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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on Jul. 30, 2025 has been entered.
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Jul. 30, 2025. Claims 1-20 are pending and currently examined.
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.
Claims 1-20 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 pre-AIA the applicant regards as the invention.
Base claim 1 is directed to method of manufacturing a plurality of error mitigated target polynucleotides of a desired sequence, said method comprising:
- obtaining a quantity of target polynucleotides of a desired sequence;
- amplifying the target polynucleotides via the polymerase chain reaction (PCR) to create initial sets of double stranded amplicons which inherently contain areas of single stranded sequence errors at one or more points in the amplicons;
- denaturing the initial sets of double stranded amplicons to create denatured amplicons;
- annealing the denatured amplicons to create additional sets of double stranded amplicons wherein said additional sets of double stranded amplicons contain areas of single stranded sequence errors at the point in the amplicons where the nucleotide sequence differs from the desired nucleotide sequence;
- reacting the additional sets of double stranded amplicons with a single strand specific nuclease to create cuts at the points of the single strand sequence errors in the additional sets of double stranded amplicons to remove the sequence errors not complementary to the desired sequence; and
- reacting the additional sets of double stranded amplicons containing cuts at the points of the single strand sequence errors with a polymerase to introduce the desired sequence at the cut point thereby mitigating the sequence errors.
Claim 1 recites “reacting the additional sets of double stranded amplicons with a single strand specific nuclease to create cuts at the points of the single strand sequence errors in the additional sets of double stranded amplicons to remove the sequence errors not complementary to the desired sequence”. This step is not clear since there is no evidence in either the instant specification or knowledge in the art that a single-strand-specific nuclease exists that does both the cutting at the points of single strand sequence errors and the removing of the sequence errors.
The invention specifies a few endonucleases, including Mung Bean endonuclease, T7 endonuclease I, E. coli endonuclease V, CEL endonuclease, S1 endonuclease and P1 endonuclease, as examples for the single-strand specific nuclease. See claim 2. Fuhrmann et al. (Nucleic Acids Research, 2005, Vol. 33, No. 6 e58) teaches that a mismatch cleavage enzyme (e.g., T7 endonuclease I) cleaves at mismatch points on a double-stranded DNA heteroduplex, resulting in fragments with overhands. However, the mismatched nucleotides are not removed. See Figure 1 of Fuhrmann. Fuhrmann teaches that the resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3’–5’-exonuclease, e.g. E. coli exonuclease I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3’–5’-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. See legend of Figure 1 of Fuhrmann. Fig. 1 is shown below:
PNG
media_image1.png
200
400
media_image1.png
Greyscale
Based on the teachings of Fuhrmann, the instant claim 1 is considered incomplete for omitting an essential element, i.e., an exonuclease that removes the mismatched nucleotides. Such omission amounting to a gap between the elements. See MPEP § 2172.01.
Base claim 8 is directed to a method of manufacturing a plurality of error mitigated polynucleotides of a desired sequence, said method comprising:
- obtaining a quantity of target polynucleotides of a desired sequence;
- amplifying the target polynucleotide via the polymerase chain reaction (PCR) to create initial sets of double stranded amplicons which inherently contain areas of single stranded sequence errors at one or more points in the amplicons;
- assembling the initial sets of double stranded amplicons with a DNA scaffold via seaming PCR to create additional sets of double stranded amplicons comprising the target polynucleotide and scaffold DNA;
- denaturing the additional sets of double stranded amplicons to create denatured additional sets of double stranded DNA amplicons;
- reacting the denatured additional sets of double stranded DNA amplicons with one or more complementary or partially complementary DNA sequences to form 2-D or 3-D DNA structures wherein the complimentary target polynucleotide sequences are forced to hybridize in the 2-D or 3-D DNA structure and wherein the additional sets of double stranded amplicons contain areas of single stranded sequence errors at the point in the target polynucleotide sequence where the sequence differs from the desired polynucleotide sequence;
- reacting the 2-D or 3-D DNA structures with a single strand specific nuclease to create cuts at the points of the single strand sequence errors thereby removing the sequence error in the target polynucleotide sequence are mitigated by reacting the 2-D or 3-D DNA structures containing cuts at the points of single strand sequence errors with a polymerase to introduce the desired sequence at the cut point thereby mitigating the sequence errors; and
- releasing the target polynucleotide comprised of the desired sequence from the 2-D or 3-D DNA structures.
