.S.C. 119(e) to provisional application No. 63/337,386 filed on 05/02/2022 and 63/358,306 filed on 07/05/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/31/2024 is acknowledged.
The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the
information disclosure statements are being considered by the examiner.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Hyperlinks are found in paragraph [0283] on page 82, paragraph [0287] on page 85, paragraph [0321] on page 99, paragraph [0420] on page 136, paragraph [0716] on page 240, paragraph [0717] on page 240, paragraph [0708] on page 237, paragraph [0760] on page 253, paragraph [0225] on page 64, paragraph [0287] on page 84, and paragraph [0438] on page 140. Correction is required.
Claim Objections
Claims 4, 5, 8, 27-30, 33, 35, 39, 48 and 52 are objected to because of the following informalities:
Claims 4, 5, 29, and 30 are objected to in the recitation of “TPR-CHAT”. “TPR-CHAT” should be replaced with “tetracopeptide repeat (TPR) – Caspase HetF Associated with TPRs (CHAT)” in its first appearance in the claim set. Appropriate correction is required.
Claim 8 is objected to in the recitation of “Cas 7”. Claim 7 which claim 8 is dependent therefrom, recites “Cas7”. Applicants should amend to be consistent with claim 7. Appropriate correction is required.
Claims 27, 28 and 35 are objected to in the recitation of “peptidase recognition motif” and “protease recognition motif”. The terms should be used consistently or expressly identified as synonymous. Appropriate correction is required.
Claim 33 is objected to in the recitation of “Cas 7”. Claim 32 which claim 33 is dependent therefrom, recites “Cas7”. Applicants should amend to be consistent with claim 32. Appropriate correction is required.
Claim 39 is objected to in the recitation of “elements (i) and (iii), of the detection composition”. Please revise to remove the comma so the claim reads, “elements (i) and (iii) of the detection composition”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-3, 28, 32-33, 42, 48-50, 52 and 54 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 2 (claim 3 dependent therefrom) in the recitation of “in effective proximity to”. The phrase is a relative term for which the claim does not provide an objective boundary. It is unclear what spatial, sequence, structural, or functional relationship between the target polypeptide interaction site and the peptidase recognition motif constitutes “effective proximity”. Appropriate correction is required.
Claim 3 is indefinite in the recitation of “comprises or consists of SEQ ID NO: 3 or a sequence therein”. The expression does not define the boundaries of the alternative “a sequence therein”. It is unclear whether the phrase encompasses any contiguous fragment of SEQ ID NO: 3, only fragments having peptidase-recognition activity, fragments of a particular length, or other sequences. Accordingly, the scope of the recited peptidase recognition motif cannot be determined with reasonable certainty. Appropriate correction is required.
Claim 28 is indefinite in the recitation of “comprises or consists of SEQ ID NO: 3 or a sequence therein”. The expression does not define the boundaries of the alternative “a sequence therein”. It is unclear whether the phrase encompasses any contiguous fragment of SEQ ID NO: 3, only fragments having peptidase-recognition activity, fragments of a particular length, or other sequences. Accordingly, the scope of the recited peptidase recognition motif cannot be determined with reasonable certainty. Appropriate correction is required.
Claim 32 (claim 33 dependent therefrom) recites the limitation “The composition of claim 31”. Claim 31 was canceled. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
Claim 42 is indefinite in the recitation of “along with any elements of the detection composition not already expressed by the engineered cells”. The phrase fails to identify which elements (i)-(iv) must be introduced, whether every nonexpressed element is required, and how or when each element is provided relative to the engineered cells. Therefore, the method steps and scope of the claimed screening method cannot be determined with reasonable certainty. Appropriate correction is required.
Claim 48 (claims 49-50, 52 and 54 dependent therefrom) recites the limitation "programmable nuclease system". Claim 1, from which claim 48 depends from, recites “a programmable nuclease-peptidase composition”. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
Claim 48 (claims 49-50, 52 and 54 dependent therefrom) recites “wherein the substrate of the peptidase is optionally tethered to a cellular structure and wherein the substrate the peptidase is coupled to an effector”. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
Claim 52 recites the limitation "programmable nuclease system". Claim 1, from which claim 48 (claim 52 dependent therefrom) depends from, recites “a programmable nuclease-peptidase composition”. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
Claim 52 recites “cleaving the substrate by the peptidase”. Claim 1, from which claim 48 (claim 52 dependent therefrom) depends from, does not recite a substrate. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
Claim 54 recites “substrate is tethered”. Claim 1, from which claim 48 (claim 54 dependent therefrom) depends from, does not recite a substrate. There is insufficient antecedent basis for this limitation in the claim. Appropriate correction is required.
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 1-5, 7-8, 10-11, 13, 15, 17, 18-20, 22, 25, 48-50, 52 and 54 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.
Claim 1 recites “A programmable nuclease-peptidase composition comprising:
a repeat-associated mysterious protein (RAMP) polypeptide; a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence specific binding of the complex to a target polynucleotide; and a peptidase capable of binding to the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide”.
Claim 1 is not limited to a particular RAMP polypeptide, peptidase, guide molecule, target polypeptide or target polynucleotide. Rather, it encompasses RAMP polypeptides and peptidase from any source or CRISPR subtype, as well as any target polypeptide with which the peptidase binds or interacts upon target polynucleotide binding.
The specification, however, does not provide adequate written description for this broad genus. The specification describes a Type III-E gRAMP/CHAT system from Desulfonema ishimotonii including target RNA dependent interaction with and cleavage of an Up1 substrate, the scope of claim 1 is not limited to that Type III-E gRAMP/TPR-CHAT/Up1 system, to a TPR-CHAT peptidase, to a peptidase having SEQ ID NO: 1 or a defined level of identity thereto, or to an Up1 derived target polypeptide. Claim 1 instead encompasses any RAMP polypeptide combined with any peptidase capable of binding the RAMP polypeptide. guide molecule, or RAMP guide molecule complex, in which target polynucleotide initiates binding and/or interaction of the peptidase with any target polypeptide.
The specification does not describe a representative number of species across this broader claimed genus. The working examples are directed to the D.ishimotonii Type III-E gRAMP-CHAT system and its Up1 substrate. The specification does not identify additional RAMP/peptidase families outside the disclosed Type III-E/TPR-CHAT context that exhibit the claimed target polynucleotide initiated peptidase target polypeptide interaction and does not identify structural features common to full claimed set of RAMP-peptidase pairs that correlate with the claimed function.
Accordingly, the specification does not reasonably convey to a person of ordinary skill in the art that the inventor had possession of the full genus of RAMP/peptidase/target polypeptide combinations recited in claim 1. Claims 2-5, 7-8, 10-11, 13, 15, 17, 18-20, 22, 25, 48-50, 52 and 54 are dependent directly or indirectly therefrom claim 1.
Claims 5, 7 and 8 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.
Claim 5 recites a programmable nuclease-peptidase composition in which (a) the peptidase is a TPR-CHAT peptidase derived from Desulfonema ishimotonii or a homolog, ortholog, or a variant thereof, (b) the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof; or both (a) and (b). The claim therefore encompasses compositions in which either or both of the RAMP polypeptide and peptidase are replaced by homologs, orthologs, or variants of the disclosed D. ishimotonii proteins.
The specification identifies the D. ishimotonii gRAMP and CHAT proteins and provides experimental evidence that the D. ishimotonii gRAMP-CHAT complex cleaves Up1 in response to target RNA. The specification also names additional organisms reported to encode TPR-CHAT/CHAT proteins including Candidatus Jettenia caeni, Candidatus Scalindua brodae, Deltaproteobacterium, and Candidatus Brocadia fulgida. However, naming those organisms does not convey possession of functional gRAMP-CHAT pairs from those organisms.
