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 .
This action is in response to the amendment filed 05/01/2026, in which claim 7 is currently amended and claims 1-6 and 8-29 were previously presented with claims 23-29 being withdrawn due to a previous restriction requirement acknowledged in the Office action mailed on 02/03/2026. Claims 1-22 are currently pending and under examination.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn. This action is a NON-FINAL.
Priority
Acknowledgment is made of applicant’s claim for priority based on a provisional application filed as 63/089,909 on 10/09/2020.
All claims are given the priority date of 10/09/2020.
Specification
The previous objection of the specification has been withdrawn in view of Applicant’s submission of the substitute specification filed on 05/01/2026.
Claim Objections
The previous objection of claim 7 has been withdrawn in view of Applicant’s Amendments to the claims filed on 05/01/2026.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4, 6-11 and 22 are rejected under 35 U.S.C. 102(a)(1)/(a)(2) as being anticipated by Buerckstuemmer et al (US 2019/0323037 Al; Cited in prior Office Action). This is a NEW Rejection.
Regarding claims 1, 2 and 11, Buerckstuemmer teaches a) a "copy and paste" transposase; and b) a construct comprising a DNA sequence or gene of interest flanked by a "copy and paste" transposon terminal sequence, such as an LTS or RTS (the LTS and RTS are the helitron recognition sequences) wherein the "copy and paste" transposases include transposases of the Helitron family including the Helraiser transposase [0018]. Buerckstuemmer teaches the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases (FIG. lA, FIGS. 9A and l0AA; [0094 and 0180]). Buerckstuemmer teaches that when one aims to amplify genomic content in eukaryotic cells and LTS and RTS have been introduced by conventional genome engineering technologies such as TALEN, CRISPR/Cas, zinc finger nucleases or meganucleases [0021]. Buerckstuemmer teaches CDK4 and CD81 cells comprising a Cas9, Helitron transposon and a donor polynucleotide comprising donor sequence flanked by the LTS and RTS [0238-0244].
Regarding claim 3, Buerckstuemmer teaches the helitron is derived from the helibat1 (Page 17, Lines 31-34).
Regarding claims 4 and 6, Buerckstuemmer teaches the donor construct comprising a donor polynucleotide for insertion into a target polynucleotide and the donor construct is a circular DNA molecule (Page 3, Figure 1B and [0120]).
Regarding claims 7 and 8, the instant specification teaches the LE sequence as SEQ ID NO: 54 and the RE sequence as SEQ ID NO: 55 [01012].
Buerckstuemmer teaches a) a "copy and paste" transposase; and b) a construct comprising a DNA sequence or gene of interest flanked by a "copy and paste" transposon terminal sequence, such as an LTS or RTS (the LTS and RTS are the helitron recognition sequences) wherein the "copy and paste" transposases include transposases of the Helitron family including the Helraiser transposase [0018]. Buerckstuemmer teaches the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases (FIG. lA, FIGS. 9A and 10AA; [0094 and 0180]). Buerckstuemmer teaches the LTS sequence as SEQ ID NO: 3 which is 100% identical to instant SEQ ID NO: 54 as well as the RTS sequence as SEQ ID NO: 4 which is 100% identical to instant SEQ ID NO: 55 (See Appendices I and II, respectively). Buerckstuemmer teaches the full nucleic acid sequence of the helitron transposase as SEQ ID NO: 5 which shows 100% identity to the LTS and RTS sequences at the 5’ and 3’ ends, respectively (See Appendices III and IV, respectively).
Regarding claim 9, Buerckstuemmer teaches the order of the donor polynucleotide, from left to right, as the LTS-EF1aPr-TurboGFP-polyA-RTS (Page 29, Figure 13A).
Regarding claim 10, Buerckstuemmer teaches the donor polynucleotide as TurboGFP which is 711 base pairs long alone; and with the addition of the EF1a promoter to the left of the TurboGFP and the polyA tail to the right of the TurboGFP the sequence would be 2,095 base pairs long (Pages34-35, Table 5).
Regarding claim 22, Buerckstuemmer teaches providing a first expression vector comprising an isolated nucleic acid providing a gene of interest and providing a second expression vector comprising a nucleic acid sequence encoding a transposase wherein the Cas9 is transfected separately [0077-0078 and 0228].
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.
Claims 12-20 are rejected under 35 U.S.C. 103 as being unpatentable by Buerckstuemmer et al (US 2019/0323037 Al; Cited in a prior Office Action) in view of Chen et al (The CRISPR Journal Vol 2, No 6, Pgs: 376-394; 2019; Cited in a prior Office Action). This is a NEW Rejection.
