DETAILED ACTION
The Response of 27 Oct. 2025 has been entered. Claims 1-18 are currently pending.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of the invention of Group I, claims 1-9 and 14-18, in the reply filed on 27 Oct. 2025 is acknowledged. The traversal is on the ground(s) that the common technical feature is not taught by Kay as cited in the restriction requirement. This is not found persuasive because the linear ssDNA yielded by the elected method, which is the common technical feature linking Groups I-III, does not make a contribution over the art cited in the 103 rejection below (e.g., the linear ssDNA yielded by the combination of CN106086012 in view of Ducani 2013 and Ping, and/or the linear ssDNA yielded by the method of Ping which comprises a GOI flanked by ITRs and is free of bacterial sequences). Applicant has not responded to the species election requirement applicable to Group III. In the interest of compact prosecution, the species election is herein withdrawn.
The requirement is still deemed proper and is therefore made FINAL.
Claims 10-13 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 27 Oct. 2025.
Claims 1-9 and 14-18 are considered here.
Claim Objections
Claims 1-9 and 16-18 are objected to because of the following informalities:
Claim 1, step d) should replace “joint” with “joined”.
Claim 1, step e) should be amended as follows: “at least two covalently linked
Claim 4, element h) should be amended as follows: “a recognition sequence for a first restriction enzyme”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) (indefiniteness)
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.
Claims 1-9 and 14-18 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites that the linear ssNA yielded by the method is “essentially free of bacterial nucleic acid sequences”. The term “essentially” is a relative term which renders the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The instant specification does not expressly define the term “essentially free” but does define “substantially free” as meaning that “the nucleic acid fragments according to the present invention do not contain any bacterial nucleic acid sequences, namely, nucleic acid sequences stemming from the bacterial plasmid or contain only very few bacterial nucleic acid bases, like at most 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleic acid bases” (Published Spec. US20220380750, [0060]). Even assuming that the above definition applies to “essentially free”, the definition rests on the meaning of “only very few”, which is not defined (the recitation “like at most” is considered to be exemplary and non-limiting). Thus, the scope of the linear ssNA produced by the claimed method is unclear.
Step b) of claim 1 recites “generating the linear double stranded nucleic acid fragment with terminal compatible ends”. There is no prior recitation of a “linear double stranded nucleic acid fragment with terminal compatible ends” and the limitation thus lacks antecedent basis.
Step c) of claim 1 recites “the linear double stranded nucleic acid fragment containing the nucleic acid fragment with viral ITR sequences and the GOI”. There is no prior recitation of a “linear double stranded nucleic acid fragment containing the nucleic acid fragment with viral ITR sequences and the GOI” and the limitation thus lacks antecedent basis.
Claim 4 recites “the plasmid containing viral nucleic acid fragments”, which lacks antecedent basis in claim 1. The rejection can be overcome by amending as follows: “the plasmid
Claims 4 and 14 recite multiple instances of “downstream” with respect to various elements of the plasmid without indicating what the elements are downstream relative to, making the arrangement of elements within the plasmid unclear.
Claim 14 recites “which generate a 3'-OH towards the functional ITR directly upstream or inside of the functional ITR sequence of element c)”. It is unclear what location is being referred to. The rejection can be overcome by amending as follows: “which generate a 3'-OH
Claim 18 recites “wherein the generated ends are sticky ends.” Claim 18 depends from claim 4, which recites multiple instances of “generated ends”, making it unclear which generated ends are being limited by claim 18.
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 1-8 and 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of CN106086012 (citations below are to attached English translation) in view of Ducani et al., Nucleic acids research 42.16 (2014): 10596-10604 (Ducani 2014) and Ping et al., Molecular Biotechnology 57.4 (2015): 382-390 (cited in IDS of 6 April 2022).