The specification does not identify a representative number of homologous, orthologous, or variant RAMP-peptidase pairs that retain the claimed target polynucleotide-initiated peptidase target polypeptide interaction. Nor does it identify structural features, sequence motifs, identify thresholds tied to function, conserved domain requirements, or pairing rules that would allow a person of ordinary skill in the art to recognize which homologs, orthologs or variants of the disclosed RAMP and CHAT proteins will form a functional RAMP-guide-peptidase complex and interact with a target polypeptide in response to target polynucleotide binding.
The specification does not reasonably convey that the applicants possessed the full scope of claim 5. Claims 7 and 8 are dependent directly or indirectly from claim 5.
Claims 26-30, 32-33, 35, 37-40, 42-44 and 46-48 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.
The specification describes a specific detection system based on the Desulfonema ishimotonii Type III-E gRAMP-CHAT complex and Up1 derived substrates. For example, the specification describes a gRAMP-CHAT/Up1 substrate containing an N-terminal avidin/biotin component and a C-terminal FAM component, in which target RNA-dependent Up1 cleavage permits visual detection on a biotin/FAM flow strip. The specification also describes engineered Up1 reporters and cell-based reporter outputs (see paragraphs [0062], [0067] and [0072]).
However, claim 26 is not limited to the disclosed Type III-E gRAMP-CHAT system, an Up1 derived substrate, the disclosed recognition region, a biotin/FAM flow-strip architecture or a specified reporter format. Instead claim 26 encompasses any RAMP polypeptide, any associated peptidase, any associated peptidase, and any detection construct, provided that target polynucleotide binding initiates peptidase mediated modification resulting in a detectable signal.
The specification does not identify a representative number of RAMP-peptidase detection construct combinations spanning the full claimed genus. Nor does it identify common structural features that distinguish detection constructs that will be recognized and modified by the full claimed range of peptidases and that will generate a detectable signal after that modification. The described detection embodiments remain centered on the disclosed gRAMP-CHAT/Up1 system. The specification does not reasonably convey possession of the full claimed range of RAMP-peptidase detection construct combinations.
Claim 30 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 30 recites a detection composition in which the peptidase is a TPR-CHAT peptidase and wherein the TPR-CHAT peptidase is derived from Desulfonema ishimotonii or a homolog, ortholog, or variant thereof; (b) wherein the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof; or both (a) and (b).
Although the specification identifies the D. ishimotonii gRAMP-CHAT system, describes Up1-derived detection substrates, and provides experimental evidence of target RNA dependent cleavage using D. ishimotonii proteins, the specification does not identify a representative number of homologous, orthologous, or variant RAMP-CHAT pairs that retain the claimed target polynucleotide initiated peptidase mediated modification of a detection construct and generation of a detectable signal.
The specification names additional organisms reported to encode TPR-CHAT/CHAT proteins but does not identify functional RAMP-CHAT pairs from those organisms, demonstrate that their RAMP and CHAT components interact, or show that target polynucleotide binding activates their peptidase activity to modify a detection construct and generate a detectable signal. Nor does the specification provide pairing, rules, identity thresholds tied to function, conserved structural features, or other guidance sufficient to convey possession of the full claimed genus.
Accordingly, the specification does not reasonably convey that the applicants had possession of the full scope of RAMP-CHAT detection construct combinations encompassed by claim 30.
Claims 5, 7 and 8 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 programmable nuclease-peptidase composition comprising the disclosed Delsulfonema ishimotonii Type III-E gRAMP polypeptide, guide molecule and the disclosed Delsulfonema ishimotonii TPR-CHAT/CHAT peptidase, wherein binding of the RAMP guide molecule complex to a target polynucleotide initiates interaction with the disclosed Up1 target polypeptide, does not reasonably provide enablement for the use of homologs, orthologs or variants of the disclosed RAMP and CHAT peptidase. 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.
Factors to be considered in determining whether undue experimentation is required are
summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2nd 1400 (Fed. Cir. 1988)) as follows: 1) quantity of experimentation necessary, 2) the amount of direction or guidance presented, 3) the presence and absence of working examples, 4) the nature of the invention, 5) the state of 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. The factors which have led the Examiner to conclude that the specification fails to teach how to make and/or use the claimed invention without undue experimentation, are addressed in detail below.
The breadth of the claims. Claim 5 encompasses RAMP polypeptides and TPR-CHAT peptidases derived from Delsulfonema ishimotonii or any homolog, ortholog or variant thereof, in any combination.
The amount of direction or guidance presented and existence of working examples. The specification provides direction for the Delsulfonema ishimotonii gRAMP-CHAT/Up1 system but provides no pairing rule, functional identity threshold, conserved-domain requirement, or other guidance for selecting functional homolog/ortholog/variant pairs. No working examples demonstrate a functional RAMP-CHAT pair comprising a homolog, ortholog or variant of either component.
The quantity of experimentation required to practice the claimed invention based
on the teachings of the specification. Substantial screening of RAMP and CHAT homologs/orthologs/variants would be required to identify pairs that form functional complexes, respond to target polynucleotide binding, and interact with or modify target polypeptide.
Claim 30 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a detection composition comprising the disclosed Delsulfonema ishimotonii Type III-E gRAMP polypeptide, guide molecule and the disclosed Delsulfonema ishimotonii TPR-CHAT/CHAT peptidase, wherein binding of the RAMP guide molecule complex to a target polynucleotide initiates interaction with the disclosed Up1 target polypeptide, does not reasonably provide enablement for the use of homologs, orthologs or variants of the disclosed RAMP and CHAT peptidase. 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.
Factors to be considered in determining whether undue experimentation is required are
summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2nd 1400 (Fed. Cir. 1988)) as follows: 1) quantity of experimentation necessary, 2) the amount of direction or guidance presented, 3) the presence and absence of working examples, 4) the nature of the invention, 5) the state of 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. The factors which have led the Examiner to conclude that the specification fails to teach how to make and/or use the claimed invention without undue experimentation, are addressed in detail below.
The breadth of the claims. Claim 30 encompasses RAMP polypeptides and TPR-CHAT peptidases derived from Delsulfonema ishimotonii or any homolog, ortholog or variant thereof, in any combination.
The amount of direction or guidance presented and existence of working examples. The specification provides direction for the Delsulfonema ishimotonii gRAMP-CHAT/Up1 detection system but provides no pairing rule, functional identity threshold, conserved-domain requirement, or other guidance for selecting functional homolog/ortholog/variant pairs. No working examples demonstrate a functional RAMP-CHAT detection composition comprising a homolog, ortholog or variant of either component or detection-construct architecture.
The quantity of experimentation required to practice the claimed invention based
on the teachings of the specification. Substantial screening of RAMP and CHAT homologs/orthologs/variants as well as detection construct architecture and would be required to identify pairs that form functional complexes, respond to target polynucleotide binding, and interact with or modify target polypeptide.
.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 13, 15, 37 and 38 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by van Beljouw et al. (Science, published 09/17/2021).
Claim 13 is directed to a polynucleotide encoding a programmable nuclease-peptidase composition or component thereof as in claim 1, optionally further comprising one or more regulatory elements and wherein the polynucleotide encoding a programmable nuclease-peptidase composition or component thereof is operatively coupled to one or more of the one or more regulatory elements. Claim 15 is directed to a vector or vector system comprising one or more polynucleotides according to claim 13, optionally wherein the vector or vector system is a viral vector or vector system, optionally an adeno-associated virus vector or vector system. Claim 37 is directed to a polynucleotide encoding one or more elements (i)-(iv) of the detection composition of claim 26. Claim 38 is directed to a vector system comprising one or more vectors encoding one or more of elements (i)-(iv) of the detection composition of claim 26.
van Beljouw et al. teaches for the expression of Candidatus “Scalindua brodae” gRAMP (Sb-gRAMP), a two plasmid expression (vector) system was used: a plasmid containing the gRAMP protein (pGRAMP) and a plasmid containing the CRISPR RNA (pCRISPR-1, pCRISPR-3 or pCRISPR-WT). pGRAMP, a coding sequence corresponding to an E. coli codon-optimized gRAMP protein variant containing a N-terminal Twin-Strep-Tag II and a SUMO-tag. Therefore, the teachings of van Beljouw et al. anticipate instant claims 13, 15, 37 and 38 as written/interpreted.
