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 makes reference to or appears to claim subject matter disclosed in U.S.
Provisional Application No. US 63/001,614, filed March 30, 2020, U.S. Provisional Application No. 62/906,003, filed September 25, 2019, and U.S. Provisional Application No. 62896494, filed 5 September 2019. Applicants have noted in the response filed 21 June 2025 that applicants will not file a petition to obtain the benefit of the earliest of 3 provisional applications filed in 2019. Therefore, the effective filing date of the current application is 09/05/2020.
Application Status
The Amendments and Remarks filed 02 January 2026 in response to the Office Action 02 July 2025 are acknowledged and have been entered. Claims 1, 2, 3, 7, 10, 12, 14, and 22 are amended. Claim 15 is canceled. Claims 1-14 and 16-26 are being examined on the merits.
Any objection or rejection not reiterated herein has been withdrawn in view of applicant’s claim amendments.
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 1-14 and 16-26 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. This is a new rejection necessitated by applicant’s claim amendments
Claims 1 recites “a nucleotide sequence comprising an insertion target site for site-specific cut-and-paste transposon” and further recites “wherein said nucleotide sequence comprises a target sequence comprising a … marker sequence operably-linked to a sequence comprising a specific target sequence for recognition and insertion of said site-specific cut-and-paste transposon that encodes a truncated or extended inactive polypeptide which is extended or truncated, respectively, after transposition”. The specification does not describe an “insertion target site”. It is unclear, from the claim language, if the insertion target site is the same as the target sequence, is only an insertion sequence (i.e., insertion target site), comprises both the recognition sequence and insertion sequence, and/or encompasses the entire fused marker construct. Furthermore, The specification teach that a target site is a site in the nucleotide sequence where the site-specific transposon inserts itself and could only contain a few nucleotides [pg. 65, para 2; pg. 9, para 2]. It is unclear how a target site of such few nucleotides can comprise a target sequence comprising a marker sequence.
Claims 1 and 2 recites “that encodes a truncated or extended inactive polypeptide which is extended or truncated, respectively, after transposition”. It is unclear which sequence this limitation is referring to: the marker sequence or the specific target sequence for recognition and insertion of said site-specific cut-and-paste transposon.
Claims 1 and 2 recite “a sequence comprising a specific target sequence for recognition and insertion of said site-specific cut-and-paste transposon that encodes a truncated or extended inactive polypeptide which is extended or truncated, respectively, after transposition”. It is unclear what portion of the nucleotide sequence encodes the inactive polypeptide, the marker sequence or the specific target sequence.
Those claims identified in the statement of rejection but not explicitly referenced in the rejection are also rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
Response to Arguments
Applicant's arguments filed 02 January 2026 have been fully considered but they are not persuasive. Applicant’s responses do not address the merits of the rejection.
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 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.
Claims 1-5, 16, ad 25-26 are rejected under 35 U.S.C. 103 as being unpatentable over Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) and as applied to claims 1-2 in view of Weng (US 20170253938 A1, 9/7/2017). This rejection is modified to clarify the motivation and reasonable expectation of success in view of Applicant’s arguments, and continues to rely on the same references, findings, and combination set forth in the prior Office Action.
Regarding claims 1-2, 16, and 25-26 Choi teaches mini-Tn7 vectors, which contain selection markers and Tn7 attachment, or attTn7, sites. [pg. 443, col. 2, para 1; Fig. 1b; pg. 445, col. 1, para 3 – pg. 445, col. 2, para 1; Fig. 3]. Choi teaches that the makers can be transcriptionally fused selectable markers [pg. 444; col.1 para 1]. Choi teaches that these constructs aid in generating broad-range cloning and expression systems, to include site- and orientation-specific Tn7 insertions occurred at a single attTn7 site [abstract].
Choi do not teach wherein said translationally fused marker sequence encodes a truncated or extended inactive polypeptide which is extended or truncated, respectively, after transposition to create a composite target sequence that encodes an active fusion product that changes the phenotype of a cell.
