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 .
Priority
This application is a Continuation of Application No. 18/288,545 filed 10/26/2023. Applicant’s claims to priority from PCT/EP2022/061630 filed 04/29/2022 and EP21382377.6 filed 04/29/2021 is hereby acknowledged.
Application Status
This Office Action is in response to claim amendments and arguments filed 05/25/2026.
Amendments to claims filed 05/25/2026 are hereby acknowledged. Claims 1-16, 18-20 and 38 are cancelled. Claims 17, 23-24 and 39 are currently amended. Claims 17, 21-37 and 39 are currently pending. However, claims 27-34 are still withdrawn since they are drawn to a non-elected invention.
Therefore, claims 17, 21-26, 35-37 and 39 are under consideration in this office action.
Any objection or rejection not reiterated herein has been overcome by Applicant amendments and is therefore withdrawn.
Applicant’s amendments and arguments have been thoroughly reviewed but are not persuasive to place the claims in condition for allowance for the reasons that follows.
Claim Interpretation
Applicant has amended the claims 17, 23-24 and 36 on 05/25/2026, to recite a limitation, i.e., “nuclease-resistant nucleotides” instead of the previous terms “protected nucleotides” in claims filed 01/15/2026.
The rejections in the previous Office Action dated 02/25/2026 were made in view
of the larger scope of the terms “protected nucleotides”, which is also encompassed within the definition presented in the Specification, page 17, third paragraph: “As used herein the term “protected nucleotide” or “nuclease-resistant nucleotide” is intended to encompass any type of molecule that provides or enhances resistance to nuclease digestion (especially exonuclease digestion)” and “The linear double-stranded DNA product may comprise a peptide, polypeptide or protein that provides or increases resistance to nucleases (e.g. exonucleases) digestion.”
The Specification’s definition encompasses peptide DNA chimera, but also “allows” for functional regions within the DNA product, encompassing covalently or non-covalently bound peptide, polypeptide or protein that provides or increases resistance to nucleases.
In Examiner’s view, the definition of “protected nucleotide” is well described within the Specification, on page 17, third paragraph, first line, and the Broad and Reasonable Interpretation (BRI) encompasses “any type of molecule that provides or enhances resistance to nuclease digestion (especially exonuclease digestion).” The BRI of the terms justified the previous rejections.
However, although this definition includes “nuclease-resistant nucleotide”, Examiner interprets the terms differently than stated in first line of said paragraph.
Examiner interprets the terms “nuclease-resistant nucleotides” specifically in the context of the claims, as would an ordinary person skilled in the art, with a reasonable interpretation, as being more restrictive and specific, i.e., Examiner interprets the terms as “nucleotides that are structurally resistant themselves to nuclease”.
Examiner interprets that the Example provided in said paragraph of the Specification (page 17, third paragraph), as being more in agreement with Examiner’s definition of the terms: “For Example, the linear double-stranded DNA product may comprise nuclease resistant nucleotides i.e. modified nucleotides that provide or increase resistance to nucleases (e.g. exonucleases).”
Examiner interprets that the scope of the claims has changed and is more limited, therefore new rejections are necessitated by the amendments.
New Rejections as necessitated by Applicant’s amendments filed 05/25/2026:
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 17, 24, 25 and 39 are rejected under 35 U.S.C. §103 as being unpatentable over Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase, downloaded from URL: https://www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase ; Phusion® Hot Start Flex DNA Polymerase | NEB; previously cited), in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited).
Regarding claim 17, Zhu teaches DNA templates that are linear, i.e., Linear Expression Templates (LETs), for crude extract-based cell-free protein synthesis (CFPS) (see abstract). Zhu teaches that this LET contains a T7 promoter and a T7 terminator (see section 2.2., page 3851, right column).
A DNA expression template molecule, obtained by PCR and used for protein expression (see page 3850, “Introduction” section, left column, first paragraph), inherently comprises a sense and antisense strand, therefore Zhu teaches these elements; and the double strands are shown in drawings in Figure 1(a).
Zhu teaches stabilization of the LETs using different strategies for comparison:
Protection of LET via terminal protection using double strand DNA-binding protein, adding a scCro-binding site (ORC) at each end (see Figure 1); or
Protection using LET methylation with dam or CpG methyltransferases (see section 2.3, page 3851); or
Protection adding three phosphorothioate bonds modifications at the 5’ ends to prevent hydrolysis (see section 2.2., right column).
