DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Interpretation
Under the broadest reasonable interpretation consistent with the specification, a “degradation domain (DD) sequence” is an amino acid sequence that, when fused or tagged to a protein or protein fragment, promotes degradation of the tagged protein/fragment in the absence of a corresponding stabilizing factor, and permits stabilization in the presence of that factor. The specification states that a protein having a DD is degraded by the proteasome, but that degradation is suppressed when a DD-specific stabilizing substance is present. See [0013]. Examples include DHFR-derived DD/TMP, FKBP-derived DD/Shield-1, FKBP12 mutant-derived DD/Shield-1 or Shield-2, FKBP12 mutant-derived DD/rapamycin, UnaG-derived DD/bilirubin, and mouse DHFR-derived DD/methotrexate. See [0013]. Thus, the term is not interpreted as any ligand-binding domain; it requires a degradation/destabilization function.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1–10 and 13–15 are rejected under 35 U.S.C. 103 as being unpatentable over Dunn et al. (WO 2018/094195 A1, published 5/24/2018 “Dunn”) in view of Wandless et al. (US 2009/0215169 A1, published 8/27/2009 “Wandless”).
Regarding claim 1, Dunn discloses split protein systems comprising first and second fusion proteins, each comprising a fragment of a protein of interest fused to a ligand-binding or interactive domain, wherein the fragments dimerize or fold together to restore activity of the protein. Dunn discloses compositions and kits comprising first and second fusion proteins whose protein fragments can dimerize or fold together. See Dunn, pp. 4–6 and 50–55. Dunn further describes templated assembly of functionally active proteins by dimerization or folding from protein fragments associated with modified oligonucleotides, and states that template-mediated dimerization or folding is applicable to activation of specific proteins. See Dunn, pp. 9–10 and 22–23. Dunn also provides working examples using split Gaussia luciferase fragments fused to FKBP/FKBP mutant domains, where assembly/dimerization restores luciferase activity. See Dunn, pp. 113–118.
Dunn does not expressly teach that each protein fragment is tagged with a degradation domain sequence.
Wandless teaches ligand-dependent destabilizing/degradation domains derived from FKBP and DHFR, wherein a protein of interest is fused in-frame to a stability-affecting/destabilizing protein. Wandless teaches that the destabilizing protein destabilizes the protein of interest in the absence of ligand and stabilizes the protein in the presence of ligand. See Wandless [0056]–[0065]. Wandless specifically teaches FKBP-derived destabilizing domains stabilized by FKBP ligands, including Shield-1, and DHFR-derived destabilizing domains stabilized by DHFR ligands, including trimethoprim. See Wandless [0068]–[0076]. Wandless further explains that a genetic fusion of a destabilizing domain to a protein of interest results in degradation of the entire fusion protein and that addition of ligand protects the fusion protein from degradation. See Wandless [0088] and [0142]. Wandless also teaches that degradation is mediated at least in part by the proteasome. See Wandless [0147] and [0212].
It would have been prima facie obvious to one of ordinary skill in the art as of the effective filing date to modify Dunn’s split protein fragments by tagging each fragment with a ligand-dependent degradation/destabilizing domain as taught by Wandless. The motivation would have been to provide rapid, reversible, ligand-dependent control over the stability and abundance of each split protein fragment, thereby controlling the amount of assembled active protein. Dunn teaches that split protein fragments tolerate fusion to ligand-binding/dimerization domains and recover activity upon folding or dimerization. See Dunn, pp. 22–23, 50–55, and 113–118. Wandless teaches that destabilizing domains are modular and may be fused to the N-terminus or C-terminus of many different proteins to regulate protein stability with small molecules. See Wandless [0142]–[0152] and [0177]–[0184]. The modification therefore represents application of a known protein-stability control technique to known split-protein fusion constructs, with predictable results and a reasonable expectation of success.
Regarding claims 2–5, Dunn teaches split protein fragments having binding regions, including ligand-binding domains and interactive protein domains, that bring protein fragments into proximity and promote folding or dimerization. See Dunn, pp. 16–18, 22–24, and 50–52. Dunn further teaches exogenous interactive protein domains, including c-Jun/c-Fos, c-Myc/Max, NZ/CZ, and related leucine zipper or dimerization domains. See Dunn, pp. 18–22, 33–35, and 50–52. Dunn repeatedly discloses systems using two protein fragments, including first and second fusion proteins and N-terminal and C-terminal split protein fragments. See Dunn, pp. 22–23, 50–55, and 113–118. Thus, the additional limitations of claims 2–5 are taught or rendered obvious by Dunn.
Regarding claims 6 and 14, Dunn teaches reporter-based split proteins, including sfGFP, Renilla luciferase, and Gaussia luciferase, and also discloses cytotoxic/pro-apoptotic and transcription-related protein embodiments. See Dunn, pp. 51–55 and 113–118. Wandless also teaches destabilizing-domain fusion proteins involving diverse proteins, including luciferase, CREB, Oct3/4, Sox2, Nanog, c-Myc, Klf4, and other proteins. See Wandless [0151]–[0152] and [0218]. At least the reporter-based and reprogramming-related alternatives are therefore taught or rendered obvious.