Weng teaches the application of a nucleic acid sequence that comprises a divided selectable/screenable marker with splice donor and acceptor sequences [0060]. Weng teaches a recombinant nucleic acid construct comprising in order from upstream to downstream and/or operably connected, a promoter sequence a nucleic acid sequence encoding a first portion of a reporter protein including an N-terminus (i.e., truncated inactive reporter), wherein said first portion is insufficient to provide reporter expression, a splice donor site, a heterologous nucleic acid sequence, a splice acceptor site, a nucleic acid sequence encoding a second portion of a reporter protein including a C-terminus; and a poly(A) signal sequence [0030]. Weng teaches that after the action of a recombinase, which can be a Sleeping beauty transposase, the reporter gene is activated [0060, 0033]. Although Wang teaches the specifics of splice donor and acceptor sequences or uses of a Sleeping beauty transposase, Wang teaches reporter constructs in which a reporter or selectable sequence is intentionally disrupted by an inserted DNA sequence and subsequently restored or activated following a site-specific DNA modification event.
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the nucleotide sequence of Choi with a divided translationally fused selectable marker, i.e., inactive, truncated polypeptide capable of conferring selectable phenotype (i.e., using the reporter activation strategy taught by Weng). One of ordinary skill would be motivated to make the modification for the advantage of generating a nucleotide sequence that is capable of changing the phenotype of a cell, shown by the lack of marker expression, as a method of determining if a transposon based nucleic acid modification and cloning has occurred or provide a direct phenotypic indicator of successful site-specific transposition.
One of ordinary skill would be motivated with an expectation of success because Choi teaches predictable site-specific insertion of DNA into defined target sites, Weng teaches predictable restoration of reporter function through site-specific DNA modification events, and using the known reporter function of Weng in the transposition events of Choi would merely involve applying a known reporter activation strategy to another known site-specific DNA modification to obtain the predictable result of identifying successful modification events through restoration or alternative marker activity.
Claims 6 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) in view of Weng (US 20170253938 A1, 9/7/2017) as applied to claims 1-5, and further in view of Suzuki (US 2002/0132350 A1, 9/19/2002).
The teachings of Choi and Weng are discussed above as applied to claims 1-5 and similarly apply to claims 6 and 8.
Regarding claims 6, Choi nor Weng teaches wherein the selectable marker sequence encodes an inactive bacterial chloramphenicol acetyl transferase (CAT) fusion protein.
Suzuki teaches mini-Mu plasmids where Mu end recognition sequences flank an internal nucleotide sequence, which can further comprise, a scorable marker gene, and/or a sequence of interest to be targeted into the host genome 0077]. Although Suzuki Mu is not a site-specific cut-and-paste transposon, Suzuki does teach a nucleotide sequence comparisons a target site for a transposon and a marker sequence. Suzuki teaches where the reporter gene, selectable marker, can be a chloramphenicol acetyltransferase (CAT) [0106] or a NPT-II gene [0128].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to substitute selection marker in the nucleotide sequence as taught and suggested by Choi and Weng with a chloramphenicol acetyl transferase (CAT) fusion protein. One of ordinary skill would be motivated to make the modification because Suzuki teaches nucleotide sequences comprising transposon sites with selectable markers or a gene of interest, i.e., a CAT fusion protein or a NPT-II fusion protein [0128]. One of ordinary skill would have a reasonable expectation of success since both Choi and Suzuki teach nucleotide sequences comprising transposon sites with selectable markers.
Regarding claim 8, the composite selectable marker sequence is a sequence that is generated after transposition occurs in the nucleotide sequence and therefore the nucleotide sequence as claimed is not distinguished from the prior art as taught above.
Claims 3-4, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) in view of Weng (US 20170253938 A1, 9/7/2017) as applied to claims 1-5, and further in view of Landrette (Landrette et al. 2011 PLoS One Volume 6, Issue 10, e26650).