Zhu teaches that adding ORC binding sites at each ends or adding dam/CpG methylation lead to increased stability of the LET and protein yield from expression (see Figure 2).
Zhu teaches that the phosphorothioate linkage modification at the 5’ end alone did not protect the LET to produce large protein yield (LET5; 5-10 µg/ml ( see page 3854, right column, last sentence)), as adding ORC sites alone (LET3) (see Figure 3). Zhu teaches that adding buffering sequences (2x 800 bp) to the sfGFP coding region, and ORC sequences (2x 18 bp) , (LET2), contributes to increased stability as shown in Figure 2. Zhu teaches that 64 nM of LET2 was able to yield 0.324 mg/ml of sfGFP protein (see page 3855,Figure 4 and section 3.3, right column).
As shown in Figure 2b, the total length of the LET2 is about 3,000 bp. The protected nucleotides number after binding of scCro would be 36. Therefore, the ratio of protected nucleotides would be 36/3,000 = 0.012, which is higher than 0.0025, but lower than 0.1.
Zhu is silent on whether the end of the linear double-stranded DNA product comprises an overhang or a blunt end. However, Zhu teaches that the DNA polymerase used to obtain the LET is a DNA polymerase (Phusion®) obtained from New England Biolabs (NEB) (see section 2.1, page 3851).
According to NEB-pdf, the Phusion® DNA polymerase produces blunt ends (“Product Information” section, first paragraph).
Regarding claim 24, Zhu teaches LET5, which is a linear DNA template molecule having three 5’ phosphorothioate bonds (SSS) at each end (see Figure 3).
Regarding claim 25, Zhu teaches a functional portion in the linear double-stranded DNA product, which function is to bind scCro protein, and which is named ORC as shown in Figure 2.
Regarding claim 39, Zhu teaches a linear double-stranded DNA product comprising protected nucleotides at a ratio of 0.012, which is between 0.1 and 0.01.
As shown in Figure 2b, the total length of the LET2 is about 3,000 bp. The protected nucleotides number after binding of scCro would be 36. Therefore, the ratio of protected nucleotides would be 36/3,000 = 0.012, which is higher than 0.01, but lower than 0.1.
Zhu teaches preference towards a linear DNA template LET2 with a sequence that is a functional portion, a binding molecule, since LET5 (DNA product having 3 phosphorothioate modifications on nucleotides at each end) provides less yield as far as mRNA and protein production.
However, Watzele teaches that combining methods/modalities to protect a double-stranded DNA templates for protein expression is acceptable (see title, abstract and [0001]-[0007]).
Watzele teaches that “the stability of the linear short DNA is improved by one or several of the following measures to protect the double-stranded DNA from exonucleases:
Incorporation of exonuclease resistant nucleotides analogues or other exonuclease-resistant molecules at the 3’ end of the template,
Use of PCR primer pairs which contain exonuclease-resistant nucleotides to produce a linear short DNA,
Protection of a template produces by a PCR reaction by connecting the 5’ end to the 3’ end of the complementary strand,
Protecting the template by DNA sequence-specific binding molecules which bind to both ends of the linear template,
Inactivation of the exonucleases by adding competitive or non-competitive inhibitors,
Circularization of the template to for a ring-shaped closed template.”
See [0001]-[0007]).
Ciafrè teaches the incorporation of phosphorothioated (PS) nucleotides
using a PCR reaction as suggested by Watzele in options (a) and (b) is possible.
Ciafrѐ teaches the incorporation of nuclease-resistant nucleotides as cap on 5’ end of strands of an amplification product is possible (see page 4139, left column, “[S]ynthesis of PS-modified genes by PCR” section, lines 1-8), but also the incorporation of modified nucleotides using PS-dNTPs in the amplification mix to generate modified nucleotides in short DNA molecules (72bp) (see same section, lines 21-26 and Figure 4).
Ciafrè teaches that 5’ end capping with modified nucleotides of a long double-stranded DNA product may not be enough to protect the gene product (see page 4140, left paragraph, lines 5-9).
Ciafrè teaches in Figure 6 that the minigene contains the T7 promoter sequence in the minigene. Ciafrè also teaches that a minigene obtained via PCR for incorporating PS-dNTPs can also contain a promoter. Ciafrè teaches that this promoter can incorporate PS-modified nucleotides (see Figure 6).