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, 4, 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021).
Cheng et al. teaches a CRISPR-Cas system of CLUST.019911 (Type III-E) comprising a Type III-E RNA guide and at least one Type III-E effector protein (see claim 1, pg. 154). Cheng et al. teaches the Type III-E CRISPR-Cas effector protein comprises at least one RAMP domain (see claim 4, pg. 154). Cheng et al. teaches that the system has a protease domain and protease activity is a peptidase activity (see claims 10 and 12, pg. 154). to target nucleic acid, thereby exhibiting protease activity (see claim 1, pg. 154). Cheng et al. teaches that Type III-E effector A comprises an N-terminal region matching a TPR domain and a C-terminal CHAT domain (see paragraph [0055]). Cheng et al. teaches a Type-III-E CRISPR-Cas system that is present within a cell (see paragraph [0018], pg. 2). Cheng et al. teaches the Type-III-E CRISPR-Cas system can be used as a medicament (see paragraph [0032], pg. 2).
Cheng et al. does not disclose a target polypeptide.
Claim 1 is directed to a programmable nuclease-peptidase composition comprising:
a repeat-associated mysterious protein (RAMP) polypeptide; a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence specific binding of the complex to a target polynucleotide; and a peptidase capable of binding to the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide. Claim 4 is directed to the programmable nuclease-peptidase composition of claim 1, wherein the peptidase is a TPR-CHAT peptidase. Claim 17 is directed to a cell or cell population comprising a programmable nuclease-peptidase composition of claim 1. Claim 18 is directed to a pharmaceutical formulation comprising: (a) a programmable nuclease-peptidase composition or component thereof as in claim 1; (b) a polynucleotide encoding (a); (c) a vector or vector system comprising one or more polynucleotides of (b); (d) a cell or cell population comprising (a), (b), (c), or any combination thereof; or (e) any combination of (a)-(d);
and a pharmaceutically acceptable carrier.
It would have been obvious to one of ordinary skill in the art before the effective filing
date of the claimed invention to employ the target-nucleic acid activated peptidase activity of Cheng et al. Type III-E CRISPR-Cas system to act on a target polypeptide.
A person of ordinary skill in the art would have been motivated to use the activated peptidase in Cheng’s system with a target polypeptide because Cheng expressly teaches that target nucleic acid binding activates the protease domain and that the activated activity is peptidase activity. A peptidase is selected and used on peptide or protein substrates. Thus, a person of ordinary skill in the art would have a reason to provide a polypeptide substrate so that target-nucleic acid recognition produces the known biochemical output of peptidase activity and modification of the polypeptide substrate.
One of ordinary skill in the art has a reasonable expectation of success because Cheng already teaches each operative component of the proposed programmable nuclease-peptidase composition: a Type III-E guide, a guide-binding Type III-E effector containing RAMP domains, sequence-specific targeting of a complementary target nucleic acid, a protease domain that has peptidase activity that is activated upon target-nucleic acid binding. 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.
Claim 2, 19 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Blake et al. (US 10,421,953, published 09/07/2017).
The teachings of Cheng et al. regarding claim 1 have been discussed above.
Blake et al. teaches proteins with protease-recognition sequences (see abstract). Blake et al. teaches protease-mediated cleavage of a recognition sequence (see column 25, lines 48-49).
Claim 2 is directed to the programmable nuclease-peptidase composition of claim 1, wherein target polypeptide interaction and/or binding occurs at, or in effective proximity to, a peptidase recognition motif in the target polypeptide. Claim 19 is directed to a method of modifying a polypeptide comprising: introducing into a sample having one or more target polynucleotides and target polypeptides, the programmable nuclease-peptidase compositions of claim 1; and activating the peptidase via sequence specific binding of the complex to the one or more target polynucleotides such that the peptidase then binds or interacts with the one or more target polypeptides resulting in modification of the one or more target polypeptides. Claim 20 is directed to the method of claim 19, wherein (a) the target polypeptide modification is cleavage of the target polypeptide; (b) introducing into the sample comprises in vitro, ex vivo, or in vivo delivery of the programable nuclease-peptidase composition into a cell or cell population; or (c) both (a) and (b).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use an engineered target polypeptide containing an appropriate protease recognition motif as taught by Blake et al. as the substrate for Cheng’s target-nucleic acid activated peptidase. The modification would retain Cheng’s guide-dependent recognition of the target nucleic acid while activating peptidase to cleave or modify the engineered target polypeptide at the designed recognition site.
A person of ordinary skill in the art is motivated to provide Cheng’s target-nucleic acid responsive peptidase with a targeted polypeptide substrate containing a recognition site to achieve defined and selective proteolytic modification after target-nucleic acid recognition.
One of ordinary skill in the art has a reasonable expectation of success because Cheng supplies the conditional activation event and Blake et al. supplies the established engineered substrate design. The proposed combination applies each component for its known function. 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.
Claims 5, 7 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of van Beljouw et al. (Science, published 09/17/2021) and further in view of Özcan et al. (Nature, published 09/30/2021).
The teachings of Cheng et al. regarding claims 1 and 4 have been discussed above.
van Beljouw et al. teaches a Type III-E effector from Scalindua brodae called Sb-gRAMP (see second column, second paragraph, pg. 1). Beljouw et al. teaches Sb-gRAMP physically combines with TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) (see abstract). van Beljouw et al. teaches gRAMP comprises Cas7-like and Cas11-like proteins (see Figure 1E).
Özcan et al. teaches a subtype III-E effector Cas7-11 originating from the from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D from Desulfonema ishimotonii (DiCas7-11) (see abstract). Özcan et al. teaches that the multiple Cas7-like domains of DiCas7-11 include domains in the Csm3/Cas7 family (see Extended Data Figure 1).
Claim 5 is directed to the programmable nuclease-peptidase composition of claim 1, wherein (a) the peptidase is a TPR-CHAT peptidase and wherein the TPR-CHAT peptidase is derived from Desulfonema ishimotonii, or a homolog, ortholog, or variant thereof; (b) the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof; or both (a) and (b). Claim 7 is directed to the programmable nuclease-peptidase composition of claim 5, wherein (a) the RAMP polypeptide comprises a Cas11 domain and multiple Cas7 domains or (b) the RAMP polypeptide is a Type III-E Cas polypeptide. Claim 8 is directed to the programmable nuclease-peptidase composition of claim 7, wherein the RAMP polypeptide comprises a Cas11 domain and multiple Cas 7 domains and further comprises a Csm3, Csm4, or Csm6 domain.
It would have been obvious to a person of ordinary skill in the before the effective filing date of the claimed invention to modify Cheng’s Type III-E guide-programmed TPR-CHAT peptidase system by employing the DiCas7-11 effector taught by Özcan as the Type III-E RAMP polypeptide, in view of van Beljouw’s teachings of a functional Type-III-E gRAMP-TPR-CHAT system.
A person of ordinary skill in the art is motivated to make the proposed combination because Cheng, van Beljouw and Özcan address closely related Type III-E CRISPR systems. A person of ordinary skill in the art would have understood DiCas7-11 to fall within the recited “RAMP polypeptide” because van Beljouw teaches the gRAMP contains Cas7-like and Cas11-like proteins that is functional associated with the TPR-CHAT peptidase.
One of ordinary skill in the art has a reasonable expectation of success because the DiCas7-11 taught by Özcan and the RAMP taught by van Beljouw share the relevant Type III-E architecture. The proposed combination consequently applies a known Type III-E RAMP-family effector for the same established role in a known Type III-E RAMP-TPR-CHAT arrangement. The expected result of an operative Type III-E composition comprising Cheng’s TPR-CHAT peptidase and Di-derived RAMP polypeptide would have been a predictable result. 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.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017).
The teachings of Cheng et al. regarding claim 1 have been discussed above.
