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 .
Election/Restrictions
Applicant’s election without traverse of Invention I, encompassing claims 1-15, 17-22 and 24-25 and the species of leucine for the anticodon, proline for the acceptor stem, and SEQ ID NO: 12 in the reply filed on 09/14/2026 is acknowledged. However, the specification identifies SEQ ID NO: 12 as SWTX51, which suppresses a proline codon and incorporates leucine [p. 21], whereas the leucine-codon/proline-acceptor embodiments are identified with SEQ ID NOs: 3 and 4 [p. 20]. The sequence identifier and functional description therefore identify different disclosed species. For purposes of the present examination, applicants’ express identification of SEQ ID NO: 12 is understood to control the species election. Examination therefore proceeds on the SEQ ID NO: 12 species, namely a sstRNA having an anticodon specific for a proline codon and leucine-accepting specificity. The inconsistent reference to leucine-codon recognition and proline acceptance is not treated as an additional election of the species of SEQ ID NOs: 3 or 4. Clarification is required as to the species elected and the claims encompassing that species.
Applicants should identify any disagreement with this interpretation in their reply.
Claims 1-34 are pending. Claims 2-4, 5-6, 16, 8-9, 23, and 26-34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention and species, there being no allowable generic or linking claim. Claims 1, 5-7, 10-15, 17-22, and 24-25 are being examined on the merits.
Priority
The application is a 371 PCT of US2021/065508 filed 12/29/2021 that claims priority to applications 63/132,932 filed 12/31/2020, 63/151,436 filed 02/19/2021 and 63/151,416 filed 02/19/2021.
Information Disclosure Statement
The information disclosure statements filed 10/09/2024 and 01/02/2026 have been considered.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
1. Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c). Sequence identifiers for sequences (i.e., “SEQ ID NO:X” or the like) must appear either in the drawings or in the Brief Description of the Drawings (see Fig. 1).
Required response – Applicant must provide:
Amended drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers (i.e., “SEQ ID NO:X” or the like) into the Brief Description of the Drawings, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
2. Specific deficiency - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1), 1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete.
Required response - Applicant must:
• Provide a substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Specification
The disclosure is objected to because of the following informalities:
The p. 2 summary uses substitution wording opposite to the ordinary reading of the detailed mechanism. For example, the proline-accepting/leucine-decoding embodiment says it can “substitute leucine for proline,” while p. 7 explains replacement of leucine by proline. It may be remedial to recite the direction as “incorporates [amino acid] at a [codon]” to avoid reversal.
SWTX51 is assigned SEQ ID NO: 12 on p. 21, but the SWTX51 cassette, SEQ ID NO: 23 on p. 23, contains the printed tRNA sequence of SEQ ID NO: 11, assigned to SWTX50. Conversely, the SWTX52 cassette, SEQ ID NO: 24, contains SEQ ID NO: 12, assigned to SWTX51. This is especially important since species 12 is elected: an experimental label “51” cannot uncritically be treated as proof that sequence 12 was the construct tested.
Examples 4 and 6 describe SWTX2/3 as separate-plasmid counterparts of SWTX51/52 [p. 25]. The corresponding sequence/name assignments at pp. 19–24 need reconciliation before attributing an individual experimental effect to the elected sequence.
SWTX55/57 have lowercase agg in SEQ ID NOs: 14/15 but aag in SEQ ID NOs: 25/26 [pp. 21–22, 24].
The p. 12 statement that the sstRNA is “in the form of cDNA” should distinguish RNA from DNA encoding it.
The p. 1 statement “DNA is translated by ribosomes” should be corrected to distinguish transcription of DNA and translation of mRNA.
The specification indicated a “red asterisk” in figure 21; however, drawing are not in color.
Appropriate correction is required.
Claim Objections
Claims 7 and 9 are objected to because of the following informalities: Each recites “wherein the the sstRNA.” It would be remedial to delete the repeated “the.” Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 7, 11, and 13 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.
Regarding claim 7, the claim recites an sstRNA encoded by “a sequence as set forth in” multiple SEQ ID NOs joined by “and.” It is unclear whether the listed sequences are alternatives defining individual sstRNAs, whether multiple sstRNAs encoded by all listed sequences are required, or whether a single encoding molecule must contain all listed sequences. The specification separately identifies the sequences with named SWTX constructs [pp. 19–22, Examples 1–2]. Applicant should clarify the relationship, for example by reciting “any one of SEQ ID NOs: …” if alternatives are intended, or expressly defining a required combination.