Ciafrè also teaches the synthesis of PS (phosphorothioated) modified minigenes (72bp) by PCR in presence of 1, 2 or 3 modified dNTPs (see page 4139, left column, “[S]ynthesis of PS-modified genes by PCR” section, lines, 21-26). Ciafrè teaches a total of 14%, 26% or 37.5% modified nucleotides incorporated in the minigene (see Figure 6). Therefore, Ciafrè teaches a linear double-stranded DNA with both a sense and antisense strand, that has protected nucleotides (see Figure 5, endonuclease digestion), i.e. 100% stability of oligonucleotides modified with 1, 2 or 3 PS-dNTPs, with ratios of 0.14, 0.26 and 0.37 for the minigene.
Ciafrè teaches a linear double-stranded DNA product that comprises the protected nucleotides at a ratio of at least 0.01 to total nucleotides.
As shown above, Zhu teaches two of the mentioned approaches, i.e., protecting using modified nucleotides with phosphorothioate modification (LET5 molecule (option (a) of Watzele) and protecting the template by DNA sequence-specific binding molecules which bind to both ends of the linear templates (LET2 molecule (option (d) of Watzele). As shown above, Ciafrѐ teaches option (b) of Watzele.
Therefore, it would have been obvious to one with ordinary skills in the art before the effective fling date of the claimed invention to have combined modifying three nucleotides at each end of the DNA product, as in LET5 of Zhu (option (a)), with option (b) of Watzele, and add further modified nucleotides by PCR as taught by Ciafrѐ. One with ordinary skills in the art motivated in strengthening the stability of the linear double-stranded DNA template LET5 and increasing yield in protein expression in in vitro transcription – translation assays, could have performed this modification with a reasonable expectation of success and would have arrived at the claimed invention.
Response to Arguments
Applicant’s arguments, see page 5 of Remarks filed 05/26/2026, with respect to the rejection(s) of claims 17, 25 and 39 under 35 U.S.C. §102 have been fully considered and are persuasive (see claim interpretation). Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection under U.S.C. §103 is made in view of Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase, downloaded from URL: https://www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase ; Phusion® Hot Start Flex DNA Polymerase | NEB; previously cited), in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited).
Therefore, Zhu may fail to teach the element of claim 17, as stated in the rejection under U.S.C §103. However, the combination of references Zhu as evidenced by NEB-pdf1, Watzele and Ciafrѐ teaches the elements of claim 17 as amended.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Claims 21-23 and 26 are rejected under 35 U.S.C. §103 as being unpatentable over Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase, downloaded from URL: https:// www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase; previously cited) , in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited), as applied to claim 17 above and in further view of Chaudhary (Chaudhary, V.K. et al. “Rapid restriction enzyme-free cloning of PCR products: a high-throughput method applicable for library construction”. PLOS One, Vol. 9, No. 10 (2014), p: e111538; previously cited).
The rejection of claim 17 is described above. The elements of claim 17 are rendered obvious by the combination of references Zhu, NEB-pdf, Watzele and Ciafrѐ.
Regarding claims 21-23 and 26, the combination of references does not teach a double-stranded DNA product wherein the antisense strand has a 5’-overhang (claim 21), wherein the antisense strand has a 5’-overang of 4 to 8 nucleotides (claim 22), nor that the 5’-overhang comprises one or more protected nucleotides (claim 23).
However, Chaudhary teaches a cloning strategy for PCR-amplified DNA which employs type II restriction endonuclease BsaI to create a linearized vector with 4 base-long 5’-overhangs (see Abstract).
Chaudhary teaches the use of stuffer sequences and BsaI to create linear DNA compatible with cloning for creating large libraries and clones (see abstract).
It would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have combined the teachings of Zhu, NEB-pdf, Watzele and Ciafrѐ with the teachings of Chaudhary, and used the linear LET5, modified with phosphorothioate nucleotides as taught by Watzele and Ciafrѐ, and add a stuffer DNA to the construct, and used BsaI to produce a 5’-overhang as taught by Chaudhary. One with ordinary skills in the art, motivated in preserving the LET5 in a library, could have performed this modification with a reasonable expectation of success since the level of skills is high in the art of cloning and library construction. One motivated in preserving and storing the LET5 described in Zhu modified by Watzele and Ciafrѐ, in stable clones, would have performed this modification and arrived at the claimed invention.