Shu et al. teaches genetically encoded protease reporters comprising a reporter polypeptide coupled to a protease-cleavable sequence, wherein cleavage by caspase produces a detectable fluorescent output (see claim 9, column 137).
Claim 10 is directed to the programmable nuclease-peptidase composition of claim 1, wherein the target polypeptide comprises, consists of, or is coupled to an effector wherein the effector is optionally a. a reporter polypeptide; b. a signal amplification polypeptide; c. an engineered prodrug; d. a cargo polypeptide; or e. a pathogenic polypeptide.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use a protease-cleavable reporter polypeptide of Shu et al. as the target polypeptide or coupled to a target polypeptide, in Cheng’s programmable target-nucleic acid activated peptidase composition. The resulting composition would comprise a target polypeptide that comprises, consists of, or is coupled to a reporter polypeptide effector.
A person of ordinary skill in the art would have been motivated to use a reporter polypeptide as a substrate for Cheng’s activated peptidase to convert target-nucleic acid recognition into a detectable signal. Incorporating that known reporter substrate into Cheng’s programmable system would permit the presence or identity of the selected target
One of ordinary skill in the art has a reasonable expectation of success because Cheng’s composition provides a nucleic acid activated peptidase and Shu et al. teaches the established use of a polypeptide reporter designed to generate a fluorescent output after proteolysis. The modification would use the reporter polypeptide for its known cleavage-reporting function and would not alter Cheng’s guide-dependent target-nucleic acid recognition mechanism. A person of ordinary skill in the art would expect that placing an appropriate peptidase recognition sequence in the reporter-coupled target polypeptide would yield a detectable signal upon Cheng’s target-nucleic acid dependent proteolysis. 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.
Claims 11 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Hoppenz et al. (Front. Chem., published 07/06/2020).
The teachings of Cheng et al. regarding claim 1 have been discussed above. Cheng et al. teaches a Type-III-E CRISPR-Cas system used in the treatment or prevention of cancer or an infectious disease (see paragraph [0033], pg. 2).
Hoppenz et al. teaches that epidermal growth factor receptor (EGFR) is a cell-surface polypeptide overexpressed in multiple cancers (see pg. 4, Epidermal Growth Factor Receptor (EGFR)). Thus. EGFR is a disease-associated polypeptide associated with cancer.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to select EGFR as the target polypeptide of Cheng’s target-nucleic acid activated peptidase in an implementation directed to cancer treatment or prevention.
A person of ordinary skill in the art is motivated to use EGFR as a target polypeptide because Cheng expressly teaches applying its programmable composition to treatment or prevention of cancer and Hoppenz teaches EGFR is an established cancer-associated cell-surface protein whose overexpression is linked to tumor-cell proliferation and which was already targeted in cancer therapy.
One of ordinary skill in the art has a reasonable expectation of success because Cheng provides a target-nucleic acid dependent peptidase and a person of ordinary skill in the art would have understood a peptidase to act on peptide bonds in a polypeptide substrate. EGFR is an accessible cell surface polypeptide and an established cancer target. 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.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Blake et al. (US 10,421,953, published 09/07/2017) and in further view of Freiburghaus et al. (Pancreatology, published 11/08/2021).
The teachings of Cheng et al. regarding claim 20 have been discussed above.
The teachings of Blake et al. regarding claim 20 have been discussed above.
Freiburghaus et al. teaches Enteropeptidase is responsible for the activation of trypsinogen (proenzyme) to trypsin (active enzyme).
Claim 22 is directed to the method of claim 20, wherein (a) the one or more target polypeptides are proenzymes and the modification results in conversion of the proenzyme into an active enzyme; (b) modification of the one or more target polypeptides results in activation or deactivation of one or more cell-signaling proteins; (c) wherein the one or more target polynucleotides are a specific transcript or set of transcripts and wherein modification of the one or more target polypeptides triggers cell death upon activating the peptidase in response to binding of the nuclease-peptidase to the specific transcript or set of transcripts; or (d) any combination of (a)-(c).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to select a proenzyme substrate, such as trypsinogen, having a known protease-recognition/activation motif, as the target polypeptide for the peptidase output of Cheng in view of Blake et al.
A person of ordinary skill in the art is motivated to select trypsinogen, or another known proenzyme bearing a known protease-recognition/activation motif, because this would provide a defined and predictable protein-processing output of Cheng’s programmable system.
One of ordinary skill in the art would have a reasonable expectation of success because the relevant biological functions were established and compatible. 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.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Blake et al. (US 10,421,953, published 09/07/2017) and in further view of Abudayyeh et al. (US 2022/0073891 A1, published 03/10/2022).
The teachings of Cheng et al. regarding claim 20 have been discussed above.
The teachings of Blake et al. regarding claim 20 have been discussed above.
Abudayyeh et al. teaches that CHAT domains are a caspase family protease that is associated with programmed cell death (see paragraph [0219]). Abudayyeh et al. teaches correction of Cluc W85X (target transcript) mRNA by dDisCas7-11 (see paragraph [0365]). Abudayyeh et al. teaches guide design for RNA editing of Cluc W85A (see Fig. 22A).
Claim 25 is directed to the method of claim 22, wherein the guide molecule is configured to detect one or more mutations in the specific transcript or set of transcripts.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the RNA-guided RAMP/nuclease-peptidase system of Cheng et al. so that guide-mediated binding to a selected transcript activates the associated CHAT peptidase and produces controlled modification of a selected target polypeptide containing a compatible protease-cleavage site, as taught by Blake et al. It would further have been obvious to configure the guide to recognize a mutation-bearing version of the selected transcript in view of Abudayyeh’s disclosure of guide-based RNA editing and correction of the Cluc W85X target mRNA.
A person of ordinary skill in the art would have been motivated to make this modification to create a programmable and selective cell-elimination system in which an intracellular molecular marker acts as the input for a defined polypeptide output.
One of ordinary skill in the art would have had a reasonable expectation of success because the proposed combination uses each reference according to its disclosed, conventional function and does not require a change in the fundamental operating principle of any reference. The predictable result would be transcript-conditional peptidase activity and target-polypeptide processing. 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.
Claims 26, 29, 39 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017).
Cheng et al. teaches a CRISPR-Cas system of CLUST.019911 (Type III-E) comprising a Type III-E RNA guide and at least one Type III-E effector protein (see claim 1, pg. 154). Cheng et al. teaches the Type III-E CRISPR-Cas effector protein comprises at least one RAMP domain (see claim 4, pg. 154). Cheng et al. teaches that the system has a protease domain and protease activity is a peptidase activity (see claims 10 and 12, pg. 154). to target nucleic acid, thereby exhibiting protease activity (see claim 1, pg. 154). Cheng et al. teaches that Type III-E effector A comprises an N-terminal region matching a TPR domain and a C-terminal CHAT domain (see paragraph [0055]). Cheng et al. teaches transfection of cells with vectors
expressing the CRISPR enzyme, guide RNAs, and guide RNAs that target the nucleic acid encoding the CRISPR enzyme can lead to efficient disruption of the nucleic acid encoding the CRISPR enzyme and decrease the levels of CRISPR enzyme, thereby limiting the genome editing activity (see paragraph [0100], pg. 7).
Cheng et al. does not disclose a target polypeptide.
Shu et al. teaches genetically encoded protease reporters comprising a reporter polypeptide coupled to a protease-cleavable sequence, wherein cleavage by caspase produces a detectable fluorescent output (see claim 9, column 137).
Claim 26 is directed to a detection composition comprising: (i) a RAMP polypeptide;
(ii) a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence-specific binding of the complex to a target polynucleotide; (iii) a peptidase capable of binding the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide complex; and (iv) a detection construct, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates peptidase mediated modification of the detection construct resulting in generation of a detectable signal. Claim 29 is directed to the composition of claim 26, wherein the peptidase is a TPR-CHAT peptidase. Claim 39 is directed to an engineered cell modified to express elements (i) and (iii), of the detection composition of claim 26. Claim 40 is directed to the engineered cell of claim 39, wherein the engineered cell is (a) further modified to express element (iv); (b) further modified to express element (ii); or (c) both (a) and (b).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the programmable RAMP-guide-peptidase composition of Cheng to further include a protease-responsive detection construct as taught by Shu et al. and engineer a cell to express the elements of the RAMP-guide-peptidase detection composition.
