DETAILED ACTION
The amendment filed on 11/24/2025 has been entered.
Claims 1, 13, 19, 25-27, and 31 were amended in the claim set filed on 11/24/2025.
Applicant’s election without traverse of Group I (Claims 1-2, 5-6, and 12-13), drawn to a nucleic acid molecule product and a bacterial cell product containing said molecule having at least one mutation for modulating interaction strength of the molecule with 16S ribosomal RNA in the reply filed on 07/21/2025 is acknowledged.
Claims 19-21, 24-31 (Group II), and 37-39 (Group III) are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Groups II and III, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/21/2025.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 21, 24, and 28 are canceled in the claim set filed on 11/24/2025.
Claims 42-44 were added in the claim set filed on 11/24/2025. No new matter was added.
Claims 1-2, 5-6, 12-13 and 42-44 in the claim set filed on 11/24/2025 are currently under examination.
Response to the Arguments
Objections to the drawings and claims in the previously mailed non-final have been withdrawn in light of applicants Drawing and Claim amendments.
Applicant’s arguments regarding previous rejection(s) of claim(s) 1-2, 5-6 and 12-13 under 35 U.S.C. 103 have been fully considered and are persuasive. Applicant’s argument on Pg. 12, states that “Ramseier is interested only in translation initiation which occurs around the first codon. There is therefore no motivation whatsoever in Ramseier to modify a sequence outside of this region.” The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 11-12. However, upon further consideration and search, new grounds of rejection are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 3-9.
The objection and rejections for claims 1-2, 5-6, 12-13 and 42-44 are documented below in this Final Office Action are necessitated by claim amendments filed on 11/24/2025.
Priority
This application is a continuation of PCT Patent Application No. PCT/IL2020/050367 having International filing date of March 26, 2020, which claims the benefit of priority of U.S. Provisional Patent Application No. 62/825,143 filed March 28, 2019. The priority date of claim set filed on Sept. 28, 2021, is determined to be March 28, 2019.
Claim Objections
Claim 1 is objected to because of the following informalities: “TSS” acronym is not defined in the claimed before using the acronym. Appropriate correction is required.
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 1-2, 5-6, 12-13 and 42-44 are rejected under 35 U.S.C. 103 as being unpatentable over Ramseier et al. (“Ramseier”; Patent App. Pub. No. WO 2009020899 A1, Feb. 12, 2009).
Claim Interpretation: The claims with the limitations comprising “or” in claim 2 (ln 5), claim 6 (ln 4), claim 13 (ln 6) indicate each of the alphabets prefixes is alternative of one another.
Ramseier discloses “The present invention provides methods and compositions for producing heterologous protein with improved yield and/or quality. A library of randomized ribosomal binding site sequences is provided for the identification of a translation initiation region sequence optimal for expression of the heterologous protein. Also provided are novel ribosomal binding site sequences, and vectors and host cells having those sequences. The library of randomized sequences is useful for screening for improved expression of any protein of interest, including therapeutic proteins, hormones, a growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like.” (Abstract).
Claims 2, 5-6 ,12, and 42-44 depend on claim 1. Claim 13 depends on claim 12, which depends on claim 1.
Regarding claims 1 and 42-44, Ramseier teaches a nucleic acid molecule comprising “Modulating translation strength by altering the translation…region” (Pg. 3 ln. 25). Ramseier teaches a nucleic acid molecule comprising “The RBS (also referred to herein as the Shine- Dalgarno sequence) is located on the … Because of the role of the RBS sequence in translation, there is a direct relationship between the efficiency of translation and the efficiency (or strength) of the RBS sequence” (Pg. 4 ln. 10-16). Ramseier teaches a nucleic acid molecules comprising “In addition to altering the RBS sequence for optimizing expression, several additional approaches are also encompassed that can be used to control protein translation levels. For example, using promoters with a range of translation strengths, modulating promoter activity by titrating induction, using plasmids with different copy numbers, improving transcript stability, and manipulating sequences other than the RBS sequence in the translation initiation region” (Pg. 12 ln. 13-18). Ramseier teaches a nucleic acid molecule comprising “A single base pair mutation was introduced by PCR amplification to create the silent codon mutation” (Pg. 3 ln. 4-5; Figure 1). Ramseier teaches a nucleic acid molecule comprising “oligonucleotide sequences are useful for optimizing expression of a heterologous protein in a host cell where the translation efficiency is decreased when compared to the translation efficiency of the protein encoded by a gene comprising the canonical RBS sequence” (Pg. 5 ln.12-15). “Modulating translation strength by altering the translation…site” reads on any type of change (e.g. mutation) relative to any position of the coding sequence within a region from upstream or downstream of the TSS causing any type of change in the interaction strength of the nucleic acid molecule to a 16s rRNA. “manipulating sequences other than the RBS sequence in the translation initiation region” reads on wherein said region is a position as recited in claim options a, b, or c. Thus, Ramseier teaches a nucleic acid molecule comprising a coding sequence, wherein said nucleic acid molecule comprises at least one mutation within a region of said molecule, wherein said mutation modulates interaction strength of said nucleic acid molecule to a 16S ribosomal RNA (rRNA); and wherein said region is selected from the group consisting of: a) positions 26 downstream of a TSS of said coding sequence through position -13 upstream of a translational termination site (TTS) of said coding sequence and said mutation modulates interaction strength to an intermediate interaction strength; b) positions -8 through -17 upstream of a TTS of said coding sequence and said mutation increases interaction strength; and c). a position downstream of a TTS of said coding sequence and said mutation increases interaction strength.