Claim 11 requires “a total length of less than 150 nucleotides or 300 nucleotides.” It is unclear whether “less than” applies to both numerical alternatives, or whether the second alternative is exactly 300 nucleotides. These constructions differ for lengths of 150–299 nucleotides and for exactly 300 nucleotides. Claims 12 and 13 incorporate the ambiguity. Clarify the intended limits. If the alternatives are less than 150 and less than 300, the broader union is simply less than 300; a separate dependent claim can retain the narrower limit.
Claim 13 requires an RNA oligonucleotide that encodes an sstRNA. It is unclear whether the RNA oligonucleotide is intended to be the mature sstRNA itself, a precursor RNA from which the sstRNA is processed, or an RNA template from which the sstRNA is produced. These alternatives involve different structural or functional relationships between the claimed oligonucleotide and the sstRNA. The statement that the encoding oligonucleotide may be RNA does not resolve that relationship. [Specification, p. 2, final paragraph.] Applicants are required to clarify the relationship intended by “encodes” in the RNA embodiment.
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 24 and 25 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 the preparation and expression of identified sense suppressor tRNA constructs and reporting cell death following transfection with a SWTX 2 and SWTX3, does not reasonably provide enablement for the full scope of the claimed methods without undue experimentation. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Nature and Scope of the Invention
Claim 24 requires contacting cells with the sstRNA of claim 1 in an amount effective to kill cells, and claim 25 requires such contacting in an amount effective to reduce cell survival. Through claim 1, the claims encompass a genus of sstRNAs having an acceptor stem and a noncognate-codon anticodon; they are not limited to any particular SEQ ID NO, the exemplified amino-acid exchanges, the tested cells, or the disclosed expression conditions. This rejection concerns the full scope of the claimed methods and does not assert that no operative embodiment is disclosed or can be practiced.
For purposes of this analysis, contacting cells with “the sstRNA of claim 1” requires delivery of the RNA species, including carrier-assisted delivery; delivery of an encoding plasmid is not, without further explanation, identical to that step. The claims encompass the materially broader range of sstRNA structures and cell settings discussed below. No particular death mechanism, clinical benefit, selective tumor killing, or numerical efficacy threshold is required by this rejection.
Guidance and working examples.
The specification expressly describes the killing and survival-reduction methods and supplies specific encoding sequences, delivery options, and screening instructions. [pp. 4–5; pp. 10–12; pp. 19–24.] Example 6 expressly reports cell death by 48 hours and widespread death at 72 hours for the SWTX2+3 condition, which is credited as affirmative support. [p. 25, Example 6, lines 20–28; Fig. 20.] That experiment is described in the context of Fugene-transfected HEK293 cells and tRNA expression constructs; the “tRNA alone” designation refers to constructs separated from the reporter plasmid in the preceding explanation. [p. 7, Fig. 20 description; p. 25, Example 4.] The additional experiments report GFP, tdTomato, NanoLuc, or mTOR effects, rather than establishing that those effects necessarily provide a killing or survival-reducing exposure across the claimed genus. [pp. 24–26, Examples 3–7.]
State of the art
The relevant artisan would have been familiar with tRNA engineering, transfection, cell culture, and cellular assays. Direct RNA transfection was known: Ahern (WO 2019/090154 A1) describes synthetic tRNA delivery and an RNA-transfection protocol in HEK293 cells. p. 53, first Discussion paragraph; p. 62, “RNA transfection.”] Berg (Berg et al. Genetics, Vol. 206, 1865–1879. 2017) also demonstrates the biological plausibility of toxic sense-codon mistranslation and functional leucine tRNAs retargeted to proline codons. [pp. 1873–1875, leucine-tRNA Results and Fig. 8.] These teachings weigh in favor of practicing focused embodiments and are not disregarded. They do not, however, provide a generally applicable selection and exposure rule for achieving the claimed endpoints across the sequence and cell breadth of the present claims.