A person of ordinary skill in the art would have been motivated to combine Cheng and Shu to convert Cheng’s sequence-specific target-polynucleotide recognition event into a direct, objective and readily measurable diagnostic output. The combination would allow a user to determine whether a particular target polynucleotide is present by detecting the signal produced by peptidase-mediated modification of the reporter polypeptide and express the composition in an engineered cell.
One of ordinary skill in the art would have had a reasonable expectation of success in combining Cheng and Shu because each reference would be used according to its established and intended function. This would generate a detectable signal when the targeted polynucleotide is recognized, and the peptidase is activated. 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.
Claims 27 and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017) and in further view of Blake et al. (US 10,421,953, published 09/07/2017).
The teachings of Cheng et al. regarding Claim 26 have been discussed above.
The teachings of Shu et al. regarding Claim 26 have been discussed above.
Blake et al. teaches proteins with protease-recognition sequences (see abstract). Blake et al. teaches protease-mediated cleavage of a recognition sequence (see column 25, lines 48-49).
Claim 27 is directed to the composition of claim 26, wherein the detection construct comprises a peptidase recognition motif recognized by the peptidase. Claim 35 is directed to the composition of claim 26, wherein the detection construct comprises a polypeptide comprising a protease recognition motif recognized by the peptidase, optionally wherein the polypeptide is a fluorescent protein protease reporter.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the detection construct of Cheng-Shu composition to comprise a peptidase-recognition motif recognized by the peptidase, as taught by Blake.
A person of ordinary skill in the art would have been motivated to include a defined peptidase-recognition motif in the reporter/detection construct to ensure that the construct is selectively and efficiently modified by the peptidase and to provide a reliable relationship between peptidase activation and reporter signal generation.
One of ordinary skill in the art would have had a reasonable expectation of success because the proposed modification applies each reference according to its disclosed purpose. A skilled artisan would reasonably have expected the activated peptidase to recognize and modify the motif-containing detection construct and thereby generate Shu’s detectable signal. 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.
Claims 30, 32 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017), van Beljouw et al. (Science, published 09/17/2021) and further in view of Özcan et al. (Nature, published 09/30/2021).
The teachings of Cheng et al. regarding Claim 26 have been discussed above.
The teachings of Shu et al. regarding Claim 26 have been discussed above.
van Beljouw et al. teaches a Type III-E effector from Scalindua brodae called Sb-gRAMP (see second column, second paragraph, pg. 1). Beljouw et al. teaches Sb-gRAMP physically combines with TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) (see abstract). van Beljouw et al. teaches gRAMP comprises Cas7-like and Cas11-like proteins (see Figure 1E).
Özcan et al. teaches a subtype III-E effector Cas7-11 originating from the from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D from Desulfonema ishimotonii (DiCas7-11) (see abstract). Özcan et al. teaches that the multiple Cas7-like domains of DiCas7-11 include domains in the Csm3/Cas7 family (see Extended Data Figure 1).
It would have been obvious to a person of ordinary skill in the before the effective filing date of the claimed invention to modify the detection construct ofCheng-Shu’s Type III-E guide-programmed TPR-CHAT peptidase system by employing the DiCas7-11 effector taught by Özcan as the Type III-E RAMP polypeptide, in view of van Beljouw’s teachings of a functional Type-III-E gRAMP-TPR-CHAT system.
A person of ordinary skill in the art is motivated to make the proposed combination because Cheng, van Beljouw and Özcan address closely related Type III-E CRISPR systems. A person of ordinary skill in the art would have understood DiCas7-11 to fall within the recited “RAMP polypeptide” because van Beljouw teaches the gRAMP contains Cas7-like and Cas11-like proteins that is functional associated with the TPR-CHAT peptidase.
One of ordinary skill in the art has a reasonable expectation of success because the DiCas7-11 taught by Özcan and the RAMP taught by van Beljouw share the relevant Type III-E architecture. The proposed combination consequently applies a known Type III-E RAMP-family effector for the same established role in a known Type III-E RAMP-TPR-CHAT arrangement. The expected result of an operative Type III-E composition comprising Cheng’s TPR-CHAT peptidase and Di-derived RAMP polypeptide would have been a predictable result. 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.
Claim 42 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017) and in further view of Dixit et al. (Cell, published 12/15/2016).
The teachings of Cheng et al. regarding claim 39 have been discussed above.
The teachings of Shu et al. regarding claim 39 have been discussed above.
Dixit et al. teaches Perturb-Seq, a pooled CRISPR screen combined with single-cell RNA sequencing. Dixit et al. teaches infecting cells with lentiviral constructs encoding sgRNAs, assigning perturbations to cells, and analyzing transcriptional effects of genetic manipulations on genes, processes and cell states (see abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the engineered RAMP-peptidase reporter cells of Cheng and Shu in the cell-perturbation screening method taught by Dixit et al.
A person of ordinary skill in the art would have been motivated to combine these references to provide a screening assay in which a perturbation’s effect on a selected transcript is converted into a readily measurable reporter signal.
One of ordinary skill in the art would have had a reasonable expectation of success because each reference would be used according to its established function. A skilled artisan would have expected that in cells expressing the RAMP and peptidase, a guide directed to a selected transcript would produce a reporter signal whose magnitude changes when the perturbation changes abundance of that transcript. 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.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017) and in further view of Abudayyeh et al. (US 2022/0073891 A1, published 03/10/2022).
The teachings of Cheng et al. regarding claim 26 have been discussed above.
The teachings of Shu et al. regarding claim 26 have been discussed above.
Abudayyeh et al. teaches RNA-gudie Cas7-11/RAMPS systems (see paragraph [0218], pg. 9). Abudayyeh et al. teaches detection a bacterium in a sample by adding Cas7-11 composition/nucleic acid/vector and a detecting reporter (see paragraph [0023], pg. 2). Abudayyeh et al. teaches a reporter signal generated after target directed activity (see Fig. 4E).
Claim 43 is directed to a method of detecting target polynucleotides in samples comprising: combining a sample or a component thereof with the detection composition as in claim 26; and activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is produced, thereby detecting the target polynucleotide in the sample.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the RAMP-guide-peptidase detection composition of Cheng et al. and the protease-responsive reporter of Shu et al. in the sample-detection method taught by Abudayyeh et al.
A person of ordinary skill in the art would have been motivated to combine Cheng, Shu and Abudayyeh to provide a direct nucleic acid detection assay in which target recognition produces an objective optical readout. The combination would provide the expected benefit of a sequence responsive diagnostic assay.
One of ordinary skill in the art would have had a reasonable expectation of success because the proposed assay applies each reference according to its disclosed function. A skilled artisan would expect that a sample containing the target polynucleotide would activate the peptidase, modify a compatible reporter construct and produce a detectable signal, whereas the sample lacking the target would not produce a signal. 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.
Claim 44 is rejected is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017), Abudayyeh et al. (US 2022/0073891 A1, published 03/10/2022) and in further view of Gootenberg et al. (Science, published 04/13/2017).
The teachings of Cheng et al. regarding claim 43 have been discussed above.
The teachings of Shu et al. regarding claim 43 have been discussed above.
The teachings of Abudayyeh et al. regarding claim 43 have been discussed above.
Gootenberg et al. teaches recombinase mediated pre-amplification of DNA or RNA followed by Cas13 or Cas12 mediated detection through fluorescence or colorimetric readout. Gootenberg et al. teaches amplification brings target DNA or RNA to a level detectable by the CRISPR assay (see abstract).