The teachings of Buerckstuemmer as described and applied above.
Regarding claims 12-17, the specification recites the modified cas protein is a dead cas9 without the activity of either the HNH domain and/or the RuvC domain [0070]. Therefore, the “deletion” of the catalytic domain is interpreted as the removal of its activity by substitution or mutation of the amino acid sequence in order to modify the Cas9 protein to be catalytically inactive or “dead” resulting in nickase activity.
Buerckstuemmer does not teach a Cas9 protein that is a nickase or is catalytically inactive.
Chen teaches transposases that naturally insert transposons randomly can be fused to catalytically dead Cas9 (dCas9) for targeted transposition specifically a synthetic Hsmar1 transposase–dCas9 fusion protein (Hsmar1 is known for its copy-and-paste mechanisms; Page 376, 2nd Column and Page 377, Figure 1 description) enabled directed transposition in cell-free reactions (Page 377, 2nd Column). Chen teaches the transposon element (Himar1C9) C-terminus fused to the N-terminus of the dCas9 using a flexible protein linker (XTEN) (Page 385, 2nd Column).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the zinc finger-like motif of Buerckstuemmer to include a dCas9 nickase as taught by Chen because Buerckstuemmer teaches it is within the ordinary skill in the art to use the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases and Chen teaches transposases that naturally insert transposons randomly can be fused to catalytically dead Cas9 (dCas9) for targeted transposition to enable directed transposition in cell-free reactions.
One would have been a reasonable expectation of success to make such a substitution in order to receive the expected benefit of substituting the zinc finger nuclease for a dCas9 nickase as taught by Buerckstuemmer and Chen.
Regarding claims 18-20, Buerckstuemmer teaches a) a "copy and paste" transposase; and b) a construct comprising a DNA sequence or gene of interest flanked by a "copy and paste" transposon terminal sequence, such as an LTS or RTS (the LTS and RTS are the helitron recognition sequences) wherein the "copy and paste" transposases include transposases of the Helitron family including the Helraiser transposase [0018]. Buerckstuemmer teaches the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases (FIG. lA, FIGS. 9A and l0AA; [0094 and 0180]). Buerckstuemmer teaches that when one aims to amplify genomic content in eukaryotic cells and LTS and RTS have been introduced by conventional genome engineering technologies such as TALEN, CRISPR/Cas, zinc finger nucleases or meganucleases [0021]. Buerckstuemmer teaches CDK4 and CD81 cells comprising a Cas9, Helitron transposon and a donor polynucleotide comprising donor sequence flanked by the LTS and RTS [0238-0244].
Buerckstuemmer does not teach a Cas9 protein that is a nickase or is catalytically inactive.
Chen teaches transposases that naturally insert transposons randomly can be fused to catalytically dead Cas9 (dCas9) for targeted transposition specifically a synthetic Hsmar1 transposase–dCas9 fusion protein (Hsmar1 is known for its copy-and-paste mechanisms; Page 376, 2nd Column and Page 377, Figure 1 description) enabled directed transposition in cell-free reactions (Page 377, 2nd Column). Chen teaches the transposon element (Himar1C9) C-terminus fused to the N-terminus of the dCas9 using a flexible protein linker (XTEN) (Page 385, 2nd Column).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the zinc finger-like motif of Buerckstuemmer to include a dCas9 nickase as taught by Chen because Buerckstuemmer teaches it is within the ordinary skill in the art to use the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases and Chen teaches transposases that naturally insert transposons randomly can be fused to catalytically dead Cas9 (dCas9) for targeted transposition to enable directed transposition in cell-free reactions.
One would have been a reasonable expectation of success to make such a substitution in order to receive the expected benefit of substituting the zinc finger nuclease for a dCas9 nickase as taught by Buerckstuemmer and Chen.
Claims 5 is rejected under 35 U.S.C. 103 as being unpatentable over Buerckstuemmer et al (US 2019/0323037 Al; Cited in a prior Office Action) in view of Shedlock et al (WO 2019/046815 A1). This is a NEW Rejection.
The teachings of Buerckstuemmer are described above and applied as before.
Regarding claim 5, Buerckstuemmer does not teach the donor construct is a linear single-stranded (ssDNA) or double-stranded (dsDNA) molecule.