Response to Arguments
Applicant’s arguments, see pages 6 and 7 of Remarks filed 05/26/2026, with respect to the rejection(s) of claims 21-23 and 26 under 35 U.S.C. §103 have been fully considered and are persuasive (see claim interpretation). Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection under U.S.C. §103 is made in view of Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase, downloaded from URL: https://www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase ; Phusion® Hot Start Flex DNA Polymerase | NEB; previously cited), in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited), as applied to claim 17 and in further view of Chaudhary (Chaudhary, V.K. et al. “Rapid restriction enzyme-free cloning of PCR products: a high-throughput method applicable for library construction”. PLOS One, Vol. 9, No. 10 (2014), p: e111538; previously cited).
Therefore, the combination of references Zhu as evidenced by NEB-pdf1, Watzele and Ciafrѐ, in further view of Chaudhary teaches the elements of claims 21-23 and 26.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Claim 37 is rejected under 35 U.S.C. §103 as being unpatentable over Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase , downloaded from URL: https:// www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase; previously cited), in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited), as applied to claim 17 above and in further view of NEB-pdf2 (“PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (NEB#M0273)”- downloaded from Internet from URL: https://www.neb.com/en-us/protocols/taq-dna-polymerase-with-standard-taq-buffer-m0273#; previously cited).
The rejection of claim 17 is described above. The elements of claim 17 are rendered obvious by the combination of references Zhu, NEB-pdf, Watzele and Ciafrѐ.
Regarding claim 37, Ciafre teaches using Taq polymerase with either the phosphorotioated primers or with phosphorothiated dNTPs (see abstract; see page 4135, left column, first paragraph).
However, regarding claim 37, the combination of references does not render elements of claim 37 obvious, i.e. “The linear double-stranded DNA product of claim 17, wherein the linear double-stranded DNA product comprises:
A 5’ overhang and a blunt end;
Two 5’ overhangs;
A 3’ overhang and a blunt end;
Two 3’ overhangs; or
A 5’ overhang and a 3’ overhang.”
However, NEB-pdf2 provides for specification on the DNA polymerase that is used since 1985, as taught in “References” section, page 2 of NEB-pdf2.
NEB-pdf2 teaches that Taq DNA polymerase leads to PCR products that contain dA overhangs (see page 2, paragraph 11, “PCR product” section).
NEB-pdf2 teaches that Taq polymerase is powerful and sensitive and widely used in PCR for routine applications (see page 1, “Overview” section).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have substituted the DNA polymerase used by Zhu, a DNA polymerase that is used for cloning ideally, with a DNA polymerase that is widely known and used routinely for product that may not require a step of cloning, a Taq polymerase that is also used by Ciafrѐ. One with ordinary skills in the art motivated in obtaining a product easily with a powerful and sensitive enzyme that is used routinely and readily available in any Molecular Biology laboratory, could have performed this substitution, and as a result obtained a PCR product comprising a 3’ overhangs. One with ordinary skills in the art could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant’s arguments, see page 7 of Remarks filed 05/26/2026, with respect to the rejection(s) of claim 37 under 35 U.S.C. §103 have been fully considered and are persuasive (see claim interpretation). Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection under U.S.C. §103 is made in view of Zhu (Zhu, B. et al. “Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins”. Biotechnology and Bioengineering, Vol. 117 (2020), pp: 3849-3857; previously cited), as evidenced by NEB-pdf1 (Phusion® Hot Start Flex DNA Polymerase, downloaded from URL: https://www.neb.com/en-us/products/m0535-phusion-hot-start-flex-dna-polmerase ; Phusion® Hot Start Flex DNA Polymerase | NEB; previously cited), in view of Watzele (Watzele, M. et al. US 2004/0259081 A1, published December 23, 2004) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited), as applied to claim 17 and in further view of NEB-pdf2 (“PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (NEB#M0273)”- downloaded from Internet from URL: https://www.neb.com/en-us/protocols/taq-dna-polymerase-with-standard-taq-buffer-m0273#; previously cited).
Therefore, the combination of references Zhu as evidenced by NEB-pdf1, Watzele and Ciafrѐ, in further view of NEB-pdf2 teaches the elements of claim 37.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
The following rejections are maintained from previous Office Action dated 02/25/2026:
Claims 17, 24 and 25 are rejected under 35 U.S.C. §103 as being
unpatentable over Gutierrez-Triana (Gutierrez-Triana, J.A. et al. eLife, Vol. 7 (2018), p: e39468; previously cited), as evidenced by NEB-pdf3 (“PCR using Q5® High-Fidelity DNA polymerase (NEB#M0491)”; URL: https:// www.neb.com/en-us/protocols/pcr-using-q5-high-fidelity-dna-polymerase-m0491?pdf=true; downloaded from internet 02/20/2026’ previously cited) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited).