Claim 44 is directed to the method of claim 43, further comprising amplifying and/or enriching the target polynucleotide, optionally wherein activating the peptidase further results in activation or generation of one or more signal amplification molecules.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the sample-detection method to further comprise amplyfying and/or enriching the target polynucleotide as taught by Gootenberg et al. It would have been obvious to amplify the target polynucleotide before combining it with or during detection by RAMP guide-peptidase detection composition thereby increasing the amount of target available to activate the peptidase and generate the reporter signal.
A person of ordianry skill in the art would have been motivated to amplify the target polynucleotide to improve the sensitivity and reliability of the sample-detection assay. Combining these teachings would provide the expected benefit of detecting lower abundance target polypeptides while retaining the sequence responsive reporter output.
One of ordinary skill in the art has a reasonable expectation of success because amplification would increase the quantity of target polynucleotide available for detection without changing the underlying guide-directed recognition, peptidase activity, reporter modification or signal generation steps. 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.
Claims 46 and 47 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017) and in further view of Boulware et al. (PNAS, published 05/03/2006).
The teachings of Cheng et al. regarding claim 26 have been discussed above.
The teachings of Shu et al. regarding claim 26 have been discussed above.
Boulware et al. teaches detecting protease-cleavage dependent fluorescence in cells by flow cytometry and sorting with a FACSAria based on the signal (see abstract and substrate library screening).
Claim 46 is directed to A method of labeling cells comprising: introducing the detection composition as in claim 26 into a population of cells, wherein the guide molecule is configured to detect one or more target transcripts associated with a particular cell type or cell state; and activating the peptidase via binding of the complex to the one or more target transcripts such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is generated, thereby labeling cells within the cell population expressing the one or more target transcripts. Claim 47 is directed to the method of claim 46, wherein labeled cells are further sorted or isolated based on production of the detectable product and/or signal.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the composition of Cheng in view of Shu with the protease cleavable fluorescent reporter and cell-sorting methodology taught by Boulware et al.
A person of ordinary skill in the art would have been motivated to combine Cheng, Shu and Boulware to achieve a cell-sorting type or cell state specific method for labeling and isolating cells. A person of ordinary skill in the art would be motivated to use the target RNA activated peptidase system of Cheng with a guide molecule directed to a transcript expressed in a desired cell type or state, thereby producing a detectable signal only in cells expressing that transcript and use FACS to identify and isolate labeled cells.
One of ordianry skill in the art would have had a reasonable expectation of success in combining Cheng, Shu and Boulware to arrive at the claimed method.because Cheng teaches the target binding activated peptidase function, Shu teaches delivery and guide selection and Boulware teach detection and sorting of cells based on protease cleavage dependent fluorescent signal. 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.
Claims 48-50, 52 and 54 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. (US2021/0198644 A1, published 07/01/2021) published 07/01/2021) in view of Shu et al. (US10,201,282 B2, published 07/06/2017) and in further view of Vlahos et al. (Nature, published 02/17/2022).
The teachings of Cheng et al. regarding claim 1 have been discussed above.
Shu et al. teaches genetically encoded protease reporters comprising a reporter polypeptide coupled to a protease-cleavable sequence, wherein cleavage by caspase produces a detectable fluorescent output (see claim 9, column 137).
Vlahos et al. teaches ER retained protein with a transmembrane anchor, a protease cleavable linker and an effector/cargo cleavage by a cognate protease controls secretion or cell-surface display of the cargo. Vlahos et al, teaches membrane tethered GFP and protease-controlled release/display (see Engineering RELEASE for protein secretion and expression).
Claim 48 is directed to a method of in vivo effector activation or delivery comprising:
introducing a programmable nuclease system of claim 1 into a cell comprising a substrate of the peptidase, wherein the substrate of the peptidase is optionally tethered to a cellular structure and wherein the substrate the peptidase is coupled to an effector. Claim 49 is directed to . The method of claim 48, wherein the effector a. is capable of producing a detectable signal when activated, b. is a therapeutic molecule or prodrug; c. is a genetic modifying molecule; d. or any combination thereof. Claim 50 is directed to the method of claim 48, wherein the effector is inactive when coupled to an uncleaved substrate or wherein the effector is inactive when coupled to a cleaved substrate portion. Claim 52 is directed to the method of claim 48, further comprising cleaving the substrate by the peptidase in response to a target RNA and activation of the peptidase of the programmable nuclease system, optionally wherein the target RNA is endogenous to the cell or is exogenous to the cell. Claim 54 is directed to the method of claim 48, wherein the substrate is tethered to a cell membrane or a nuclear membrane.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the programmable nuclease-peptidase system of Cheng in view of SHu with the protease-controlled effector release platform taught by Vlahos et al. It would have been obvious to couple an effector to a substrate of Cheng's CHAT peptidase, tether that substrate to a cellular structure using the membrane anchor or retention strategy of Vlahos et al. and use target RNA binding activated peptidase cleavage to release or activate the effector.
A person of ordinary skill in the art is motivated to combine Cheng, Shu and Vlahos to produce a traget RNA dependent system for controlled activation or delivery of an effector. A person of ordinary skill in the art would have been motivated to couple an effector to a CHAT peptidase substrate, tether the substrate within a cell, and use target RNA dependent peptidase activation to release or activate an effector. This would provide a programmable, RNA triggered effector activation or delivery system which was a recognized objective in the fields of synthetic biology, cell engineering and controlled therapeutic delivery.
One of ordianry skill in the art would have had a reasonable expectation of success in combining Cheng, Shu and Vlahos et al. as the proposed modification would replace Vlahos protease with Cheng's target RNA activated CHAT peptidase and couple its substrate to an effector, while retaining the known tether/cleavable linker architecture. Because both systems rely on proetase recognition and cleavage of a peptide susbtrate to control an associated cargo, one of ordinary skill would have expected that target RNA dependent activation of Cheng's peptidase would cleave a compatable substrate and release or activate the couple effector. 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.
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 1-5, 7-8, 10, 13, 17-20, 22, 25-26, 32-33, 35, 37-40, 42-43, 46-50, 52 and 54 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5, 8, 15, 26, 28, 33-35, 43, 52, 63, 65-67, 70-71, 76 and 78 of copending Application No. 19/089,389 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because they cover the same scope.
Claim 1 of the instant application is directed to a programmable nuclease-peptidase composition comprising: a repeat-associated mysterious protein (RAMP) polypeptide; a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence specific binding of the complex to a target polynucleotide; and a peptidase capable of binding to the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide.
Claim 2 of the instant application is directed to the programmable nuclease-peptidase composition of claim 1, wherein target polypeptide interaction and/or binding occurs at, or in effective proximity to, a peptidase recognition motif in the target polypeptide.
Claim 3 of the instant application is directed to the programmable nuclease-peptidase composition of claim 2, wherein the peptidase recognition motif comprises or consists of SEQ ID NO: 3 or a sequence therein, optionally MKKD (SEQ ID NO: 25).
Claim 4 of the instant application is directed to The programmable nuclease-peptidase composition of claim 1, wherein the peptidase is a TPR-CHAT peptidase.
Claim 5 of the instant application is directed to the programmable nuclease-peptidase composition of claim 1, wherein (a) the peptidase is a TPR-CHAT peptidase and wherein the TPR-CHAT peptidase is derived from Desulfonema ishimotonii, or a homolog, ortholog, or variant thereof; (b) the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof; or both (a) and (b).
Claim 7 of the instant application is directed to the programmable nuclease-peptidase composition of claim 5, wherein (a) the RAMP polypeptide comprises a Cas11 domain and multiple Cas7 domains or (b) the RAMP polypeptide is a Type III-E Cas polypeptide.
Claim 8 of the instant application is directed to the programmable nuclease-peptidase composition of claim 7, wherein the RAMP polypeptide comprises a Cas11 domain and multiple Cas 7 domains and further comprises a Csm3, Csm4, or Csm6 domain.