The teachings of Choi and Wang are discussed above as applied to claims 1-2 and similarly apply to claims 3-4, and 10.
Regarding claims 3-4 and 10, Suzuki do not teach wherein said fused marker sequence encodes an extended, inactive polypeptide. The recitation of “which is truncated after transposition to form a composite target sequence which encodes an active, polypeptide conferring a selectable phenotype upon the cell” is a functional limitation and does not further limit the structure of the product of the nucleotide sequence.
Landrette teaches a somatic mutagenesis with activated luciferase reporter that contains a PB mutator transposon (i.e., extended, inactive polypeptide) [Fig. 1A]. Landrette teaches that during mutagenesis active PB transposase (PBase) catalyzes the precise excision of the PB mutator transposon and a full-length luciferase product is reconstituted [Figure 1A; pg. 2, col. 2, para 1]. Landrette teaches that this strategy marks cells in which transposition has occurred [pg. 2, col. 2, para 1].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the nucleic acid structure as taught and suggested by Choi and Weng with an extended, inactive polypeptide, i.e., inactive polypeptide capable of conferring selectable phenotype, where the marker is expressed upon a transposon excision by a transposase. One of ordinary skill would be motivated to make the modification for the advantage of generating a nucleotide sequence that is capable of changing the phenotype of a cell, shown by marker expression, as a method of determining if a transposon based nucleic acid modification or cloning has occurred.
Claims 11, 13, and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) in view of Weng (US 20170253938 A1, 9/7/2017) and Landrette (Landrette et al. 2011 PLoS One Volume 6, Issue 10, e26650) as applied to claim 1-5 and 10, and further in view of Suzuki (US 2002/0132350 A1, 9/19/2002).
The teachings of Choi, Landrette, and Weng are discussed above as applied to claims 1-5 and 10 and similarly apply to claims 11, 13 and 21.
Regarding claim 11 and 21, Choi, Weng nor Landrette teaches wherein the selectable marker sequence encodes an inactive NPT-II fusion protein.
Suzuki teaches mimi-Mu plasmids where Mu end recognition sequences flank an internal nucleotide sequence, which can further comprise, a scorable marker gene, and/or a sequence of interest to be targeted into the host genome 0077]. Although Suzuki Mu is not a site-specific cut-and-paste transposon, Suzuki does teach a nucleotide sequence comparisons a target site for a transposon and a marker sequence. Suzuki teaches where the reporter gene, selectable marker, can be an NPT-II gene [0128].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to substitute selection marker in the nucleotide sequence as taught and suggested by Choi, Weng, and Landrette with a NPT-II fusion protein. One of ordinary skill would be motivated to make the modification because Suzuki teaches nucleotide sequences comprising transposon sites with selectable markers or a gene of interest, i.e., NPT-II fusion protein. One of ordinary skill would have a reasonable expectation of success since both Choi and Suzuki teach nucleotide sequences comprising transposon sites with selectable markers.
Regarding claim 13, the composite selectable marker sequence is a sequence that is generated after transposition occurs in the nucleotide sequence and therefore the nucleotide sequence as claimed is not distinguished from the prior art as taught above.
Claims 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) in view of Weng (US 20170253938 A1, 9/7/2017) as applied to claims 1-5 and 16, and further in view of Lee (US005348886A, 9/20/1994).
The teachings of Choi and Wang are discussed above as applied to claims 1 and 16 and similarly apply to claims 17-20.
Regarding claims 17-20, Choi do not teach where the vector is a baculovirus shuttle vector capable of propagating in bacteria and in Lepidopteran insect cells susceptible to infection by the baculovirus or in Escherichia coli and insect cells selected from the group consisting of Spodoptera frugiperda, Trichoplusia ni cells, and Bombyx mori cells.