Regarding claims 7 and 15, Wandless teaches FKBP-derived and FKBP12 mutant-derived destabilizing domains stabilized by Shield-1 and DHFR-derived destabilizing domains stabilized by trimethoprim. See Wandless [0068]–[0076], [0142]–[0147], and [0167]–[0176]. Thus, the recited DHFR-derived, FKBP-derived, and FKBP12 mutant-derived DD sequence alternatives are taught by Wandless.
Regarding claims 8 and 9, Dunn teaches coding sequences and expression of fusion proteins comprising protein fragments fused to ligand-binding domains. See Dunn, pp. 106–112 and 113–116. Dunn also teaches cloning and expression constructs for fusion proteins. See Dunn, pp. 106–107 and 111–116. Wandless teaches nucleic acids encoding fusion proteins comprising proteins of interest fused to destabilizing domains, as well as expression vectors and cells comprising nucleic acids encoding destabilizing-domain fusion proteins. See Wandless [0057]–[0064], [0082]–[0086], [0191], [0195], and [0199]–[0200]. It would have been obvious to provide nucleic acids and expression vectors encoding each modified DD-tagged split protein fragment in order to express the fusion proteins in cells.
Regarding claim 10, Dunn teaches compositions and kits comprising split fusion proteins and components for directed protein assembly. See Dunn, pp. 4–6, 56–57, and 99–100. Wandless teaches kits comprising DD-fusion protein systems, including nucleic acids, ligands, and instructions for use, and also teaches expression vectors and cells comprising nucleic acids encoding DD-fusion proteins. See Wandless [0080]–[0082], [0191], and [0195]. The recitation of a “genome editing kit” is interpreted as an intended use of the claimed kit and does not further limit the structure of the kit absent additional recited genome-editing-specific components.
Regarding claim 13, Dunn teaches methods for directed assembly of proteins using split fusion proteins and haplomer systems. See Dunn, pp. 57–62, 100–105, and 116–118. Wandless teaches introducing nucleic acids encoding destabilizing-domain fusion proteins into cells and regulating fusion protein stability by addition or removal of ligand such as Shield-1 or trimethoprim. See Wandless [0082]–[0086], [0142]–[0149], and [0191]. It would have been obvious to regulate protein activity in a cell by introducing the modified DD-tagged split protein fragments of Dunn as modified by Wandless, and introducing a DD-specific stabilizing factor to stabilize the fragments and allow assembly/reconstitution of activity.
Claims 11–12 are rejected under 35 U.S.C. 103 as being unpatentable over Dunn et al. (WO 2018/094195 A1; “Dunn”) in view of Wandless et al. (US 2009/0215169 A1; “Wandless”), as applied to claim 1 above, and further in view of Schmelas C, Grimm D. “Split Cas9, Not Hairs - Advancing the Therapeutic Index of CRISPR Technology.” Biotechnol J. 2018 Sep;13(9):e1700432 (“Schmelas”), and Senturk et al. “Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.” Nat Commun. 2017 Feb 22;8:14370 (“Senturk”).
Regarding claim 11, Dunn and Wandless teach the DD-tagged split-protein system as set forth above with respect to claim 1. Dunn and Wandless do not expressly teach that the DD-tagged split protein fragments are Cas9 nuclease fragments.
Schmelas further teaches split Cas9 systems in which Cas9 is separated into fragments and reconstituted into functional Cas9 in cells, including rapamycin-controlled FKBP/FRB split Cas9, light-regulated split Cas9, sgRNA-scaffold split Cas9, and intein split Cas9 systems. See Schmelas, Abstract and sections 3.1–3.4. Senturk teaches DD-Cas9, in which Cas9 is fused to an FKBP12-derived destabilizing domain, specifically FKBP12 F36V/L106P, and stabilized by Shield-1, providing rapid, reversible, dose-dependent control of Cas9 expression and genome editing. See Senturk, Abstract, Fig. 1A–1C, and Methods—Plasmids.
It would have been prima facie obvious to one of ordinary skill in the art as of the effective filing date to apply the DD-tagging strategy taught by Wandless and Senturk to the split Cas9 nuclease fragments taught by Schmelas, in view of Dunn’s split-protein framework, to obtain chemically controllable genome-editing fragments. Therefore, claim 11 is rendered obvious.
Regarding claim 12, Dunn and Wandless teach the use of nucleic acids and expression constructs/vectors encoding fusion proteins, as set forth above with respect to claims 8–9. Schmelas teaches vector-compatible split Cas9 systems, including AAV/split Cas9 concepts. See Schmelas, Abstract and sections 2–3. Senturk teaches lentiviral vectors encoding DD-Cas9 and demonstrates ligand-dependent genome editing. See Senturk, Fig. 1A–1C and Methods—Plasmids.
It would have been prima facie obvious to one of ordinary skill in the art as of the effective filing date encode DD-tagged split Cas9 fragments in expression vectors for genome-editing use because expression vectors were conventional for delivering Cas9 and DD-fusion constructs, and the art provided a clear motivation to chemically control Cas9 activity.
Conclusion
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/CHRISTOPHER M BABIC/ Supervisory Patent Examiner, Art Unit 1633
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