Claim 10 of the instant application is directed to the programmable nuclease-peptidase composition of claim 1, wherein the target polypeptide comprises, consists of, or is coupled to an effector, wherein the effector is optionally
Claim 13 of the instant application is directed to a polynucleotide encoding a programmable nuclease-peptidase composition or component thereof as in claim 1, optionally further comprising one or more regulatory elements and wherein the polynucleotide encoding a programmable nuclease-peptidase composition or component thereof is operatively coupled to one or more of the one or more regulatory elements.
Claim 17 of the instant application is directed to a cell or cell population comprising a programmable nuclease-peptidase composition of claim 1.
Claim 18 of the instant application is directed to a pharmaceutical formulation comprising: (a) a programmable nuclease-peptidase composition or component thereof as in claim 1; (b) a polynucleotide encoding (a); (c) a vector or vector system comprising one or more polynucleotides of (b); (d) a cell or cell population comprising (a), (b), (c), or any combination thereof; or (e) any combination of (a)-(d); and a pharmaceutically acceptable carrier.
Claim 19 of the instant application is directed to a method of modifying a polypeptide comprising: introducing into a sample having one or more target polynucleotides and target polypeptides, the programmable nuclease-peptidase compositions of claim 1; and
activating the peptidase via sequence specific binding of the complex to the one or more target polynucleotides such that the peptidase then binds or interacts with the one or more target polypeptides resulting in modification of the one or more target polypeptides.
Claim 20 of the instant application is directed to the method of claim 19, wherein (a) the target polypeptide modification is cleavage of the target polypeptide; (b) introducing into the sample comprises in vitro, ex vivo, or in vivo delivery of the programable nuclease-peptidase composition into a cell or cell population; or (c) both (a) and (b).
Claim 22 of the instant application is directed to the method of claim 20, wherein
(a) the one or more target polypeptides are proenzymes and the modification results in conversion of the proenzyme into an active enzyme; (b) modification of the one or more target polypeptides results in activation or deactivation of one or more cell-signaling proteins;
(c) wherein the one or more target polynucleotides are a specific transcript or set of transcripts and wherein modification of the one or more target polypeptides triggers cell death upon activating the peptidase in response to binding of the nuclease-peptidase to the specific transcript or set of transcripts; or (d) any combination of (a)-(c).
Claim 25 of the instant application is directed to the method of claim 22, wherein the guide molecule is configured to detect one or more mutations in the specific transcript or set of transcripts.
Claim 26 of the instant application is directed to a detection composition comprising:
(i) a RAMP polypeptide; (ii) a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence-specific binding of the complex to a target polynucleotide; (iii) a peptidase capable of binding the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide complex; and
(iv) a detection construct, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates peptidase mediated modification of the detection construct resulting in generation of a detectable signal.
Claim 32 of the instant application is directed to the composition of claim 31, wherein (a) the RAMP polypeptide comprises a Cas11 domain and multiple Cas7 domains or (b) the RAMP polypeptide is a Type III-E Cas polypeptide.
Claim 33 of the instant application is directed to the composition of claim 32, wherein the RAMP polypeptide comprises a Cas11 domain and multiple Cas 7 domains and further comprises a Csm3, Csm4, or Csm6 domain.
Claim 35 of the instant application is directed to the composition of claim 26, wherein the detection construct comprises a polypeptide comprising a protease recognition motif recognized by the peptidase, optionally wherein the polypeptide is a fluorescent protein protease reporter.
Claim 37 of the instant application is directed to a polynucleotide encoding one or more elements (i)-(iv) of the detection composition of claim 26.
Claim 38 of the instant application is directed to a vector system comprising one or more vectors encoding one or more of elements (i)-(iv) of the detection composition of claim 26.
Claim 39 of the instant application is directed to an engineered cell modified to express elements (i) and (iii), of the detection composition of claim 26.
Claim 40 of the instant application is directed to the engineered cell of claim 39, wherein the engineered cell is (a) further modified to express element (iv); (b) further modified to express element (ii); or (c) both (a) and (b).
Claim 42 of the instant application is directed to a method for screening cell perturbations comprising; introducing a perturbation to a cell population comprising engineered cells of claim 39, along with any elements of the detection composition not already expressed by the engineered cells, and wherein the guide molecules are configured to detect one or more target transcripts associated with a specific cell type or cell state; activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase to produce a detectable product and/or signal; detecting an ability of the perturbation to modify expression of the one or more target transcripts by measuring a change in the detectable product and/or signal relative to a control.
Claim 43 of the instant application is directed to a method of detecting target polynucleotides in samples comprising: combining a sample or a component thereof with the detection composition as in claim 26; and activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is produced, thereby detecting the target polynucleotide in the sample.
Claim 46 of the instant application is directed to a method of labeling cells comprising:
introducing the detection composition as in claim 26 into a population of cells, wherein the guide molecule is configured to detect one or more target transcripts associated with a particular cell type or cell state; and activating the peptidase via binding of the complex to the one or more target transcripts such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is generated, thereby labeling cells within the cell population expressing the one or more target transcripts.
Claim 47 of the instant application is directed to the method of claim 46, wherein labeled cells are further sorted or isolated based on production of the detectable product and/or signal.
Claim 48 of the instant application is directed to a method of in vivo effector activation or delivery comprising: introducing a programmable nuclease system of claim 1 into a cell comprising a substrate of the peptidase, wherein the substrate of the peptidase is optionally tethered to a cellular structure and wherein the substrate the peptidase is coupled to an effector.
Claim 49 of the instant application is directed to the method of claim 48, wherein the effector a. is capable of producing a detectable signal when activated b. is a therapeutic molecule or prodrug; c. is a genetic modifying molecule; d. or any combination thereof.
Claim 50 of the instant application is directed to the method of claim 48, wherein the effector is inactive when coupled to an uncleaved substrate or wherein the effector is inactive when coupled to a cleaved substrate portion.
Claim 52 of the instant application is directed to the method of claim 48, further comprising cleaving the substrate by the peptidase in response to a target RNA and activation of the peptidase of the programmable nuclease system, optionally wherein the target RNA is endogenous to the cell or is exogenous to the cell.
Claim 54 of the instant application is directed to the method of claim 48, wherein the substrate is tethered to a cell membrane or a nuclear membrane.
Claim 1 of ‘389 is directed to a programmable nuclease-peptidase composition comprising: a repeat-associated mysterious protein (RAMP) polypeptide, wherein the RAMP polypeptide is capable of forming a RAMP-guide molecule complex with a guide molecule capable of sequence specific binding with a target polynucleotide thereby directing sequence specific binding of the RAMP-guide molecule complex to the target polynucleotide; and a peptidase capable of binding to the RAMP polypeptide, the guide molecule, the target polynucleotide, and/or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide.
Claim 5 of ‘389 is directed to the programmable nuclease-peptidase composition of claim1any one of the preceding claims, wherein target polypeptide interaction and/or binding occurs at, or in effective proximity to, a peptidase recognition motif in the target polypeptide, optionally wherein the peptidase recognition motif comprises or consists of a Csx30 polypeptide, a polypeptide according to SEQ ID NO: 2 or a sequence therein, a polypeptide having a sequence according to SEQ ID NO: 3 or a sequence therein, optionally wherein the peptidase recognition motif comprises or consists of MKKD, a Csx3025o-565 polypeptide, a Csx30396-565 polypeptide, a Csx304o7-565, and/or a Csx304o7-56o polypeptide.
Claim 8 of ‘389 is directed to the programmable nuclease-peptidase composition of claim 1, wherein the peptidase is a TPR-CHAT peptidase, optionally wherein the TPR-CHAT peptidase is derived from Desulfonema ishimotonii, or a homolog, ortholog, or variant thereof.
Claim 15 of ‘389 is directed to the programmable nuclease-peptidase composition of claim 1, wherein the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof, optionally wherein the RAMP polypeptide comprises a Casll domain and multiple Cas7 domains, optionally wherein the RAMP polypeptide further comprises a Csm3, Csm4, or Csm6 domain, optionally wherein the RAMP polypeptide is a Type III-E Cas polypeptide.