Lee teaches a method for producing infectious recombinant baculoviruses in bacteria is described. A novel baculovirus shuttle vector (bacmid) was constructed that contains a low-copy-number bacterial replicon, a selectable drug resistance marker, and a preferred attachment site for a site-specific bacterial transposon, inserted into a nonessential locus of the baculovirus genome. This shuttle vector can replicate in E. coli as a plasmid and is stably inherited and structurally stable after many generations of growth. Bacmid DNA isolated from E. coli is infectious when introduced into susceptible lepidopteran insect cells [abstract]. Lee teaches that susceptible host insect cells are derived from Spodoptera frugiperda or from Trichoplusia ni, Plutella xylostella, Manduca sexta, or Mamestra brassicae [col. 8, lines 14-18]. Lee teaches that Bacmids containing target sites for site-specific transposons are recipients for transposons carried on other genetic elements (col. 3, lines 5-7). This approach not only greatly facilitates the use of baculovirus vectors for the expression of cloned foreign genes, but also permits the development of new strategies for rapid protein engineering of eukaryotic proteins and expression cloning of previously uncharacterized genes from cDNA libraries (col. 3, lines 7-13).
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the vector as taught and suggested by Choi and Weng where the vector is a bacmid. One of ordinary skill would be motivated to make the modification for the advantage of using the vectors for expression of cloned foreign genes and for rapid protein engineering.
Response to Arguments
Applicant's arguments filed 02 January 2025 with respect to the rejections under 35 USC§ 103. Applicant’s arguments have been fully considered and are not persuasive.
Applicant argues that Weng is directed to splice donor and splice acceptor sequences acted upon by spliceosomes, that recombinases operate differently from transposases, that Sleeping Beauty and piggyBac transposons are random transposition systems, and that Tn7 utilizes distinct target recognition and insertion mechanisms requiring multiple transposition proteins. Applicant further argues that the pending claims are directed to site-specific insertion of Tn7 elements into gene fusions comprising TnsD binding and insertion sites and that such insertions cannot be accomplished using recombinases or random transposons.
The arguments are not persuasive because they do not address the basis of the rejection. The rejection does not rely upon Weng for teaching Tn7 transposition, TnsD-mediated target recognition, Tn7 insertion machinery, or insertion into attTn7 target sites. Furthermore, the claims are not limited to these specific embodiments. Choi is relied upon for teaching site-specific Tn7 transposition systems, insertion target sites recognized by transposition machinery, and nucleotide sequences associated with site-specific transposition events. Weng is relied upon for teaching the broader concept of constructing a reporter or marker sequence that changes activity following a DNA modification event. Weng teaches reporter constructs that are initially inactive because a target sequence interrupts or separates functional portions of the reporter and that become active following modification of the target sequence. Weng therefore teaches the general principle of detecting successful DNA modification events through activation of a previously inactive marker. The rejection proposes applying the reporter activation strategy taught by Weng to the site-specific transposition system taught by Choi. Applicant’s discussion regarding mechanistic differences among spliceosomes, recombinases, Sleeping Beauty transposases, and Tn7 transposition proteins does not overcome the rejection because the rejection does not rely upon equivalence of those mechanisms. Rather, the rejection relies upon the common principle that a targeted DNA modification event can be linked to activation of a reporter or selectable marker.
Applicant argues that Suzuki merely teaches functional CAT reporter genes contained within Mu vectors and does not disclose inactive CAT fusion proteins that become active following insertion of a site-specific transposon. The argument is not persuasive.
