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 .
Information Disclosure Statement
Initialed and dated copy of Applicant’s information disclosure statement (IDS) filed on 11/18/2023 is attached to the instant Office action. The submission is in compliance with the provisions of 37 C.F.R. § 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of the Claims
Claims 1, 3, 10, 13-15, 28-29, 31, 37, 43-44, 68, 72-73, 75-76, 81, 86-87, 92, and 103 are pending and are examined in this Office action.
Claim Objections
Claims 81 and 103 recite in step a. the phrase “an expression construct” when referencing the construct of preceding claim 1. It is suggested to replace the phrase “an expression construct” with ---the expression construct---.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. § 112(b):
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.
Indefiniteness
Claims 10, 14, 29, 31, 43, 68, and 76 are rejected under 35 U.S.C. § 112(b), as being indefinite for failing to particularly point out and distinctly claim the subject matter which Applicants regard as the invention.
Claims 10 and 29 recite the limitation “an amino acid sequence” of SEQ ID NO:… It is unclear if the claims require the whole sequence or only a fragment, for example a dimer. The phrase “an amino acid sequence” rather than “the amino acid sequence” encompasses oligopeptides or polypeptides that comprise the full-length sequence or any portion of the recited SEQ ID NOs including dimers or larger (oligo)peptides, the specific utility of which is unclear.
Claims 14, 31, 43, 68, and 76 recite the limitation “a nucleic acid sequence” of SEQ ID NO:… It is unclear if the claims require the whole sequence or only a fragment, for example a dimer. The phrase “a nucleic acid sequence” rather than “the nucleic acid sequence” encompasses oligonucleotides or polynucleotides that comprise the full-length sequence or any portion of the recited SEQ ID NOs including dimers or larger (oligo)nucleotides, the specific utility of which is unclear.
In the interest of compact prosecution, the claims are nonetheless examined.
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 of this title, 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, 3, 10, 13-15, 28-29, 31, 37, 43-44, 68, 72-73, 75-76, 81, 86-87, 92, and 103 are rejected under 35 U.S.C. § 103 as being unpatentable over ROSBASH (Rosbash et al., United States Patent Application Publication No. 2019/0390186 A1, published December 26, 2019) in view of KEENE (Keene and Lager, United States Patent Application Publication No. 2017/0327866 A1, published November 16, 2017), DICKINSON (Dickinson et al., PCT International Patent Application Publication No. WO 2020/142676 A1, published 09 July 2020), and BONG (Bong et al., Korean Patent Application Publication No. KR 101394333 B1, published May 1, 2014; English machine translation attached to this Office action).
The claims are drawn to an expression construct for tethering a protein of interest to a target RNA molecule, which comprises a promoter operably linked to a nucleic acid sequence encoding an engineered protein, the engineered protein comprising an RNA-binding polypeptide comprising RNA recognition motifs (RRMs) A-B of a Bruno-like RNA-binding protein, wherein the RNA-binding polypeptide is fused to a protein or polypeptide of interest, wherein the nucleic acid sequence encoding the engineered protein comprises: a. a first nucleic acid sequence encoding the RNA-binding polypeptide and a second nucleic acid sequence encoding the protein or polypeptide of interest upstream or downstream of the first nucleic acid sequence; or b. a first nucleic acid sequence encoding the RNA-binding polypeptide, and a second nucleic acid sequence comprising a cloning sequence upstream or downstream of the first nucleic acid sequence, whereby a third nucleic acid sequence encoding the protein or polypeptide of interest cloned into the cloning sequence generates the nucleic acid sequence encoding the engineered protein; wherein an amino acid sequence comprising an RRM C of the Bruno-like RNA-binding protein is absent from the RNA-binding polypeptide and wherein the RNA-binding polypeptide recognizes and specifically binds an RNA sequence comprising one or more Bruno-like protein binding sites in the target RNA molecule to thereby tether the protein or polypeptide of interest to the target RNA molecule.