Regarding claim 17, Gutierrez-Triana teaches a linear double-stranded DNA product that contains a cassette with gfp/ insert, i.e. coding sequence (see abstract, and Figure 1).
Regarding claim 17 reciting “wherein the linear double-stranded DNA product comprises the nuclease-resistant nucleotides at a ratio of between 0.0025-0.1 to total nucleotides”, Gutierrez-Triana teaches that a modified oligonucleotide modified with phosphorothioate bonds in the first five nucleotides can be used to perform the 5’ extension (see Figure 1; page 11, “Donor amplification” section and page 10, Key Resources Table). When only 5 nucleotides are modified in a strand, compared to total nucleotides (GFP insert: about 700 nucleotides + 2 Homology Flank regions: about 400x2 nucleotides (nt) =about 1,500 nt), the ratio would be 0.0033. If both sides (strands) are protected within the same DNA product, the ratio would be 10: (1,500x2) = 0.0033, which falls within the range claimed (0.0025-0.1). Gutierrez-Triana also teaches that they do not have problem integrating linear donor DNA fragments larger than 2 kb (see page 6 of 15, “Discussion” section). Therefore, 10 nucleotides modified and protected versus 2000 (2 kb), give ratio of 0.0025, when both strands (2,000x2) are considered.
Gutierrez-Triana teaches modification of nucleotides of 5’ end of the DNA
product on both sense and antisense strands (see figure 1A). Gutierrez-Triana teaches modification of the first 5 nucleotides in 5’ end by using 5’ moiety extension (see page 11, “[D]onor amplification” section, lines 4-6).
Gutierrez-Triana teaches PCR using Q5® High-Fidelity DNA polymerase (see page 11 of 15, “Donor plasmids” section). According to NEB-pdf3, Q5® High-Fidelity DNA polymerase leads to a PCR product with blunt ends (see page 3 of NEB-pdf3, paragraph 13). Therefore, the linear DNA product comprises blunt ends.
Regarding claim 17(a), Gutierrez-Triana does not teach a sense strand that comprises at least two protected nucleotides upstream of the cassette and at least two protected nucleotides downstream of the cassette. Gutierrez-Triana does not teach an antisense strand that comprises at least two nuclease-resistant nucleotides upstream of the cassette and at least two nuclease-resistant nucleotides downstream of the cassette. Gutierrez-Triana is also silent on the cassette comprising a promoter.
However, Ciafrè teaches the incorporation of phosphorothioated (PS) nucleotides as cap on 5’ end of strands of an amplification product is possible (see page 4139, left column, “[S]ynthesis of PS-modified genes by PCR” section, lines 1-8), but also the incorporation of modified nucleotides using PS-dNTPs in the amplification mix to generate modified nucleotides in short DNA molecules (72bp) (see same section, lines 21-26 and Figure 4).
Ciafrè teaches that 5’ end capping with modified nucleotides of a long double-stranded DNA product may not be enough to protect the gene product (see page 4140, left paragraph, lines 5-9).
Ciafrè teaches in Figure 6 that the minigene contains the T7 promoter sequence in the minigene. Ciafrè also teaches that a minigene obtained via PCR for incorporating PS-dNTPs can also contain a promoter. Ciafrè teaches that this promoter can incorporate PS-modified nucleotides (see Figure 6).
Ciafrè also teaches the synthesis of PS (phosphorothioated) modified minigenes (72bp) by PCR in presence of 1, 2 or 3 modified dNTPs (see page 4139, left column, “[S]ynthesis of PS-modified genes by PCR” section, lines, 21-26). Ciafrè teaches a total of 14%, 26% or 37.5% modified nucleotides incorporated in the minigene (see Figure 6). Therefore, Ciafrè teaches a linear double-stranded DNA with both a sense and antisense strand, that has protected nucleotides (see Figure 5, endonuclease digestion), i.e. 100% stability of oligonucleotides modified with 1, 2 or 3 PS-dNTPs, with ratios of 0.14, 0.26 and 0.37 for the minigene.
Ciafrè teaches a linear double-stranded DNA product that comprises the protected nucleotides at a ratio of at least 0.01 to total nucleotides.
It would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to modify the linear double-stranded DNA product of Gutierrez-Triana with the short DNA sequence of Ciafrè and ligate a short DNA sequence comprising modified nucleotides upstream and downstream of the cassette to provide better protection against endonuclease.
