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 .
Priority
This application claims domestic benefit of provisional application 63/573,241 filed on Apr. 2, 2024, and all claims are awarded this earlier filing date.
Claim Status
Claims 1-13,15-17, 30, 37, 83, 94, and 99-101 are pending and under examination. Claims 1, 2, 5, 10-12, 30, and 83 have been amended. Claim 28 is cancelled. Claims 99-101 are new. Claims 1, 2, 30, and 83 are independent claims.
Response to Arguments
Objections Withdrawn
The objection of to the abstract is withdrawn following the applicants’ amendments.
The objection to the specification is withdrawn following the applicants’ amendments.
The objection to the sequencing requirements is withdrawn.
The objection of claim 2 is withdrawn following the applicants’ amendments.
Rejections Withdrawn
The rejection of claims 1-13,15-17, and 28 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112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn following the applicants’ amendments.
The rejection of claims 1-13,15-17, 28, 30, 37, 83, and 94 are rejected under 35 U.S.C. 103 as being unpatentable over Gill in view of Kelsic and Kiani is withdrawn following the applicants’ amendments.
New Rejections
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-13,15-17,30,37,83,94 and 99-101 are rejected under 35 U.S.C. 103 as being unpatentable over Ghadessy et al. (PNAS, 2001, on IDS) in view of Fowler and Fields (Nat. Methods, 2014) and in further view of Gamble et al. (Cell 2016).
In regards to claims 1 and 2, Ghadessy teaches obtaining a plurality of cells comprising polynucleotides encoding polymerase variants (see pg. 4552, last para), depositing individual cells (including transformed cells) into partitioned droplets with reagents (see pg. 4552, last para, and 4553 1st para., ), lysing the cells in the partitions (see Fig. 2), and producing nucleic acid products whose abundance reflects polymerase activity, thereby enabling functional screening of polymerase variants (see Abstract and throughout). Ghadessy further teaches comparing variant enrichment relative to control populations to determine relative polymerase activity (see Table 1, pg. 4555, last para. – pg. 4556 1st para.).
However, Ghadessy does not teach performing sequencing to quantitively determine amounts of identifying nucleic acid products nor determining relative activity of variants by sequencing-based comparison to control variants.
Fowler remedies this deficiency by teaching sequencing-based quantification of variant populations and determining relative functional activity by comparing sequence read counts of variants to either control or reference populations (see Fig. 1, pg. 801 right col. Para 2-3, and throughout).
Accordingly, the combined teachings of Ghadessy and Fowler teach or suggest:
A method for determining the relative activity of a plurality of variants of the same polymerase, the method comprising: (i) obtaining a plurality of cells that collectively comprise a plurality of polynucleotides, wherein polynucleotides of the plurality of polynucleotides encode different variants of the same polymerase, (ii) depositing, into different partitions, (a) individual cells of the plurality of cells, and (b)reagents sufficient for an active nucleic acid modifying enzyme variant to produce an identifying
nucleic acid product that can be used to identify the active variant in the partition; (iii) lysing the individual cells in the different partitions to combine (a) a variant produced by the cell in the partition and (b) the reagents sufficient for an active variant to produce an identifying nucleic acid product; (iv) performing sequencing to determine an amount of the identifying nucleic acid products of the first variant and an amount of the identifying nucleic acid products of the second variant; and (v) determining: (a) the relative activity of the first variant by comparing the amount of the
identifying nucleic acid products of the first variant to an amount of an identifying nucleic acid
product produced in a partition comprising a control variant; and (b) the relative activity of the first variant by comparing the amount of the identifying nucleic acid products of the second variant to an amount of an identifying nucleic acid product produced in a partition comprising a control variant. As recited in claim 1.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to incorporate the sequencings-based quantification method of Fowler into the partitioned polymerase screening method of Ghadessy in order to improve quantitative accuracy, throughput and resolution of variant activity determination. A person of ordinary skill in the art would have had a reasonable expectation of success because each references relies on predictable molecular biology and sequencing techniques operating in compatible experimental frameworks. Therefore, claim 1 is unpatentable as obvious over the combined teachings of Ghadessy and Fowler.
In regards to claim 3, the claim depends on claim 1, and further recites that the first and second polynucleotides each comprise three consecutive codons arranges as a synonymous codon, non-synonymous codon, and an additional synonymous codon. Neither Ghadessy nor Fowler expressly teaches this specific codon arrangement. However, Gamble teaches that three adjacent codons act in concert to modulate functional expression and that synonymous and non-synonymous codons in consecutive positions influence protein expression and functional output as measured by sequencing-based assays (see Summary, Experimental Procedures) . Gamble further teaches randomization and analysis of three consecutive codons to evaluate functional consequences (see Fig. 4, Experimental Procedures, and throughout).
Accordingly, Gamble teaches the structural and functional significance of the specific three-codon consecutive motif recited in claim 3. It would have been obvious to one of ordinary skill in the art to apply Gamble’s teachings regarding adjacent codon order and composition to the polymerase variant screening method of Ghadessy as modified by Fowler in order to account for known codon-context effects on protein expression and activity. Therefore, claim 3 is unpatentable for the same reasons as claim 1 and further in view of Gamble.
In regards to claims 4-6 and 16, Ghadessy teaches libraries comprising numerous polymerase variants differing at multiple codon positions, as such libraries inherently comprise additional polynucleotides encoding further non-synonymous and synonymous substitutions (see pg. 4552 last para., pg. 4554 last para., and throughout).
In regards to claims 7 and 8, Fowler teaches performing sequencing to determine an amount of nucleic acid product for any number of variants (see Fig. 1, pg. 801 right col. Para 2-3, and throughout).