ROSBASH teaches compositions and methods for identifying RNA binding polypeptide targets, and teaches fusion polypeptides comprising an RNA binding polypeptide operationally linked to an RNA modifying enzyme ( e.g., adenosine deaminase, cytidine deaminase ), and methods of use therefore (entire document; see Title, Abstract, ¶0008, for example).
Regarding instant claim 1, ROSBASH teaches a vector containing a fusion polypeptide operably linked to a promoter positioned for expression in a cell (i.e., expression construct) (¶0015). The invention uses a targets of RNA-binding proteins identified by editing system, a method that couples an RBP (RNA binding protein) to the catalytic domain of the RNA-editing enzyme and expresses the fusion polypeptide in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. The system was used to identify the targets of three RBPs (¶0159). The expression vector can include a native or nonnative promoter operably linked to the nucleotide sequence encoding the fusion polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the fusion polypeptide (¶0055). By "fusion polypeptide" is meant a polypeptide or protein that combines at least two amino acid sequences that are not naturally contiguous (i.e., engineered protein comprising a RNA-binding polypeptide fused to a protein or polypeptide of interest; i.e., first, second, and third nucleic acid sequence encoding the engineered protein) (¶0058). The fusion polypeptide is preferably produced by standard recombinant DNA techniques. For example, a DNA molecule encoding the first polypeptide is ligated to another DNA molecule encoding the second polypeptide, and the resultant hybrid DNA molecule is expressed in a host cell to produce the fusion polypeptide (i.e., engineered protein comprising a RNA-binding polypeptide fused to a protein or polypeptide of interest; i.e., first, second, and third nucleic acid sequence encoding the engineered protein). The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered, e.g., the DNA molecules are ligated to each other in-frame (i.e., a first nucleic acid sequence encoding the RNA-binding polypeptide and a second nucleic acid sequence encoding the protein or polypeptide of interest upstream or downstream of the first nucleic add sequence) (¶0059).
ROSBASH teaches RNA recognition motifs (RRMs). For example, Fig.1C provides a schematic depicting how the dsRBDs of ADAR were replaced with the RBP of interest. The editing specificity of the fusion polypeptide was determined by the RNA recognition features of the RBP, and the target transcript was permanently marked by a novel editing event (¶¶0089, 0180).
ROSBASH also teaches mutations of RNA recognition motifs (i.e., an amino acid sequence comprising an RRM is absent from the RNA-binding polypeptide) (¶0236).
ROSBASAH teaches the use of various linkers, for example edited sites for Thor-ADAR and Thor-linker-ADAR fusion polypeptides (¶¶0150-0151).
ROSBASH does not explicitly teach an engineered protein comprising an RNA-binding polypeptide comprising RNA recognition motifs (RRMs) A-B of a Bruno-like RNA-binding protein. However, such claimed compositions and methods would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application for the following reasons.
KEENE teaches methods for in vivo identification of endogenous mRNA targets of microRNAs (entire document; see Title, Abstract, for example).
KEENE teaches that the ligand used to carry out the invention is an RNA binding protein selected from the RNA Recognition Motif (RRM) family of cellular proteins involved in pre-messenger RNA processing. More than 200 members of the RRM superfamily have been reported, the majority of which are ubiquitously expressed and conserved in phylogeny. They include hnRNP proteins (A, B, C, D, E, F, G, H, I, K, L), RRM proteins CArG, DT-7, PTB, K1, K2, K3, HuD, HUC, rbp9, elF4B, sxl, tra-2, AUBF, AUF, 32KD protein, ASF/SF2, U2AF, SC35, and other hnRNP proteins. Tissue-specific members of the RRM family include IMP, Bruno, AZP-RRMI, X16, and Bj6 (¶0055).
DICKINSON teaches systems and methods for modulating RNA, and provides methods for modifying a target RNA of interest (entire document; see Title, Abstract, for example).
In Table 2, DICKINSON teaches a finite number of RNA regulatory domains from proteins that include Bruno-like RNA binding proteins, specifically BRUNOL4, BRUNOL5, and BRUNOL6.