Regarding claim 1, CN106086012 teaches a method for in vitro generation of genetically engineered, linear DNA fragments containing two ITR sequences flanking a GOI and being free of plasmid-derived bacterial sequences, comprising: a) providing a bacterial plasmid containing a nucleic acid fragment containing the two viral ITR sequences flanking the GOI; b) digesting the plasmid with a first restriction enzyme to yield a linear double stranded nucleic acid fragment; c) recircularizing the linear double stranded nucleic acid fragment to obtain a double stranded circular nucleic acid fragment; d) amplifying the recircularized double stranded fragment via rolling circle amplification (RCA) to generate multiple covalently linked, reverse complement (concatemeric) copies of the circular template strand; e) digesting the concatemers with a second restriction enzyme to obtain linear nucleic acid sequences comprising the ITR sequences flanking the GOI; and f) purifying the linear nucleic acid (p. 3-4, under Summary of the Invention; p. 5, ¶3-6; p. 5-7, under Embodiment; Figs. 1-3). CN106086012 teaches that the prepared linear DNA is free of any prokaryotic DNA sequences that could lead to toxic/immunogenic effects, and that the linear DNA is useful for gene therapy applications (p. 5, ¶4, 9-11). CN106086012 further teaches that the restriction enzyme used in step b) can generate stick ends (i.e. terminal compatible ends), such that the subsequent recircularizing step joins the compatible ends (p. 3, fourth-to-last ¶; claim 6). CN106086012 further teaches that the RCA process of step d) at first yields single-stranded DNA copies followed (after strand displacement) by double-stranded copies (p. 4, 1st full ¶), such that the final product comprises linear dsDNA. Regarding the isolating step c) in claim 1, CN106086012 teaches isolating/purifying the recircularized product of step c) (p. 3, step A-3), and it would have been obvious to carry out such a purifying step after the digestion step b) (either in lieu of the purifying after step c), or in addition to). Changes in the order of steps are generally obvious in the absence of new or unexpected results (see MPEP 2144.04, IV., C.). Moreover, one would have been motivated to carry out a purifying step after the restriction enzyme step in order to separate out the unwanted bacterial plasmid sequences and the enzyme from the desired product.
Regarding claims 2 and 3, CN106086012 teaches that the restriction enzymes used in steps b) and e) can generate blunt or sticky ends (p. 3, fourth-to-last ¶; claim 6).
Regarding claims 4 and 18, CN106086012 teaches a starting plasmid comprising an expression cassette comprising the GOI flanked by ITRs wherein the cassette is flanked by restriction sites suitable for digestion of steps b) and e), and further teaches that the restriction enzymes used in steps b) and e) can generate blunt or sticky ends (see above). It would have been obvious in view of the above teachings to include within the plasmid DNA a first set of outermost restriction sites generating sticky ends to facilitate the recircularization of c), as well as a different set of inner restriction sites generating blunt ends to preclude recircularization after step e) and to allow for a stable linear end product.
Regarding claim 5, CN106086012 teaches purifying the final linear nucleic acid product using gel electrophoresis (p. 6, under 3.3.4.).
Regarding claim 6, CN106086012 teaches recircularizing the DNA with a ligase (p. 3, step A-2; p. 6, under Embodiment 2).
Regarding claim 7, 16 and 17, CN106086012 teaches the RCA is carried out as an isothermal reaction using Phi29 polymerase (p. 4, 1st full ¶; p. 5, ¶5-9; p. 6, under 3.2.).
Regarding claim 8, CN106086012 teaches that the RCA step comprises heat denaturation of the circular DNA and subsequent cooling (p. 6, under 3.3.1.).
Claims 1-8 and 16-18 differ from CN106086012 in that: the generated product comprises linear single-stranded DNA (claim 1).
Ducani 2014 teaches that RCA with Phi29 polymerase on a circular dsDNA template (as described in CN106086012) yields a mixture of ssDNA and dsDNA, and that a pure ssDNA product can be obtained by performing the reaction in the presence of a single-strand binding protein (under RESULTS; Figs. 2-3 and related text).
Ping teaches linear ssDNA constructs substantially similar to those yielded by the claimed method, comprising a gene of interest flanked by ITRs and free of any bacterial sequences (Fig. 1 and related text). Ping teaches that such linear DNA constructs can be transfected into mammalian cells and can achieve long-term transgene expression via gene transfer, and are useful as minivectors for gene therapy that are safer than traditional vectors due to the lack of immunogenic bacterial sequences (p. 385-386, under Results; p. 388, 1st ¶).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of CN106086012 to make linear DNA constructs comprising a GOI flanked by ITRs that are free of bacterial/plasmid sequences wherein the linear constructs comprise ssDNA constructs because it would have been obvious to combine prior art elements according to known methods to yield predictable results. Using the method of CN106086012 to make linear ssDNA constructs would have led to predictable results with a reasonable expectation of success because CN106086012 and Ducani 2014 teach that RCA as taught by CN106086012 yields a mixture of ssDNA and dsDNA, and it would have been obvious for one of ordinary skill to isolate the ssDNA products (e.g., from a mixture yielded by the RCA step, or by performing RCA in the presence of a single-strand binding protein as taught by Ducani 2014 to yield a pure ssDNA product). One of ordinary skill would have been motivated to carry out the method of CN106086012 to prepare and isolate ssDNA constructs because Ping teaches that substantially similar constructs can achieve long-term transgene expression via gene transfer and are useful as minivectors for gene therapy that are safer than traditional vectors due to the lack of immunogenic bacterial sequences.