Therefore, one motivated in protecting the cassette at each end could have amplify DNA fragments using PS-dNTPs, one with a promoter sequence, one without, and ligate in such as way they flank the gene of interest. One motivated in obtaining a cassette capable of independent transcription and resistant to endonucleases could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Regarding claim 24, Gutierrez-Triana teaches phosphonothioates-modified nucleotides at the 5’ end of both strands of the linear double-stranded DNA product (see Figure 1A, and Key Resource Table, page 10, page 11, “[D]onor amplification” section, line 4-6).
Regarding claim 25, Gutierrez-Triana teaches a probe, i.e. fluorescent marker, as a fusion green fluorescent protein, gfp ( see Figure 1). Gutierrez-Triana also teaches Biotin as a modification (see Figures 1 and 2).
Response to Arguments
Applicant's arguments filed 05/25/2026 have been fully considered but they are not persuasive.
Regarding Applicant’s argument on page 7 against the combination of Gutierrez-Triana and Ciafrè in rejection under 35.U.S.C. §103,
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references Gutierrez-Triana and Ciafrè, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, both references are drawn to protection of linear DNA fragments, using phosphorothioates linkage modifications (see title for Ciafrè; see Figure 1 and “Key Resource Table” on page 10 of 15, in Gutierrez-Triana).
Also, Examiner would like to point out that the claims are drawn to a structure, and a composition, that are rendered obvious by the combination of references, not the intended use of such structure or composition.
The rejections were made in view of what is known in the art, when considering the fact that phosphorothioate (PS) modifications are well known, as stated by the review in Plant Biotechnology, Sauer (Sauer, N.J. et al. "Oligonucleotide-directed mutagenesis for precision gene editing". Plant Biotechnology Journal, Vol. 14 (2016), pp: 496-502). Sauer shows in Figure 1 that three phosphorothioate linkage modifications are made at each end of an oligonucleotide designed as template donor for CRISPR/Cas9 system for gene editing (see page 497). Sauer states that “Based on peer-reviewed literature, the most frequently used designs were modified with phosphorothioate linkages on terminal bases” (see page 497, right column, lines 8-11). Sauer also states that increasing the number of PS linkages contribute to cell toxicity (see page 497, lines 11-15). But it stands to reason that such a structure with increased phosphorothioate linkages was made, as taught by a reference relied upon by Sauer, Rios (Rios, X. et al. “Stable gene targeting in Human cells using single-strand oligonucleotides with modified bases”. PLoS ONE, Vol. 7, No. 5 (2012), p: e36697). Rios shows oligonucleotides modified with up to 7 PS linkages on each end (see Table 1). It is also clear that Rios refers to the oligonucleotides as “protected oligos” as a reference to “nuclease-protected” oligos (see reference #20 in Rios).
Liang (Liang, X. et al. "Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA". Journal of Biotechnology, Vol. 241 (2017), pp: 136-146) teaches that the gene editing can be performed using a short double stranded DNA oligonucleotide with 3’overhang (linear DNA with sense and antisense strands), convenient since it allowed integration of a longer FLAG epitope tag along with a restriction site at rates up to 50% (see abstract). This shows that the modified dsDNA oligo can be manipulated for different purposes and inserted by homologous recombination, regardless of the presence of the modifications.
Liang teaches that 5’ and 3’ ends of the oligonucleotides were protected with two consecutive phosphorothioate-modified bases (see page 141, right column, lines 2-4). Liang teaches that using such phosphorothioate DNA oligo leads to more efficient gene editing ( see abstract).
Therefore, the structure, a short DNA oligonucleotide having PS protected
5’ and 3’ ends is known in the art. Their use is recommended for efficient HDR process in gene editing and is used by Gutierrez-Triana to search for other methods of increasing protection against multimerization as well. Gutierrex-Triana suggests that such modification may not be enough for enhancing the stability of the resulting dsDNA (Figure 1B, and Results section, page 2). Therefore, one with ordinary skills in the art motivated in increasing the number of PS linkage to test further, would consider the teachings of Ciafrѐ.
Ciafrѐ teaches how to obtain a longer DNA fragments having modified nucleotides and teaches that such obtained cassettes/minigenes can result in stability and functional effectiveness (see title).