Claim 8 is not clear in at least the following aspects:
A. Claim 8 specifies a step of assembling the initial sets of double-stranded amplicons with a DNA scaffold via seaming PCR to create additional sets of double stranded amplicons comprising the target polynucleotide and scaffold DNA. This limitation recites the terms “seaming PCR” and “scaffold DNA” or “DNA scaffold”. The specification mentions “seaming PCR” several times. E.g., it teaches that target polynucleotides may be derived from the assembly of various oligonucleotides into larger polynucleotides via assembly apparatuses such as the BioXp™ (SGI-DNA, United States) or seaming PCR. See e.g. [0030]. This teaching suggests that “seaming PCR” may be used to assemble oligonucleotides to generate target polynucleotides, but does not provide guidance on how a “seaming PCR” is performed. The specification also mentions the term “scaffold” throughout the disclosure. See e.g. Figs. 1-2. It teaches that the term “scaffold DNA” means noncoding DNA utilized for the purpose of forming a 2-D or 3-D DNA structure. See e.g. para [0025]. However, neither the claims nor the specification specifies structural characteristics for one of skill in the art to understand what structures must a “DNA scaffold” or “scaffold DNA” possess for it to be used in the claimed invention and how such “DNA scaffold” can be assembled into the additional sets of double-stranded amplicons via “seaming PCR”. Therefore, the claimed method lacks essential elements, such as structural elements required for “seaming PCR”, “scaffold DNA”, and “DNA scaffold”.
B. Claim 8 recites “Reacting the denatured additional sets of double stranded DNA amplicons with one or more complementary or partially complementary DNA sequences to form 2-D or 3-D DNA structures wherein the complimentary target polynucleotide sequences are forced to hybridize in the 2-D or 3-D DNA structure and wherein the additional sets of double stranded amplicons contain areas of single stranded sequence errors at the point in the target polynucleotide sequence where the sequence differs from the desired polynucleotide sequence”. This limitation is not clear. First, it is not clear what the “one or more complementary or partially complementary DNA sequences” are from, and how they can lead to the formation of “2-D or 3-D DNA structures”. Secondly, the term “the complimentary target polynucleotide sequences” does not have antecedent basis. E.g., it is not clear if the term “the complimentary target polynucleotide sequences” refers to “one or more complementary or partially complementary DNA sequences”. Moreover, it is not clear how to interpret the term “2-D or 3-D DNA structures”.
C. Claim 8 recites “Reacting the 2-D or 3-D DNA structures with a single strand specific nuclease to create cuts at the points of the single strand sequence errors thereby removing the sequence error in the target polynucleotide sequence are mitigated by reacting the 2-D or 3-D DNA structures containing cuts at the points of single strand sequence errors with a polymerase to introduce the desired sequence at the cut point thereby mitigating the sequence errors”. This limitation is not clear. First, as indicated above, the claim does not make it clear how the term “2-D or 3-D DNA structures” is to be interpreted. So, it is not clear how the single-strand specific nuclease can create cuts, and where the nuclease cuts. As indicated in the discussion about claim 1, a single-strand specific endonuclease cuts on both strands of a heteroduplex DNA double strand around mismatch points but does not remove the mismatched sequences. Secondly, the phrase “thereby removing the sequence error in the target polynucleotide sequence” is not clear since the “target polynucleotide sequence” is supposed to have the “desired sequence.” Moreover, it is not clear how the phrase “are mitigated by reacting the 2-D or 3-D DNA structures containing cuts at the points of single strand sequence errors with a polymerase to introduce the desired sequence at the cut point thereby mitigating the sequence errors” fits in the claim language.
Base claim 15 is directed to a method for producing an antigen specific immune response in a subject, said method said method comprising the steps of:
- choosing one or more desired antigens for expression within a subject;
- obtaining a quantity of target polynucleotides of a desired sequence, said target polynucleotides of a desired sequence containing one or more expression cassettes for the desired one or more antigens;
- amplifying the target polynucleotides via the polymerase chain reaction to create initial sets of double stranded amplicons which inherently contain areas of single stranded sequence errors at one or more points in the amplicons;
- denaturing the initial sets of double stranded amplicons to create denatured amplicons;
- annealing the denatured amplicons to create additional sets of double stranded amplicons wherein the additional sets of double stranded amplicons contain areas of single stranded sequence errors at one or more points the point in the amplicon, where the nucleotide sequence differs from the desired nucleotide sequence are mitigated by reacting the additional sets of double stranded amplicons with a single strand specific nuclease to create cuts at the points of the single strand sequence errors in the additional sets of double stranded amplicons to remove the errors not complementary to the desired sequence;
- reacting the additional sets of double stranded amplicons containing cuts at the points of the single strand sequence errors with a polymerase to introduce the desired sequence at the cut point thereby mitigating the sequence errors by creating a plurality of double stranded amplicons with the desired sequence containing expression cassettes for the desired one or more antigens;
- formulating the plurality of double stranded amplicons with the desired sequence containing expression cassettes for the desired one or more antigens into a therapeutic dose; and
- administering the therapeutic dose to a subject, wherein the subject produces the desired one or more antigens in response to the therapeutic dose.