Shedlock teaches a nonviral method for the ex-vivo genetic modification of an immune cell comprising delivering to the immune cell, (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme and (b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon the transposon is a Helraiser Transposon wherein the transposase is flanked by left and right terminal sequences termed LTS and RTS [07 and 0035]. Shedlock teaches linearized donor constructs were also efficiently delivered to the cell nucleus that linear transposon products can also be efficiently electroporated into primary human T cells. [0428].
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Buerckstuemmer to include the donor DNA as a double stranded linear construct as taught by Shedlock because Buerckstuemmer teaches it is within the ordinary skill in the art to use the individual components together within a cell, such as CDK4 and CD81 cells comprising a Cas9, Helitron transposon and a donor polynucleotide comprising donor sequence flanked by the LTS and RTS, to amplify genomic content in eukaryotic cells and Shedlock teaches the use of a double stranded linear construct was just as effectively delivered and utilized by the transposase as circular DNA.
One would have been motivated to make such a modification in order to receive the expected benefit of successful recombineering with double stranded linear DNA constructs as taught by Shedlock.
Claims 21 is rejected under 35 U.S.C. 103 as being unpatentable over Buerckstuemmer et al (US 2019/0323037 Al; Cited in a prior Office Action) in view of Rosser et al (Nat Commun 8, 1191; Pages 1-9 (2017); Cited in a prior Office Action). This is a NEW Rejection.
The teachings of Buerckstuemmer are described above and applied as before.
Regarding claim 21, Buerckstuemmer does not teach a degron associated with the helitron polypeptide or programmable DNA-binding polypeptide.
Rosser teaches the degron tagged dCas9-PR to induce gene expression of target genes by co-transfecting the AID-dCas9-PR with a reporter construct expressing luciferase under control of a minimal promoter containing eight recognition sites for an artificial guide RNA(gRNA-1), and observed a strong luciferase signal in the presence of gRNA-1 but no luciferase activity was detected in the absence of the gRNA-1; thus, demonstrating the potential of AID-dCas9-PR as a drug controllable transcriptional activator (Page 4, 1st Column bridging 2nd Column).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Buerckstuemmer to include the degron as taught by Rosser because Buerckstuemmer teaches it is within the ordinary skill in the art to use the individual components together within a cell, such as CDK4 and CD81 cells comprising a Cas9, Helitron transposon and a donor polynucleotide comprising donor sequence flanked by the LTS and RTS, to amplify genomic content in eukaryotic cells and Rosser teaches degron tagged dCas9-PR to induce gene expression of target genes by co-transfecting the AID-dCas9-PR with a reporter construct expressing luciferase under control of a minimal promoter containing eight recognition sites for an artificial guide RNA.
One would have been motivated to make such a modification in order to receive the expected benefit of a drug controllable transcriptional activator as taught by Rosser.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of claims 1-4, 6-20 and 22 under 35 U.S.C. 103 as being unpatentable over Buerckstuemmer et al (US 2019/0323037 Al) in view of Chen et al (The CRISPR Journal Vol 2, No 6, Pgs: 376-394; 2019) has been withdrawn.
The previous rejection of claim 5 under 35 U.S.C. 103 as being unpatentable over Buerckstuemmer et al (US 2019/0323037 Al) in view of Chen et al (The CRISPR Journal Vol 2, No 6, Pgs: 376-394; 2019) as applied to claims 1-4, 6-20 and 22 above, and further in view of Jun et al (ACS Synth. Biol. 2018, 7, 1651-1659) has been withdrawn.
The previous rejection of claim 21 under 35 U.S.C. 103 as being unpatentable over Buerckstuemmer et al (US 2019/0323037 Al) in view of Chen et al (The CRISPR Journal Vol 2, No 6, Pgs: 376-394; 2019) as applied to claims 1-4, 6-20 and 22 above, and further in view of Rosser et al (Nat Commun 8, 1191; Pages 1-9 (2017)) has been withdrawn.
Applicant’s arguments have been fully considered but have not been found to be persuasive. Applicant argues that the cited art, individually or in combination, does not teach the claimed composition. Applicant continues to argue that claim 1 requires a helitron polypeptide comprising an endonuclease domain and a helicase domain connected to or otherwise capable of forming a complex with the DNA-binding polypeptide. Applicant points to the specification and argues that the endonuclease domain is a HUH endonuclease – a single-stranded DNA-specific nuclease that forms a 5’-phosphotyrosine intermediate – and the helicase domain is an SF1B helicase that unwinds duplex DNA in the 5’ to 3’ direction to enable rolling-circle replication. Applicant continues to argue that the two domains together define helitron transposases as a mechanistic class and are what enable the rolling-circle, single-strand based insertion that the present invention exploits.