Claims 4, 9, 14, 15 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of CN106086012 in view of Ducani 2014 and Ping, as applied to claims 1-8 and 16-18 above, further in view of Ducani et al., Nature methods 10.7 (2013): 647-652 (Ducani 2013) (cited in IDS of 29 May 2024).
The teachings of CN106086012, Ducani 2014 and Ping are set forth above. Regarding claim 9, CN106086012 further teaches that the product of the RCA is heat denatured and cooled to renature the linear DNA prior to treatment with restriction enzyme (i.e. so as to form a functional restriction site) (p. 6, under 3.3.4.).
Claims 4, 9, 14, 15 and 18 differ from the combination of CN106086012 in view of Ducani 2014 and Ping, as applied to claims 1-8 and 16-18, in that: the ITRs of the plasmid are flanked by two sets of different restriction sites (claim 4); the heat denaturing and cooling of the RCA product allows hybridization of adjacent inverted terminal repeat and restriction site sequences to form a double stranded hairpin containing a functional restriction site (claim 9); the ITRs of the plasmid are flanked by two sets of different restriction sites, wherein the inner restriction site is for a type IIs restriction enzyme (claim 14); and the type IIs enzyme is selected from the group in claim 15 (which includes BseGI and BtsCI).
Ducani 2013 teaches a method of forming linear ssDNA constructs that is substantially similar to the method of CN106086012 in view of Ducani 2014 and Ping, comprising excising a gene of interest from an expression plasmid with a first restriction enzyme (BsmBI or BsaI) that generates compatible sticky ends, purifying the fragment comprising the gene of interest via gel electrophoresis, recircularizing the purified fragment, carrying out RCA to produce concatemer linear ssDNA copies of the circularized DNA, and using a second restriction enzyme to cut the concatemer copies into individual linear ssDNA strands (p. 648, under The MOSIC method and Fig. 1; ONLINE METHODS, under Pseudogene in vitro amplification). The second restriction enzyme was a type IIs enzyme (BseGI or BtsCI), and the second restriction site was a type IIs site which forms a hairpin structure via hybridization of adjacent inverted terminal repeat and restriction site sequences (Fig. 1, including Fig. 1(c) and caption; p. 648, right col., 1st full ¶). Ducani 2013 teaches that the type IIs site (wherein the enzyme cuts outside of the recognition sequence located within the stem of the hairpin) allows for elimination of the recognition site sequences from the resulting linear ssDNA product, achieves efficient digestion with no undigested byproduct and allows for the processing of different linear ssDNA products from the same circular template (Fig. 1, including Fig. 1(c) and caption; p. 648, right col., 1st full ¶; p. 649, right col., 1st full ¶).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of CN106086012 in view of Ducani 2014 and Ping to make linear DNA constructs comprising a GOI flanked by ITRs that are free of bacterial/plasmid sequences wherein the GOI/ITR fragment is a excised from the plasmid using a first restriction enzyme generating sticky compatible ends and the linear concatemeric product of the RCA is digested with a second type IIs restriction enzyme that acts on a hairpin recognition site as taught by Ducani 2013 because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use a first restriction enzyme that generates sticky compatible ends to excise the GOI/ITR fragment from the plasmid in order to facilitate the subsequent recircularization step, as taught by Ducani 2013. One of ordinary skill would have further been motivated to use a type IIs enzyme to process the RCA product because Ducani 2013 teaches that the type IIs enzyme allows for elimination of the recognition site sequences from the resulting linear ssDNA product, achieves efficient digestion with no undigested byproduct and allows for the processing of different linear ssDNA products from the same circular template. Using a first restriction enzyme generating sticky ends to excise the GOI/ITR fragment and a second type IIs enzyme to process the RCA product in the method of CN106086012 in view of Ducani 2014 and Ping would have led to predictable results with a reasonable expectation of success because Ducani 2013 teaches use of such enzymes in a substantially similar method to that of CN106086012 in view of Ducani 2014 and Ping (comprising essentially the same steps of excising a gene of interest from an expression plasmid with a first restriction enzyme, recircularizing the excised fragment, carrying out RCA to produce concatemeric linear ssDNA, and using a second restriction enzyme to cut the concatemeric DNA into linear ssDNA strands).
Conclusion
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657