The method of Claim 15 comprises the steps of claim 1, and therefore, contains the same issues as claim 1 which also make the claim 15 indefinite.
Claim Rejections - 35 USC § 102/103
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.
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, 4-18 and 20 are rejected under 35 U.S.C. 102/103 as being unpatentable over Sequeira et al. (Mol Biotechnol (2016) 58:573–584).
As indicated in the 112(b) rejection above, these claims are indefinite. To expediate examination, these claims are interpreted to encompass a process shown below:
[AltContent: textbox (Plurality of target nucleotides with a desired sequence)][AltContent: arrow][AltContent: textbox (PCR)][AltContent: textbox (Initial sets of dsDNA amplicons with errors (star))][AltContent: arrow][AltContent: arrow][AltContent: textbox (Single-strand
specific nuclease)][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: ][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: arrow][AltContent: textbox (polymerase)]
[AltContent: textbox (Denature and re-anneal)][AltContent: ]
[AltContent: textbox (dsDNA amplicons with single-stranded sequence mismatches (heteroduplex with bulges at the mismatch points) )]
[AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector][AltContent: connector]
[AltContent: textbox (Single-strand specific nucleases cut on both strands around the mismatch points and cleave the double-strand amplicon heteroduplex )]
[AltContent: textbox (dsDNA product with the desired sequence )]
Sequeira teaches that efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. The authors analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, the data reveal that incorporation of a mutation removal step including T7 endonuclease I can effectively
improve the fidelity of artificial gene synthesis. See Abstract.
Fig. 1 of Sequeira is presented below:
PNG
media_image2.png
404
766
media_image2.png
Greyscale
It teaches that overlapping oligonucleotides are assembled (PCR1) and used as template for a second PCR reaction (PCR2) to build the full-length nucleic acid. A denaturation - renaturation step is used to form hetero-duplex DNA containing mismatches. DNA mismatches are recognized and cleaved by endonucleases. Using the digestion reaction as template, a third PCR reaction is used (PCR3) to recover the error-corrected DNA fragment. See legend of Fig. 1.
Accordingly, Sequeira teaches a gene synthesis method including mismatch repair that is indistinguishable from the invention as claimed.
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 3 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Sequeira et al. (Mol Biotechnol (2016) 58:573–584) in view of Fuhrmann et al. (Nucleic Acids Research, 2005, Vol. 33, No. 6 e58), and further in view of Imai et al. (Mol Genet Genomics (2007) 278:211–220).
Claims 3 and 19 specify that the polymerase is E. coli DNA polymerase I.
Relevance of Sequeira and Fuhrmann is set forth above in the 102/103 and 112(b) rejections, respectively. Fuhrmann further teaches that the short overhangs generated by EMC dissociate at the reaction temperature of 37oC, leaving them as single-stranded overhangs, and that these single-stranded extensions are degraded by a single-strand-specific 3’–5’-exonuclease, e.g. by E. coli exonuclease I or the corresponding activity of proofreading polymerases (Figure 1). See page 2, left column, para 2. However, they are silent on E. coli DNA polymerase I.
Imai teaches that E. coli DNA polymerase I (PolI) which possesses three distinct enzymatic activities; 5´-3´ DNA polymerase activity, 3´-5´ exonuclease activity that edits 3’ terminal nucleotides of nascent DNA, and 5´-3´ exonuclease activity that removes nucleotides from the 5’ end of DNA or RNA (Kornberg and Baker 1992). Proteolytic cleavage of PolI yields two fragments; the 5´-3´ exonuclease domain and the Klenow domain, which include 3´-5´ exonuclease and 5´-3´ polymerase activities (Brutlag et al. 1969). See page 1, right column, para 3. Accordingly, Imai teaches that E. coli DNA polymerase I (PolI) possesses the functions of 3’-5’ exonuclease and 5’-3’ polymerase activities – activities needed for the synthesis of resulting error-mitigated nucleotide sequence in the process of Sequeira and Fuhrmann.
It would have been prima facie obvious before the effective filing date of the current invention to combine the teachings of Sequeira, Fuhrmann and Imai to arrive at the invention as claimed. One would have been motivated to do so, e.g., to use the 3’-5’ exonuclease activity (and the polymerase activity) of the E. coli PolI in the process Sequeira and Fuhrmann.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, JANET ANDRES, on (571) 272-0867, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/Primary Examiner, Art Unit 1671