Applicant’s argument is not persuasive because the Applicant argues requirements of the invention that are not currently within the claimed invention. Specifically, that the endonuclease domain is a HUH endonuclease – a single-stranded DNA-specific nuclease that forms a 5’-phosphotyrosine intermediate – and the helicase domain is an SF1B helicase that unwinds duplex DNA in the 5’ to 3’ direction to enable rolling-circle replication. However, in the interest of compact prosecution, Buerckstuemmer teaches the Helraiser transposase contains a putative, N-terminal nuclear localization signal (NLS) and a zinc finger-like motif, followed by a RepHel enzymatic core wherein the RepHel consists of a -300-aa-long Rep nuclease domain, characterized by the conserved HUH motif and two active site Tyr residues, and a -600-aa helicase domain containing the eight conserved motifs characteristic of the SF1 superfamily of DNA helicases (FIG. lA, FIGS. 9A and 10AA; [0094 and 0180]).
Applicant argues that Chen would not be obvious to combine with Buerckstuemmer for the substitution of the Cas9 and helitron components as well as no adequate reason to combine. However, the new rejection only relies on Chen for the substitution of the zinc finger-like motif for the dCas9 that Chen uses with the transposons. Therefore, the previous rejection and current arguments from Applicant are moot.
Applicant argues no reasonable expectation of success, specifically, Applicant continues to argue that replacing Himar1 with a helitron polypeptide is not a routine substitution. However, the new rejection only relies on Chen for the substitution of the zinc finger-like motif for the dCas9 that Chen uses with the transposons. Therefore, the previous rejection and current arguments from Applicant are moot.
Applicant argues Jun’s sgR-DNA system is designed for use with CRISPR/Cas-mediated homology directed repair (HDR), a pathway that depends on cellular repair machinery and sequence homology between the donor and the target. Applicant continues to argue that helitron transposition, by construct, proceeds via rolling-circle replication through tyrosine-mediated cleavage of single-stranded DNA – a mechanism that is independent of HDR and does not share the substrate requirements or cellular context that motivate Jun’s linearization approach. Finally, Applicant argues that the base combination of Buerckstuemmer and Chen fails to find the currently claimed invention obvious as stated in the above applicant arguments.
The Examiner acknowledges that in the context of the claim limitations as well as the specification, the rejection in view of Jun is improper and therefore, withdrawn. However, a new rejection, in view of Hickman, teaches all the limitations in combination with Buerckstuemmer as described and applied above.
Applicant argues the functional purpose of Rosser’s AID degron is to control the temporal availability of a dCas9-based transcriptional activator – a gene expression regulation tool that has no relation to transposase activity or targeted DNA insertion. Applicant continues to argue that the Examiner does not identify any recognized limitation of helitron transposition control that a degron would address, nor does the examiner establish any nexus between Rosser’s transcriptional regulation context and the claimed helitron-Cas9 insertion system. Applicant argues that a motivation to add a degron to a transcriptional activator provides no rational basis for adding a degron to a transposition system as well as the base combination fails for the reasons set forth above.
However, the currently claimed invention does not include the limitation that the degron must be associated with the helitron polypeptide but instead that the composition further comprises “a degron associated with the helitron polypeptide or programmable DNA-binding polypeptide”. Rosser is relied on for the teachings that the degron tagged dCas9-PR to induce gene expression of target genes by co-transfecting the AID-dCas9-PR with a reporter construct expressing luciferase under control of a minimal promoter containing eight recognition sites for an artificial guide RNA(gRNA-1), and observed a strong luciferase signal in the presence of gRNA-1 but no luciferase activity was detected in the absence of the gRNA-1; thus, demonstrating the potential of AID-dCas9-PR as a drug controllable transcriptional activator (Page 4, 1st Column bridging 2nd Column). Therefore, one of ordinary skill in the art would have been motivated to modify the teachings of Buerckstuemmer to include the degron as taught by Rosser for the degron tagged dCas9-PR to induce gene expression of target genes by co-transfecting the AID-dCas9-PR with a reporter construct expressing luciferase under control of a minimal promoter containing eight recognition sites for an artificial guide RNA in order to receive the expected benefit of a drug controllable transcriptional activator as taught by Rosser.
Therefore, the Applicant’s arguments are not found to be persuasive and the new rejections of record as outlined above render all Applicant’s arguments moot.
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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