DICKINSON teaches RNA recognition motifs (RRMs), and protein fusion to a nucleotide linker (¶¶0072, 0234).
DICKINSON teaches that one or more proteins of the disclosure may be operably linked to another protein through linkage to a pair of accessory proteins that have a strong affinity for each other. Such accessory proteins are known in the art. For example, the Sun Tag is one such system that includes an antibody with a strong affinity for a peptide. One protein, polypeptide, or domain of the disclosure may be linked to a SunTag peptide and another protein, polypeptide, or domain of the disclosure may be linked to an antibody to allow operable linkage of the two proteins, polypeptides, or domains through the interaction of the Sun Tag peptide and antibody. Further examples include biotin and avidin/streptavidin and spytag and spycatcher (¶0021) (cf. instant claims 3, 10, 14). DICKINSON also teaches oligonucleotide analogs with modifications that improve binding affinity, and/or improve binding specificity (¶0122).
DICKINSON teaches an RNA targeting system that comprises (1) an RNA hairpin-binding protein that serves as the core of the system and is a selective, high affinity binder to a specific RNA structure displayed on an engineered gRNA, (2) a gRNA that features both the structure that interacts with the engineered hairpin-binding protein and a sequence with complementarity to the target RNA of interest, and (3) an effector protein, such as a nuclease or epitranscriptomic regulator, that acts on the targeted RNA in a proximity-dependent manner (¶0196).
DICKINSON teaches an RNA targeting system that comprises (1) an RNA hairpin-binding protein that serves as the core of the system and is a selective, high affinity binder to a specific RNA structure displayed on an engineered gRNA, (2) a gRNA that features both the structure that interacts with the engineered hairpin-binding protein and a sequence with complementarity to the target RNA of interest, (3) a charged protein that could bind to the displayed gRNA sequence non-specifically to stabilize and protect the guiding RNA prior to target engagement, and (4) an effector protein, such as a ribonuclease or epitranscriptomic regulator, that acts on the targeted RNA in a proximity-dependent manner (FIG. 12A). While Cas13 houses all of these functional components in a single protein domain , DICKINSON envisioned engineering a system that combines multiple protein domains that each perform one of these functions, which the inventors termed CRISPR/Cas-inspired RNA targeting system (CIRTS). CIRTS vary in their module composition and are uniquely numbered as listed in FIG. 12A and FIG. 21 (¶0212) (cf. instant claims 73, 75-76, 86-87).
DICKINSON teaches the use of a flag tag in the system, as SEQ ID NO:14 (see Table with sequences), and teaches the separate delivery of each gRNA along with CIRTS-0 fused to a 3x FLAG-tag (cf. instant claims15, 28, 37).
DICKINSON teaches the use of reporter proteins (¶0200) (cf. instant claim 44).
DICKINSON further teaches and claims (claim 93 of DICKINSON, for example) a kit comprising the taught and claimed systems (cf. instant claim 103).
BONG teaches the effects of AtBRN (Arabidopsis thaliana Bruno RNA-binding protein in transgenic plants (entire document; see Title, Abstract, for example).
BONG teaches AtBRN1 (At4g03110) and AtBRN2 (At1g03457), previously reported as AtBRUL-1 or AtBRUI-2, which belong to the RNA binding protein-family with Bruno proteins. Bruno protein was first reported in Drosophila, an RNA-binding protein containing RNA-motif, which selectively splices in a variety of organisms, regulates mRNA translation inhibition and migration. Bruno protein binds to eIF4E, a synthesis initiator (Background section).
BONG further teaches that transcriptional regulation, including mRNA transport, stability and translation, plays an important role in plant growth and development, and most of these processes are directly or indirectly mediated by RNA binding proteins (RBPs). The KH- and RNA-sensing motif (RRM)-like RNA binding proteins are known to be involved in plant flowering. In the step of inhibiting translation of the target RNA, Bruno protein binds to eIF4E, a synthesis initiator, and inhibits formation of eIF4E-eIF4G complex, ultimately inhibiting 40S ribosomal formation. The Bruno protein functions as a Bruno-CUP-eIF4E complex, and Bruno genomic homologues of Arabidopsis are FCA, AtBRN1 and AtBRN2 (Background section).