Regarding Applicant’s argument on page 9 stating that “one could not then ligate the DNA fragments of Ciafrѐ to either end of the dsDNA donor due to the presence of the modifications at the ends of the dsDNA donor”, Examiner was referring to a dsDNA donor with only PS linkages as described by Ciafre, without the Biotin moiety, and as known in art such as Liang. Ciafrѐ teaches ligation of oligonucleotides modified with PS linkages with longer molecules by T4 DNA ligase (see page 4137, left column, second paragraph and right column).
Ciafrѐ’s teachings pre-dates every reference using PS linkages as a method of stabilization in oligonucleotides used as template within a CRISPR/Cas9 system for gene editing.
The structure is rendered obvious by the combination of references.
Therefore, the rejections are maintained.
Claim 26 is rejected under 35 U.S.C. §103 as being
unpatentable over Gutierrez-Triana (Gutierrez-Triana, J.A. et al. eLife, Vol. 7 (2018), p: e39468; previously cited), NEB-pdf3 (“PCR using Q5® High-Fidelity DNA polymerase (NEB#M0491)”; URL: https:// www.neb.com/en-us/protocols/pcr-using-q5-high-fidelity-dna-polymerase-m0491?pdf=true; downloaded from internet 02/20/2026; previously cited) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited) as applied to claim 17 above and in further view of Oyarzabal Santamarina (Oyarzabal Santamarina, J. et al. WO 2021/152147 A1, published August 05, 2021, with priority date January 31, 2020; previously cited).
The rejection of claim 17 is described above. The combination of references Gutierrez-Triana, NEB-pdf3, and Ciafrè renders the elements of claim 17 obvious.
Ciafrè also teaches that gene transfer is currently the basis of many emerging therapeutic strategies, emphasizing the disadvantages of using naked DNA as bio-pharmaceutical product, since it lacks persistence and long-lasting expression, therefore the main reason for its low efficacy in gene therapy (see page 4140, “Discussion” section). Ciafrè also teaches that producing long, double stranded, biologically functional DNA molecules modified with phosphorothioate substitutions, to use for in vivo gene transfer, significantly enhances longevity and stability of the molecule (see page 4141, left column, second paragraph).
Regarding claim 26, the combination of Gutierrez-Triana, NEB-pdf3 and Ciafrè does not teach a nanoparticle comprising the linear double-stranded DNA product either (claim 26).
However, Oyarzabal Santamarina teaches closed linear DNA constructs (see title and abstract). Oyarzabal Santamarina teaches that linear DNA can be delivered to cells using nanoparticles (see page 31, lines 5-11).
It would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have used the linear PCR product as taught by Gutierrez-Triana modified by NEB-pdf3 and Ciafrè and packaged it in a nanoparticle as taught by Oyarzabal Santamarina. One with ordinary skills in the art, motivated in using a non-viral delivery vector for gene transfer into cells, and motivated into a carrier that also contribute to longevity and stability of the DNA molecule, could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant's arguments filed 05/25/2026 have been fully considered but they are not persuasive.
Regarding Applicant’s argument on page 10 against the combination of Gutierrez-Triana and Ciafrè in rejection under 35.U.S.C. §103,
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references Gutierrez-Triana and Ciafrè, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). The arguments against the references are addressed above.
Claims 35-36 are rejected under 35 U.S.C. §103 as being
unpatentable over Gutierrez-Triana (Gutierrez-Triana, J.A. et al. eLife, Vol. 7 (2018), p: e39468; previously cited), as evidenced by NEB-pdf3 (“PCR using Q5® High-Fidelity DNA polymerase (NEB#M0491)”; URL: https:// www.neb.com/en-us/protocols/pcr-using-q5-high-fidelity-dna-polymerase-m0491?pdf=true; downloaded from internet 02/20/2026; previously cited) and Ciafrè (Ciafrè, S.A. et al. Nucleic Acids Research, Vol. 23 (1995), pp: 4134-4142; previously cited), as applied to claim 17 above and in further view of Sykes (Sykes, K.F. et al. “Linear expression elements: a rapid, in vivo, method to screen for gene functions”. Nature Biotechnology, Vol. 17 (1999), pp: 355-359; previously cited).
The rejection of claim 17 is described above. The combination of references Gutierrez-Triana, NEB-pdf3, and Ciafrè renders the elements of claim 17 obvious.
Ciafrè teaches that the phosphorothioate- modified molecules can be synthesized in vitro by PCR and that they can be transcribed, thus representing synthetic functional transcription units” (see page 4141, right column, last paragraph). Therefore, the PCR product is capable of producing mRNAs.