Predictability
The prefiling record provides specific reasons why a functional decoding change is not alone predictive of the claimed cell outcome. Berg 2019 (Berg et al. Genetics, Vol. 213, 849–863. 2019) reports different effects on growth for serine tRNAs retargeted to different noncognate codons, including no observed growth effect for the glutamine-targeting variant under the tested conditions and differing doubling-time effects for the other variants. [p. 858, left column, paragraph 2; Fig. 8C–D.] Berg 2019 also identifies difficulty predicting nucleotide changes that alter stability while maintaining partial activity. [p. 859, right column, paragraph 4] These results provide evidence of sequence/context dependence. Together with the limited endpoint exemplification in the application, they provide a reason to doubt that the entire claim breadth can be practiced by uniformly applying an anticodon change and adjusting the administered amount.
Required experimentation
Beyond the disclosed region, the artisan would need to select an sstRNA and cell context, establish functionally active intracellular RNA exposure, determine the relevant formulation and exposure duration, and identify conditions yielding the claimed cell outcome rather than only altered reporter expression, slowed growth, or a tolerated decoding change. The specification proposes screening and empirical dosing but supplies no comparably general correlation or selection criteria that transfer the killing or survival result across those interacting variables. [p. 5, final paragraph; p. 11; p. 25, Example 6.] Accordingly, the necessary work across the scope is not merely adjustment of a known effective dose for an already established method; it includes identifying additional operative biological combinations through renewed screening without sufficient guidance for extrapolating the desired outcome. The known individual laboratory techniques do not resolve this open-ended selection burden, and the effective-amount limitations identify success without teaching how to obtain it throughout the genus.
For claim 24, reporter suppression, reduced protein abundance, or reduced scratch closure does not by itself establish killing, although the express death report in Example 6 is acknowledged. For claim 25, the required effect may be less than complete killing, but reduced reporter signal or altered proliferation is not necessarily reduced cell survival; the specification does not establish a general relationship between those readouts and the claimed survival endpoint. Considering the breadth and nature of the claims, the relevant skill and known techniques, the substantial but localized guidance and examples, the documented variability, and the resulting experimental burden, the disclosure does not enable the full scope of either method without undue experimentation. The rejection would require reconsideration for a materially narrowed sequence/cell/delivery implementation and is not based solely on the absence of a direct-RNA or animal working example.
Claim Rejections - 35 USC § 101
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claim 22 is rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101).
Claims 22 is drawn to a “cell.” The specification does not provide a limiting definition of a cell and does not expressly exclude cells within a human organism (pages 4, 11, 13). Furthermore, claim 29 explicitly recites “where in the cells are in vivo”. Thus, the term “cell” could reasonably be interpreted as encompassing cells within a human organism, which is non-statutory subject matter. The rejection may be obviated by requiring that the cell be an isolated cell, an in vitro human cell, or a non-human cell.
Claim Rejections - 35 USC § 102
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 1, 5, 6, and 22 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Berg (Berg et al. Genetics 206:1865–1879 (2017)).
Regarding claim 1, Berg discloses engineered leucine tRNAs having an acceptor stem and an anticodon specific for a noncognate sense codon. In particular, Berg describes cloning tL(UAA)B2 and changing its anticodon from UAA to UGG to enable mistranslation of proline codons. [p. 1873, right column, last paragraph - p. 1874, left column, first paragraph.] Berg depicts the leucine tRNA acceptor stem and UGG anticodon and reports selected tL(UGG) suppressor constructs that restore the mutant-cell phenotype under the reported conditions. [p. 1875, Fig. 8A–B and legend.] These tRNAs recognize a proline sense codon while retaining leucine-accepting identity and therefore are sense suppressor tRNAs having the claimed noncognate codon specificity.
Regarding claim 5-6, the disclosed UGG anticodon recognizes a proline codon, and the disclosed molecule is a leucine tRNA retargeted to proline-codon recognition. Berg expressly discusses its aminoacylation by leucyl-tRNA synthetase and depicts the leucine-tRNA acceptor stem. [p. 1873, right column, last paragraph; p. 1875, Fig. 8B.] One selected suppressor contains a 32-base deletion downstream of the tRNA gene, rather than a deletion within the mature tRNA coding sequence. [p. 1874, left column, first paragraph.] Thus, the disclosure includes the claimed proline-reading, leucine-accepting sstRNA.