Regarding claim 2, Ramseier teaches a nucleic acid molecule comprising “Modulating translation strength by altering the translation initiation region” (Pg. 3 ln. 25). Ramseier teaches a nucleic acid molecule comprising “The translation initiation region has been defined as the sequence extending immediately upstream of the ribosomal binding site (RBS) to approximately 20 nucleotides downstream of the initiation codon…In prokaryotes, alternative RBS sequences can be utilized to optimize translation levels of heterologous proteins by providing translation rates that are decreased with respect to the translation levels using the canonical, or consensus, RBS sequence … described by Shine and Dalgarno ((1974) Proc. Natl Acad. Sci USA 71: 1342-1346)... In most prokaryotes, the Shine-Dalgarno sequence assists with the binding and positioning of the 30S ribosome component relative to the start codon on the mRNA through interaction with a pyrimidine-rich region of the 16S ribosomal RNA (Pg. 3 ln. 32-34, Pg. 4 ln 1-10). Ramseier teaches a nucleic acid molecule comprising “The RBS (also referred to herein as the Shine- Dalgarno sequence) is located on the mRNA … upstream from the start of translation, typically from 4 to 14 nucleotides upstream of the start codon, and more typically from 8 to 10 nucleotides upstream of the start codon. Because of the role of the RBS sequence in translation, there is a direct relationship between the efficiency of translation and the efficiency (or strength) of the RBS sequence” (Pg. 4 ln. 10-16). Ramseier teaches a nucleic acid molecules comprising “RBS sequence fragment wherein one or more nucleotides corresponding to the canonical RBS sequence (SEQ ID NO: 1) has been fully randomized” (Pg. 5 ln. 22-23). Ramseier teaches a nucleic acid molecule wherein “strength ribosome binding site” (Pg. 48 ln. 32). Thus, Ramseier teaches a nucleic acid molecule wherein a) said mutation modulates interaction strength of a six-nucleotide sequence containing said mutation to said 16S rRNA; b) said interaction strength to a 16S rRNA is to an anti-Shine Dalgarno (aSD) sequence of said 16S rRNA; or c) said interaction strength to a 16S rRNA is to an anti-Shine Dalgarno (aSD) sequence of said 16S rRNA and is determined from Table 3.
Regarding claim 5, Ramseier teaches a nucleic acid molecule wherein “randomized RBS sequences for optimizing heterologous expression of a polypeptide of interest in a host cell” (Pg. 2 ln. 14-16) and “useful in host cells … including bacterial cells” (Pg. 2 ln. 21-23). Ramseier teaches “the host cell can be a member of any of the bacterial taxa” (Pg. 16 ln 1). Ramseier teaches a nucleic acid molecule wherein “the RBS sequence variants described herein can be classified as resulting in high, medium, or low translation efficiency. In one embodiment, the sequences are ranked according to the level of translational activity compared to translational activity of the canonical RBS sequence” (Pg. 25 ln. 13-15). Thus, Ramseier teaches a nucleic acid molecule wherein said increasing increases interaction strength to a strong interaction strength, decreasing decreases interaction strength to a weak interaction strength and wherein strong, weak and intermediate interaction strengths are determined from Table 1.