Ciafrè also teaches that phosphorothioate modification might represent a way of protecting injected DNA molecules for gene transfer (see page 4141, right column, last paragraph).
Regarding claims 35-36, the combination of references does not render obvious elements of claims 35 and 36, i.e., “ A vaccine comprising the linear double-stranded DNA product of claim 17” (claim 35) and “ The vaccine of claim 35, wherein the linear double-stranded DNA product encodes an mRNA, and wherein the vaccine is an mRNA-based vaccine” (claim 36).
However, Sykes teaches that a PCR product, i.e., a linear expression elements (LEE), comprising a promoter and terminator regions and an open reading frame (ORF) encoding an antigen, can be delivered by gene gun into fibroblast cells in culture, and also to a mouse ear (see title, abstract, and Figure 4, and page 359, right column).
Sykes uses modified abasic nucleotides and a non-covalent linkage to produce a recombinant linear PCR product (see Figure 2A). Sykes teaches that similar gene expression is obtained with or without covalent linkage within the LEE (see Figure 3).
Sykes teaches that adding a terminator is required for gene expression (see page 357, left column, first paragraph).
Sykes teaches that a linear recombinant PCR product can be used for vaccination and production of antibodies in vivo in mice (see Figure 4).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have used the PCR product with protected nucleotides via phosphorothioate linkage modifications as taught by the combination of Gutierrez-Triana, NEB-pdf3, and Ciafrè, and added a terminator to the linear PCR product as taught by Sykes. One with ordinary skills in the art, motivated in obtaining a simplified, but nuclease-resistant linear PCR product as taught by the combination of Gutierrez-Triana, NEB-pdf3, and Ciafrè, and using it as an expression cassette to produce an mRNA encoding for antibodies as taught by Sykes, could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant's arguments filed 05/25/2026 have been fully considered but they are not persuasive.
Regarding Applicant’s argument on pages 10 and 11 against the combination of Gutierrez-Triana and Ciafrè in rejection under 35.U.S.C. §103,
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references Gutierrez-Triana and Ciafrè, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). The arguments against the references are addressed above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 17, 24-26, 35, 37 and 39 provisionally rejected on the ground of non-statutory double patenting as being unpatentable over claims 1-7 and 16 of co-pending Application No. 18/288,545 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because:
Regarding claim 17, claims 1 and 2 of co-pending application ‘545 also recite “a linear double-stranded DNA product comprising a sense strand and an antisense strand, wherein the linear double-stranded DNA product comprises a single cassette”, and “wherein the cassette comprises a promoter and a coding sequence” and wherein the sense strand comprises at least two modified nucleotides in the 5’ end region of the cassette and at least two modified in the 3’ end of the cassette, i.e. upstream and downstream of the cassette. Claim 2 of the co-pending application ‘ 545 also claims identical limitations in the antisense strand.
Regarding claim 17(b), claim 4 of co-pending app. ‘545 claims a ratio, i.e. 0.01, of modified/phosphorothioated nucleotides to total nucleotides.
Regarding claims 24, claims 1 and 2 of the copending application ‘545 also claim phosphorothioated modified nucleotides .
Regarding claim 25, claim 6 of co-pending application ‘545 also claims the same elements, i.e. “a complex molecule comprising the linear double-stranded DNA product” and “a functional portion, optionally wherein the functional portion is a binding molecule or a probe.”
Regarding claim 26, claim 7 of co-pending app. ‘545 also claims a nanoparticle comprising the linear double-stranded DNA product.
Regarding claim 35, claim 16 of co-pending app. ‘545 also claims a vaccine comprising the linear double-stranded DNA product.
Regarding claim 37, claim 3 of co-pending app. ‘545 also claims 5’ overhang, 3’ overhang, and/or blunt end comprised in the linear double-stranded DNA product.
Regarding claim 39, claim 5 of co-pending app. ‘545 also claims the same range for the ratio of modified/phosphorothioated nucleotides to total nucleotides, i.e. 0.01-0.1.
This is a provisional non-statutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
Applicants request that the double patenting rejection be held in abeyance until it is the only remaining rejection. Therefore, this rejection is maintained.
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 ALEXANDRA G DACE DENITO whose telephone number is (703)756-4752. The examiner can normally be reached Monday-Friday, 8:30-5:00EST.
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/A.D./Examiner, Art Unit 1636 /NANCY J LEITH/Primary Examiner, Art Unit 1636
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