Figure 1 of BONG depicts the protein structure of AtBRN1 or AtBRN2 (a), wherein each protein contains three RNA motifs (RRMs). It also depicts the genomic DNA structure and T-DNA insertion site (left) of the gene coding for AtBRN1 or AtBRN2, and atbrn1 / atbrn2-3 duplexes (d). See excerpts of Figure 1 below (cf. instant claims 72, 81, 92).
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The AtBRN2 protein taught by BONG is 72.6% (i.e., about 75%) similar to instant SEQ ID NO:33; see alignment below (cf. instant claim 10).
ALIGNMENT OF INSTANT SEQ ID NO:33 AND SEQ ID NO:4 FROM BONG
RESULT 24
BBH02698
ID BBH02698 standard; protein; 438 AA.
XX
AC BBH02698;
XX
DT 25-SEP-2014 (first entry)
XX
DE Arabidopsis thaliana Bruno RNA-binding protein 2 (AtBRN2), SEQ 4.
XX
KW BRN2 protein; Bruno RNA-binding protein 2; antigen; crop improvement;
KW flowering; plant; seed; transgenic plant.
XX
OS Arabidopsis thaliana.
XX
CC PN KR1394344-B1.
XX
CC PD 13-MAY-2014.
XX
CC PF 14-FEB-2013; 2013KR-00015905.
XX
PR 14-FEB-2013; 2013KR-00015905.
XX
CC PA (UYMY-) UNIV MYONGJI IND & ACAD COOP FOUND.
XX
CC PI Abbasi N, Bong CS, Sae KH;
XX
DR WPI; 2014-J95678/57.
DR N-PSDB; BBH02696.
XX
CC PT Controlling flowering time in plants, preferably dicotyledonous plants,
CC PT by isolating Arabidopsis thaliana Bruno RNA-binding protein from
CC PT Arabidopsis, introducing gene into vector, and transforming vector into
CC PT plant cell.
XX
CC PS Claim 2; SEQ ID NO 4; 20pp; Korean.
XX
CC The present invention relates to a novel method for controlling flowering
CC time in plants, preferably dicotyledonous plants. The method involves
CC isolating Arabidopsis thaliana Bruno RNA-binding protein (AtBRN) gene,
CC introducing the gene into a vector, and transforming the vector into a
CC plant cell. The invention further provides: (1) a method for producing a
CC transgenic plant for controlling flowering time of the plants; (2) a
CC method for regulating the flowering time in the transgenic plant; (3) a
CC seed of the transgenic plant; and (4) a composition for controlling
CC flowering time of the plants. The present sequence represents a
CC specifically claimed AtBRN2 protein (At1g03457), whose coding sequence is
CC used in the vector for preparing the transgenic plant of the invention
CC for controlling flowering time.