Regarding claim 6, Ramseier teaches a nucleic acid molecule comprising “The translation initiation region has been defined as the sequence extending immediately upstream of the ribosomal binding site (RBS) to approximately 20 nucleotides downstream of the initiation codon… In most prokaryotes, the Shine-Dalgarno sequence assists with the binding and positioning of the 30S ribosome component relative to the start codon on the mRNA through interaction with a pyrimidine-rich region of the 16S ribosomal RNA (Pg. 3 ln. 32-34, Pg. 4 ln 7-10). Ramseier teaches a nucleic acid molecule comprising “The RBS (also referred to herein as the Shine- Dalgarno sequence) is located on the mRNA … upstream from the start of translation, typically from 4 to 14 nucleotides upstream of the start codon … Because of the role of the RBS sequence in translation, there is a direct relationship between the efficiency of translation and the efficiency (or strength) of the RBS sequence” (Pg. 4 ln. 10-16). Ramseier teaches a nucleic acid molecules comprising “the library comprises a plurality of oligonucleotides comprising an RBS sequence fragment wherein one or more nucleotides corresponding to the canonical RBS sequence (SEQ ID NO: 1) has been fully randomized. In another embodiment ... or wherein only 1, 2, 3, 4, or 5 nucleotide positions corresponding to the canonical RBS sequence have been fully randomized” (Pg. 5 ln. 2-8; Pg. 5 ln 21-30). Thus, Ramseier teaches a nucleic acid molecule wherein a) said region from position 26 downstream of the TSS through position -13 upstream of the TTS comprises the first 400 base pairs of said region; b) said molecule comprises at least a second mutation, wherein said second mutation is in a different region than said at least one mutation; c) said at least one mutation is within said coding sequence and mutates a codon of said coding sequence to a synonymous codon; d) wherein said mutation improves the translation potential of said coding sequence; e) wherein said mutation does at least one of: increasing translation initiation efficiency, increasing translation initiation rate, increasing diffusion of the small subunit to the initiation site, increasing elongation rate, optimization of ribosomal allocation, increasing chaperon recruitment, increasing termination accuracy, decreasing translational read-through and increasing protein yield; f) said nucleic acid molecule is a messenger RNA (mRNA); or g) a combination thereof.
Regarding claim 12-13, Ramseier teaches oligonucleotides comprising “novel RBS sequence fragments useful for the heterologous expression of a protein or polypeptide of interest in a bacterial host cell” (Pg. 4 ln. 29-31). Ramseier teaches bacterial cells such as “P.fluorescens, E. coli, and the like” (Pg. 2 ln. 23). Ramseier teaches “the host cell can be a member of any of the bacterial taxa” (Pg. 16 ln 1). Thus, Ramseier teaches a cell comprising a nucleic acid molecule; and wherein a) said cell is a bacterial cell; b) said cell is a cell of a bacterium recited in Table 1; c) said cell is a cell of a bacterium selected from Escherichia Coli, Alphaproteobacteria, Spirochaete, Purple bacteria, Gammaproteobacteria, deltaproteobacteria and Betaproteobacteria; or d) wherein said cell is a bacterial cell and said bacterium is not a Cyanobacteria or Gram-positive bacteria.
Therefore, the invention as recited in claims 1-2, 5-6, 12-13 and 42-44 is prima facie obvious over the prior art Ramseier et al. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to have a nucleic acid molecule comprising a coding sequence, wherein said nucleic acid molecule comprises at least one mutation within a region of said molecule, wherein said mutation modulates interaction strength of said nucleic acid molecule to a 16S ribosomal RNA (rRNA) according to the limitations of the instant application claims 1-2, 5-6, 12-13 and 42-44 based on Ramseier et al. (Patent App. Pub. No. WO 2009020899 A1).
Response to Arguments
Applicant' s arguments filed 11/24/2025 (Pg.12-13) with respect to claim 1-2, 5-6, 12-13 and 42-44 have been considered but do not apply to the new grounds of rejections. To clarify some instances argued in the response filed 11/24/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “Ramseier does not suggest or motivate modifying any other region. Ramseier is interested only in translation initiation which occurs around the first codon. There is therefore no motivation whatsoever in Ramseier to modify a sequence outside of this region.” (Pg. 11)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as recited in the new grounds of rejections stated above, on Pg. 4-5, Ramseier teaches a nucleic acid molecules comprising “In addition to altering the RBS sequence for optimizing expression, several additional approaches are also encompassed that can be used to control protein translation levels. For example, using promoters with a range of translation strengths, modulating promoter activity by titrating induction, using plasmids with different copy numbers, improving transcript stability, and manipulating sequences other than the RBS sequence in the translation initiation region” (Pg. 12 ln. 13-18). Therefore, Ramseier does suggest or motivate modifying sequence region(s) other than the translation initiation region.
Applicants’ argument: “Ramseier not only would not motivate mutations in these regions, but a skilled artisan would have no expectation of success when making mutations in these regions as Ramseier only suggests that the mutations would affect initiation.” (Pg. 11)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as recited in the new grounds of rejections stated above, on Pg. 9, and response above, Ramseier does suggest or motivate modifying sequence region(s) other than the translation initiation region. Furthermore, the invention as recited in claims 1-2, 5-6, 12-13 and 42-44 is prima facie obvious over the prior art Ramseier et al. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to have a nucleic acid molecule comprising a coding sequence, wherein said nucleic acid molecule comprises at least one mutation within a region of said molecule, wherein said mutation modulates interaction strength of said nucleic acid molecule to a 16S ribosomal RNA (rRNA) according to the limitations of the instant application claims 1-2, 5-6, 12-13 and 42-44 based on Ramseier et al. (Patent App. Pub. No. WO 2009020899 A1).
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and above responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure: Göringer, H. U., Hijazi, K. A., Murgola, E. J., & Dahlberg, A. E. (1991). Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination. Proceedings of the National Academy of Sciences, 88(15), 6603-6607. (Entire document- claim 1)
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682