Regarding claim 22, Berg discloses yeast cells containing the foregoing sstRNAs. Figure 8A and its legend identify strains bearing tL(UGG) suppressor constructs, including the construct with the downstream 32-base deletion, and report the growth phenotype of those cells. [p. 1875, Fig. 8A and legend.] The cells therefore comprise the sstRNA required by claim 1.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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, 5-7, 10–12, 14–15, and 17–21 are rejected under 35 U.S.C. 103 as being unpatentable over Ahern (WO 2019/090154 A1, published May 9, 2019) in view of Berg (Berg et al. Genetics 206:1865–1879 (2017)).
For purposes of the following art rejection, the phrase “encoded by a sequence as set forth in SEQ ID NOs: 1, 2 and 5-13” in claim 7 is understood as identifying alternative complete encoding sequences, including the elected SEQ ID NO: 12. This construction is consistent with the specification’s separate identification of those sequences as encoding individual sstRNA constructs. [Specification, pp. 19–21, Examples 1–2.] It does not treat claim 7 as requiring one sstRNA to be encoded collectively by every listed sequence, or as encompassing arbitrary fragments solely because they occur within a listed sequence. Applicants should clarify the alternative wording if a different scope is intended.
Regarding claim 7, Ahern discloses an anticodon-edited human leucine tRNA encoded by SEQ ID NO: 319, designated LeuTAAchr6.trna135. [p. 76, sequence-table row 23, SEQ ID NO: 319.] Ahern’s listed 83-nucleotide encoding sequence is identical to applicant’s SEQ ID NO: 12 outside the anticodon. Ahern contains the DNA triplet TTA at the anticodon location, whereas applicant contains TGG. The only differences are T-to-G and A-to-G substitutions at linear sequence positions 35 and 36, respectively. [p. 76, SEQ ID NO: 319; applicant specification, p. 21, SWTX51, SEQ ID NO: 12.] The positions stated here are counted directly from the 5′ end of each listed encoding sequence.
Ahern’s disclosed TTA encoding triplet produces a UUA anticodon for stop-codon recognition and therefore does not itself disclose the elected sense-suppressor sequence.
Berg, however, expressly teaches engineering a leucine tRNA with a UGG anticodon to recognize proline codons while delivering leucine. [p. 1873, right column, last paragraph - p. 1874, left column, first paragraph; p. 1875, Fig. 8.]
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the to apply Berg’s proline-reading anticodon to a human leucine-tRNA scaffold disclosed by Ahern to obtain a human leucine-based suppressor of proline codons. Replacing Ahern’s TTA with the DNA counterpart TGG of Berg’s UGG anticodon yields applicant’s SEQ ID NO: 12 exactly, without another sequence change. Ahern teaches changing the decoding target of tRNAs by molecular editing of the anticodon and identifies human tRNA sequences as substrates for that approach. [pp. 9–10, discussion of tRNA sequences, oligonucleotide design, and anticodon editing.] Berg supplies the particular target anticodon and the leucine-to-proline-codon suppression relationship. The proposed modification preserves Ahern’s disclosed tRNA body while changing the codon-recognition triplet, rather than requiring de novo design of an undisclosed scaffold. There would have been a reasonable expectation of obtaining functional suppression because Ahern reports anticodon-editing screens identifying multiple functional suppressors for the amino-acid and stop-codon classes examined, and Berg demonstrates functional leucine tRNAs retargeted to proline codons. [p. 51, paragraph concluding that the screens identified multiple engineered tRNAs displaying potent suppression; pp. 1873–1875, leucine-tRNA Results and Fig. 8.] The references also establish that activity varies among tRNAs and that efficient mistranslation may be toxic. Those qualifications do not defeat the expectation of making a functional sense-suppressor molecule, because claim 7 does not require a specified suppression efficiency or sustained survival of cells expressing it.
Regarding claim 10, Ahern expressly discloses an oligonucleotide encoding its modified tRNA. [p. 4, first paragraph.] Incorporating the identified anticodon substitution into that encoding oligonucleotide provides an oligonucleotide encoding the resulting sstRNA.
Regarding claims 11 and 12, Ahern expressly states that the encoding oligonucleotide has a total length of less than 150 nucleotides and may be DNA. [p. 4, first paragraph.] The mapped sequence is 83 nucleotides, and the proposed two-base substitution does not change its length.