XX
SQ Sequence 438 AA;
Query Match 76.2%; Score 772.5; Length 438;
Best Local Similarity 72.6%;
Matches 151; Conservative 23; Mismatches 19; Indels 15; Gaps 2;
Qy 1 MAEAKEENREKNEEEESVKLFVGQIPKHMSESQLLTLFQEFAVVDEVNIIKDKITRASRG 60
||| | | || |||||||:||||:| ||||||:||::|:||||||:| ||| ||
Db 1 MAE------ETMENEERVKLFVGQVPKHMTEIQLLTLFREFSIVNEVNIIKEKTTRAPRG 54
Qy 61 CCFLLCPSREEADKLVNACHNKKTLPGANSLLQVKYADGELERL---------EHKLFVG 111
|||| ||:||:|||::|: |||||||||:| ||||||||||||| |||||||
Db 55 CCFLTCPTREDADKVINSFHNKKTLPGASSPLQVKYADGELERLDVLDCSCNPEHKLFVG 114
Qy 112 MLPKNVSEAEVQSLFSKYGTIKDLQILRGAQQTSKGCAFLKYETKEQAVSAMESINGKHK 171
|||||||| |||||||:||||||||||||: |||||| |||||:|||||:|||::||:|
Db 115 MLPKNVSETEVQSLFSEYGTIKDLQILRGSLQTSKGCLFLKYESKEQAVAAMEALNGRHI 174
Qy 172 MEGSTVPLVVKWADTERERHTRRLQKAQ 199
|||: |||:|||||||:|| ||| | |
Db 175 MEGANVPLIVKWADTEKERQARRLLKVQ 202
BONG teaches vectors constructed using the pRH5 'and pYESTrp3 vectors (Invitrogen) as hybrid RNA (MS2-SOC1) and fusion protein (VP16 AD-AD-AtBRNs) plasmids. The full-length SOC1 cDNA (1,293 bp, SEQ ID NO: 5) contained 5 'UTR and 3' UTR and was amplified by RT-PCR using primers 126F (SEQ ID NO: 18) and 126R (SEQ ID NO: 19) Cloning into Aat II and Sma I sites of pRH5 'vector resulted in pMS2-SOC1. PCR was performed using primers containing restriction enzyme sequences for cloning. PCR primers were 124F (SEQ ID NO: 20) and 124R (SEQ ID NO: 21) for AtBRN1, 125F (SEQ ID NO: 22) and 125R (SEQ ID NO: 23) for AtBRN2. The amplified DNA fragments to the AtBRN1 cut by treatment with restriction enzymes Eco R1 and Not I, to the AtBRN2 After a cloned into pYESTrp3 vector after cutting by treatment with restriction enzymes Hind III and Not I pVP16-AtBRN1 or pVP16-AtBRN2 (Plasmid structure section).
BONG teaches transforming plant cells with a recombinant vector comprising a gene encoding Arabidopsis thaliana Bruno RNA-binding protein (ATBRN) derived from Arabidopsis thaliana , and also teaches transgenic plants comprising double BRN1/BRN2 mutants (Description of Embodiments section).
In Example 3, BONG teaches the AtBRN1 or AtBRN2 overexpression of the gene SOC1 101-D To inhibit the extremely early flowering phenotype. This showed that AtBRN2 interacts with SOC1 mRNA in the plant body (cf. instant claim 68).
BONG specifically teaches the use of plant promoters and terminators. A "plant promoter" is a promoter capable of initiating transcription in plant cells, where constitutive promoters (i.e., UBQ10, ubiquitin) do not limit selectivity. BONG teaches that, in the plant expression vectors of the invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium and the terminator of the Octopine gene of tumefaciens (i.e., OCS), but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the invention (Description of Embodiments section) (cf. instant claims 37, 43).
Given the teachings of ROSBASH, DICKINSON, KEENE, and BONG as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by ROSBASH, DICKINSON, KEENE, and BONG, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of identification of mRNA targets and RNA binding polypeptide targets, as taught by ROSBASH, DICKINSON, KEENE, and BONG.
Although ROSBASH, DICKINSON, KEENE, and BONG do not explicitly reduce to practice every single limitations of all of the instant claims (e.g., the full-length instantly claimed SEQ ID NOs: 3, 5, 18-19, 33, 38), these particular embodiments would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art.1 The cited art teaches the instantly claimed inventive concept, and in general the recited elements of the instant claims. Thus the recited limitations (nucleic acid and amino acid sequences) would be desirable optimization(s) of the compositions and methods taught by the cited references, and merely a design choice that would be readily apparent to one of ordinary skill in the art, absent evidence to the contrary.
Summary
No claim is allowed.
Examiner’s Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRATISLAV STANKOVIC whose telephone number is (571) 270-0305. The examiner can normally be reached Monday-Friday, 08:00-17:00 h EST.
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BRATISLAV STANKOVIC, PhD, JD
Primary Patent Examiner
Art Unit 1663
/BRATISLAV STANKOVIC/Primary Examiner, Art Unit 1663
1 For example, instant SEQ ID NO:5 is only seven nucleotides long.