The rejection does not rely upon Suzuki for teaching activation of an inactive CAT fusion protein following site-specific transposition. Choi and Weng collectively teach nucleotide constructs in which a DNA modification event results in activation of a marker sequence. Suzuki is relied upon for teaching the use of chloramphenicol acetyltransferase (CAT) as a selectable or reporter marker in transposon-associated constructs. Selection of CAT from the finite number of known selectable marker genes taught by Suzuki represents the predictable substitution of one known selectable marker for another. A person of ordinary skill in the art would have been motivated to utilize CAT within the modified Choi/Weng construct because CAT was a well-known selectable marker routinely employed for monitoring successful genetic manipulation events. Applicant’s argument focuses on whether Suzuki individually teaches the claimed inactive CAT fusion construct. However, nonobviousness cannot be established by attacking references individually where the rejection is based upon a combination of references. The rejection relies upon the combined teachings of Choi, Weng, and Suzuki. Further, claim 8 recites a composite selectable marker sequence generated after transposition. The recited composite sequence merely represents the expected result of the modified construct following the insertion event and does not distinguish over the combination of references for the reasons discussed above.
Applicant argues that Landrette is directed to excision of a transposon from within a reporter gene to restore reporter function and therefore differs from the presently claimed insertion-based systems. The argument is not persuasive. Landrette is relied upon for teaching the concept of an initially inactive reporter construct that becomes active following a transposition-mediated DNA modification event. Although Landrette employs transposon excision rather than insertion, the reference demonstrates that transposition-associated modification of a DNA sequence may be linked to restoration of reporter activity and generation of a detectable phenotype. The rejection does not require Landrette to teach site-specific Tn7 insertion. Rather, Choi supplies the site-specific Tn7 transposition teachings while Landrette supplies the teaching that transposition-associated modification of a reporter construct may be used to generate a detectable phenotypic signal. Applicant’s arguments focus on differences in the specific transposition event utilized by Landrette. However, those differences do not outweigh the express teaching that reporter activity may be coupled to a transposition-mediated DNA modification event. Applying that known strategy to the site-specific Tn7 insertion system of Choi would have been well within the ordinary skill in the art and would have yielded the predictable result of identifying successful transposition events through a change in reporter activity. Claims 11 and 21 further recite NPT-II fusion proteins. Suzuki teaches NPT-II as a known selectable marker suitable for use in transposon-associated constructs. Substituting NPT-II for other known selectable markers constitutes a routine and predictable design choice. A person of ordinary skill in the art would have had a reasonable expectation of success because selectable markers such as CAT and NPT-II were conventionally interchangeable depending upon the desired selection system.
Applicant argues that Choi and Lee do not disclose insertion of Tn7 or Tn7-like elements into gene fusions comprising insertion sites for site-specific transposons and marker genes encoding inactive or active products. The argument is not persuasive. Claims 17-20 are directed to vector formats and host systems. The rejection relies upon Choi for the underlying Tn7 transposition system and relies upon Lee for teaching baculovirus shuttle vectors (bacmids) capable of propagation in bacterial hosts and susceptible lepidopteran insect cells. Lee expressly teaches bacmid vectors containing target sites for site-specific transposons and further teaches that such vectors replicate in E. coli and are useful for introduction into susceptible insect cells. Lee additionally teaches the advantages of using such vectors for recombinant gene manipulation and protein expression. The rejection does not rely upon Lee for teaching the specific marker activation mechanism recited in the base claims. Rather, Lee is relied upon for the additional vector limitations recited in claims 17-20. A person of ordinary skill in the art would have been motivated to utilize the transposition systems taught by Choi within the bacmid vector systems taught by Lee in order to obtain the known advantages of baculovirus shuttle vectors for cloning, expression, and protein engineering.
For the foregoing reasons, Applicant’s arguments have been fully considered but are not persuasive. Therefore, the rejections under 35 U.S.C. §103 are maintained.
Allowable Subject Matter
The following is a statement of reasons for the indication of allowable subject matter: Claims 7, 9, 12, 14, and 22-24 are free of the art. The closest prior art is Choi (Choi et al. 2005. Nature Methods. Vol.2 NO.6; June 2005) as discussed above. Choi nor the prior teach or provide a reasonable rationale to order the selectable marker fusions as claimed.
Conclusion
No claims 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 TIFFANY N GROOMS whose telephone number is (571)272-3771. The examiner can normally be reached M-F 830-530.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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