Regarding claim 14, Ahern discloses an expression cassette comprising a promoter and a nucleic acid encoding the modified tRNA. [p. 4, paragraph 3; p. 23, paragraphs 6-7]
Regarding claim 15, Ahern describes an expression cassette with termination signals and an expression vector having operatively linked transcriptional initiation, coding, and transcriptional termination regions. [p. 23, last paragraph; p. 29, paragraph 2] A skilled artisan would have retained suitable promoter and termination elements to express the anticodon-modified tRNA, the expression function for which those elements are disclosed.
Regarding claims 17 and 18, Ahern expressly discloses a vector comprising the encoding oligonucleotide or expression cassette and identifies viral and plasmid vectors. [p. 4, paragraph 3] Placing the modified encoding oligonucleotide in those disclosed vectors would provide delivery and expression of the resulting sstRNA using the same vector functions taught by Ahern.
Regarding claim 19, Ahern expressly discloses a composition comprising the modified tRNA and a pharmaceutically acceptable carrier. [p. 4, paragraph 5]
Regarding claims 20 and 21, Ahern expressly identifies a liposome as the carrier, providing a particle and satisfying the liposome alternative. [p. 4, paragraph 6] Ahern additionally reports functional delivery of engineered tRNA as synthetic RNA and describes lipid-mediated transfection of ACE-tRNA into HEK293 cells. [p. 53, first paragraph under “DISCUSSION”; p. 62, “RNA transfection.”] A skilled artisan would have had reason to use the disclosed liposome formulation with the anticodon-modified sstRNA to deliver the RNA cargo.
Claims 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Geslain (Geslain et al. Nucleic Acids Research, 2010, Vol. 38, No. 5 e30) in view of Ahern (WO 2019/090154 A1, published May 9, 2019) .
Regarding claims 24 and 25, Geslain teaches engineered human serine tRNAs having an acceptor stem and a modified anticodon recognizing a noncognate sense codon. In particular, the isoleucine-reading construct retains serine aminoacylation while directing serine incorporation at isoleucine codons, thereby satisfying the sstRNA limitations incorporated from claim 1 [Results, “Design of a battery of mutagenic tRNAs”; Fig. 1(a)]. Geslain further teaches that expression of the engineered tRNAs in human cells causes cell death, measured using propidium iodide staining and flow cytometry [Materials and Methods, “Cell proliferation and cell death”; Fig. 2(e)]. This disclosure supplies both the killing result of claim 24 and the reduction in cell survival of claim 25.
Geslain introduces the tRNAs through transfection of encoding plasmids rather than by contacting cells with the tRNA molecules themselves [Materials and Methods, “Cell culture”; Results, “Design of a battery of mutagenic tRNAs”].
Ahern teaches direct delivery of engineered tRNA transcripts into HEK293 cells using Lipofectamine 2000, followed by measurement of their functional codon-suppression activity [p. 62, “RNA transfection”]. Ahern additionally reports successful suppression using synthetic RNA transcripts and identifies this result as permitting development of nanoparticle formulations [p. 53, Discussion, first paragraph]. Ahern thus teaches direct RNA delivery as an operative alternative to intracellular production from an encoding construct.
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to deliver Geslain’s isoleucine-reading, serine-accepting tRNA directly to HEK293 cells using Ahern’s RNA-transfection approach. The reason for this modification would have been to provide the active tRNA directly for inducing Geslain’s proteome-stress response, bypassing the transcription step required by plasmid delivery. The proposed combination retains Geslain’s sense-codon specificity and uses Ahern’s delivery technique. A reasonable expectation of success follows from the demonstrated activity of engineered tRNAs in the same human cell type and from the fact that the proposed modification changes delivery of the active adaptor rather than its aminoacylation identity or decoding specificity. An ordinarily skilled artisan would have selected and adjusted the delivered amount to obtain sufficient intracellular mistranslation to reproduce the disclosed cytotoxic response. Direct delivery of the selected cytotoxic tRNA at an effective amount would therefore have been expected to kill cells as required by claim 24 and correspondingly reduce cell survival as required by claim 25.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY N GROOMS whose telephone number is (571)272-3771. The examiner can normally be reached M-F 830-530.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