Claim 26 of ‘389 is directed to the programmable nuclease-peptidase composition of claim 1, wherein the target polypeptide comprises, consists of, or is coupled to an effector, optionally wherein the effector is a. a reporter polypeptide b. a signal amplification polypeptide; c. an engineered prodrug; d. a cargo polypeptide; e. a transcription factor; f. a pathogenic polypeptide; or g. any combination thereof;
Claim 28 of ‘389 is directed to A polynucleotide encoding a programmable nuclease- peptidase composition or component thereof as in claim 1, optionally further comprising one or more regulatory elements and wherein the polynucleotide encoding a programmable nuclease- peptidase composition or component thereof is operatively coupled to one or more of the one or more regulatory elements.
Claim 33 of ‘389 is directed to a cell or cell population comprising a programmable nuclease-peptidase composition of claim 1.
Claim 34 of ‘389 is directed to a pharmaceutical formulation comprising: a programmable nuclease-peptidase composition or component thereof as in claim 1, a target polypeptide, a target polynucleotide, a nucleic acid and/or polypeptide detection composition or component thereof, a polynucleotide encoding the programmable nuclease- peptidase composition or component thereof, a vector or vector system comprising the polynucleotide encoding the programmable nuclease-peptidase composition or component thereof, a cell or cell population comprising the programmable nuclease-peptidase composition or component thereof, the polynucleotide encoding the programmable nuclease-peptidase composition or component thereof, a vector or vector system comprising the polynucleotide encoding the programmable nuclease peptidase composition or component thereof, or any combination thereof; and a pharmaceutically acceptable carrier.
Claim 35 of ‘389 is directed to a method of modifying a polypeptide comprising: introducing the programmable nuclease-peptidase compositions of claim 1 into a sample having one or more target polynucleotides and one or more target polypeptides; and activating the peptidase via sequence specific binding of the RAMP-guide molecule complex to the one or more target polynucleotides; and binding and/or interaction of the peptidase with the one or more target polypeptides resulting in modification of the one or more target polypeptides, optionally wherein binding and/or interacting of the peptidase further comprises binding and/or interacting with a target polypeptide or region thereof, optionally wherein the target polypeptide modification is cleavage of the target polypeptide, optionally wherein introducing comprises in vitro, ex vivo, or in vivo delivery of the programable nuclease-peptidase composition into a cell or cell population, optionally wherein the one or more target polypeptides are proenzymes and the modification results in conversion of the proenzyme into an active enzyme, optionally wherein modification of the one or more target polypeptides results in activation or deactivation of one or more cell-signaling proteins optionally wherein the one or more target polynucleotides are a specific transcript or set of transcripts and wherein modification of the one or more target polypeptides triggers cell death, modulates gene and/or protein expression, or both, upon activating the peptidase in response to binding of the nuclease-peptidase to the specific transcript or set of transcripts, optionally wherein the guide molecule is configured to detect one or more mutations in the specific transcript or set of transcripts.
Claim 43 of ‘389 is directed to a detection composition comprising: (i) a RAMP polypeptide; (ii) a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence-specific binding of the complex to a target polynucleotide; (iii) a peptidase capable of binding the RAMP polypeptide, the target polynucleotide, optionally the guide molecule, and/or further complexing with the RAMP-guide molecule complex; and (iv) a detection construct, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates peptidase mediated modification of the detection construct resulting in generation of a detectable signal.
Claim 52 of ‘389 is directed to The detection composition of claim 43, wherein the RAMP polypeptide is derived from Desulfonema ishimotonii, or a homolog, ortholog or variant thereof[[.]], optionally wherein the RAMP polypeptide comprises a Casl l domain and multiple Cas7 domains, optionally wherein the RAMP polypeptide further comprises a Csm3, Csm4, or Csm6 domain, optionally wherein the RAMP polypeptide is a Type III-E Cas polypeptide, optionally wherein the Type III-E Cas polypeptide is a Cas-7-11 polypeptide, homolog thereof, ortholog thereof, or variant thereof.
Claim 63 of ‘389 is directed to the detection composition of claim 43, wherein the detection construct comprises a polypeptide comprising a peptidase recognition motif recognized by the peptidase, optionally wherein the polypeptide is a fluorescent protein protease reporter.
Claim 65 of ‘389 is directed to a polynucleotide encoding one or more elements (i)-(iv) of the detection composition of claim 43.
Claim 66 of ‘389 is directed to a vector system comprising one or more vectors encoding one or more of elements (i)-(iv) of the detection composition of claim 43.
Claim 67 of ‘389 is directed to an engineered cell modified to express elements (i) and (iii) of the detection composition of claim 43, optionally wherein the engineered cell is further modified to express element (iv) of the detection composition, optionally wherein the engineered cell is further modified to express element (ii) of the detection composition.
Claim 70 of ‘389 is directed to A method for screening cell perturbations comprising: introducing a perturbation to a cell population comprising engineered cells of claim 67, along with any elements of the detection composition not already expressed by the engineered cells, and wherein the guide molecules are configured to detect one or more target transcripts associated with a specific cell type or cell state; activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase to produce a detectable product and/or signal; and detecting an ability of the perturbation to modify expression of the one or more target transcripts by measuring a change in the detectable product and/or signal relative to a controls optionally wherein activating the peptidase further results in activation or generation of one or more signal amplification molecules.
Claim 71 of ‘389 is directed to a method of detecting target polynucleotides in samples comprising: combining a sample or a component thereof with the detection composition as in claim 43; and activating the peptidase via binding of the RAMP polypeptide-guide molecule complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is produced, thereby detecting the target polynucleotide in the sample, optionally wherein activating the peptidase further comprises binding and/or interaction of a target polynucleotide or region thereof with the peptidase, optionally further comprising amplifying and/or enriching the target polynucleotide, optionally wherein the method does not include amplifying and/or enriching the target polynucleotide, optionally wherein activating the peptidase further results in activation or generation of one or more signal amplification molecules.
Claim 76 of ‘389 is directed to a method of labeling cells comprising: introducing the detection composition as in claim 43 into a population of cells, wherein the guide molecule is configured to detect one or more target transcripts associated with a particular cell type or cell state; and activating the peptidase via binding of the RAMP polypeptide-guide molecule complex to the one or more target transcripts such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is generated, thereby labeling cells within the cell population expressing the one or more target transcripts, optionally wherein labeled cells are further sorted or isolated based on production of the detectable product and/or signal.
Claim 78 of ‘389 is directed to a method of in vivo effector activation or delivery comprising: introducing a programmable nuclease system of claim l into a cell comprising the target polypeptide, optionally wherein the target polypeptide is optionally tethered to a cellular structure and wherein the target polypeptide is coupled to an effector, optionally wherein the effector a. is capable of producing a detectable signal when activated; b. is a therapeutic molecule or prodrug; c. is a genetic modifying molecule; d. is a transcription factor; or e. any combination thereof; optionally wherein the effector is inactive when coupled to an uncleaved target polypeptide, optionally wherein the effector is inactive when coupled to a cleaved target polypeptide portion, optionally further comprising cleaving the target polypeptide by the peptidase in response to a target RNA and activation of the peptidase of the programmable nuclease-peptidase composition, optionally wherein cleaving the target polypeptide is in response to binding of the RAMP-guide molecule complex to the target RNA, optionally wherein the target to the cell or is exogenous to the cell, optionally wherein the target polypeptide is tethered to a cell membrane, a nuclear membrane, a cytoskeleton, or other cellular structure.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DALTON KIEFER, PhD whose telephone number is (571)272-1235. The examiner can normally be reached M-F 7:30-5 EST.
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.
/DALTON EDWARD KIEFER/Examiner, Art Unit 1652
/ROBERT B MONDESI/Supervisory Patent Examiner, Art Unit 1652
Prosecution Insights