In regards to claims 9, 12, and 15, the variant libraries as taught by Ghadessy inherently comprise sequences different by multiple nucleotide substitutions, rendering the recited Hamming distance an inherent and obvious characteristic.
In regards to claims 10-11, and 13, it would have been obvious to increase library size and coverage of amino acid substations as a routine optimization of directed evolution screening, such as that taught by Ghadessy.
In regards to claim 17, Ghadessy teaches transforming cells to express the library (see pg. 4552 last para.).
In regards to claims 30, 83, and 101, Ghadessy teaches libraries comprising a plurality of polynucleotides encoding different variants of the same gene product, including polymerase variants generated by mutagenesis for functional screening (see pg. 4552 last para., pg. 4553 2nd para., pg. 4554 last para.). Fowler teaches sequencing-based functional analysis of variant libraries in which individual variants are identified and quantified by mapping sequencing reads to variant nucleotide sequences. Accurate determination of variant frequencies and functional effects requires reliable discrimination between closely related variant sequences and management of sequencing error. Accordingly, variant libraries analyzed by sequencing-based methods are designed and expected to comprise sufficient nucleotide differences among variants to permit unambiguous identification and quantification. Accordingly variant libraries designed for sequencing-based analysis are known and expected to include variants differing by multiple nucleotide substitutions (see Fig. 1-2, pg. 801 5th para., pg. 803 6th para., pg. 805 2nd para., and throughout.) Such libraries inherently comprise polynucleotides encoding variants that differ by non-synonymous codons, as well as synonymous codon substitutions due to codon degeneracy and routine mutagenesis techniques as observed in the libraries generated by Ghadessy (see pg. 4555, 4th-5th para.). The library produced by Ghadessy averaged 4-5 mutations per polynucleotide (see pg.4554 2nd para.). Fowler further teaches sequencing-based analysis of variant libraries comprising numerous distinct polynucleotide sequences (see Fig. 1-2, pg. 801 5th para., pg. 803 6th para., pg. 805 2nd para., and throughout) .
The additional limitation that at least 50% or 90% of the polynucleotides differ by a Hamming distance of at least 2 from every other such polynucleotide does not patentably distinguish the claimed library. Under standard Hamming-code principles applied to DNA sequencing, sequences that differ by only a single nucleotide cannot be reliably distinguished in the presence of even a single error. As explained by Bystrykh, “the minimal distance dmin= 2t+1 will be able to correct t substitution errors” therefore a Hamming distance of at least 2 is required to detect a single error or single-mismatch, and a distance of at least 3 is required to correct a single error (see Bystrykh, PLoS One 2012, pg. 2 ¶3). Accordingly, variant libraries generated by routine mutagenesis and designed for sequencing-based analysis are expected to comprises sequences differing by multiple nucleotide out of necessity to distinguish closely related nucleotide sequences using standard next generation sequencing technologies and ensure reliable discrimination, particularly for long genes (e.g. ~2500 bp of Taq polymerase) where sequencing errors is more likely. It would have been obvious to one of ordinary skill in the art to design or select libraries with increased nucleotide diversity in order to improve distinguishability, reduce sequencing ambiguity, and enhance robustness of downstream analysis.
The selection of a minimum Hamming distance and minimum proportion of library members meeting the distance represents an optimization of a result-effective variable related to sequence diversity and error tolerance, which would have been obvious to optimize using routine experimentation, see In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). A person of ordinary skill in the art would have had a reasonable expectation of success in generating such a library because the techniques for introducing and controlling nucleotide diversity in variant libraries were well known.
In regards to claim 37, Gamble teaches that consecutive codon arrangements influence functional expression, rendering the specific consecutive codon motif obvious, utilizing the same rationale as claim 3 above.
In regards to claim 94, synthesizing polynucleotides is a routine molecular biology techniques and would have been obvious to perform in producing the libraries (see Fowler pg. 801 3rd para.).
In regards to claims 99-100, these claims depend on claim 1 and further recite that at least 50% or 90% of the library differ by a Hamming distance of at least 2 from every other polynucleotide. The additional limitation that at least 50% or 90% of the polynucleotides differ by a Hamming distance of at least 2 from every other such polynucleotide does not patentably distinguish the claimed library. Under standard Hamming-code principles applied to DNA sequencing, sequences that differ by only a single nucleotide cannot be reliably distinguished in the presence of a even a single error. As explained by Bystrykh, “the minimal distance dmin= 2t+1 will be able to correct t substitution errors” therefore a Hamming distance of at least 2 is required to detect a single error or single-mismatch, and a distance of at least 3 is required to correct a single error (see Bystrykh, PLoS One 2012, pg. 2 ¶3). Accordingly, variant libraries generated by routine mutagenesis and designed for sequencing-based analysis are expected to comprises sequences differing by multiple nucleotide out of necessity to distinguish closely related nucleotide sequences using standard next generation sequencing technologies and ensure reliable discrimination, particularly for long genes (e.g. ~2500 bp of Taq polymerase) where sequencing errors is more likely. It would have been obvious to one of ordinary skill in the art to design or select libraries with increased nucleotide diversity in order to improve distinguishability, reduce sequencing ambiguity, and enhance robustness of downstream analysis.
The selection of a minimum Hamming distance and minimum proportion of library members meeting the distance represents an optimization of a result-effective variable related to sequence diversity and error tolerance, which would have been obvious to optimize using routine experimentation, see In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). A person of ordinary skill in the art would have had a reasonable expectation of success in generating such a library because the techniques for introducing and controlling nucleotide diversity in variant libraries were well known. Accordingly, claims 99 and 100 are unpatentable as obvious.
Conclusion
No claim is allowed.
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 Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
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, Heather Calamita can be reached at 571-272-2876. 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.
/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681