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
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.
Application Status
Receipt is acknowledged of amendment, filed 12/09/2025. Claims 1-29 are currently pending.
Election/Restriction
Applicant’s election of Group I, drawn to claims 1-22, in the reply filed on 12/09/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 23-29 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/09/2025.
Claims 1-22 are currently under examination.
Information Disclosure Statement
Receipt of acknowledgment of the information disclosure statements filed on 04/06/2023 and 06/26/2023 have been received and all references have been considered.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. The specification contains embedded hyperlinks at paragraphs [0129, 0456, 0534, 0605, 0609, 0668, 0690, 0761, 0789, 0792, 0892, 0898 and 0902].
Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 7 is objected to because of the following informalities:
Claim 7 recites “LE helitron” and “RE helitron” whereas “LE” and “RE” are abbreviations. The first appearance of an abbreviation needs to be written out fully and followed by the abbreviation in parenthesis.
Appropriate correction is required.
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 1-4, 6-20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable by Buerckstuemmer et al (US 2019/0323037 Al) in view of Chen et al (The CRISPR Journal Vol 2, No 6, Pgs: 376-394; 2019).
Regarding claim 1, 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 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 explicitly teach the fusion of the Cas9 and helitron.
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).
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 individual Cas9 and helitron components of Buerckstuemmer to include a Cas9-transposase complex as taught by Chen 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 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 amplifying genomic content in eukaryotic cells and targeted transposition to enable directed transposition in cell-free reactions as taught by Buerckstuemmer and Chen.
Regarding claim 2, Buerckstuemmer does not teach the helitron is fused at the N- or C- terminus of the programmable DNA-binding polypeptide.
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 individual Cas9 and helitron components of Buerckstuemmer to include a Cas9-transposase complex as taught by Chen 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 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 amplifying genomic content in eukaryotic cells and targeted transposition to enable directed transposition in cell-free reactions as taught by Buerckstuemmer and Chen.
Regarding claim 3, Buerckstuemmer teaches the helitron is derived from the helibat1 (Page 17, Lines 31-34).
Regarding claim 4, 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 wherein the "copy and paste" transposases include transposases of the Helitron family including the Helraiser transposase [0018]. 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 6, Buerckstuemmer teaches the donor construct is a circular DNA molecule (Page 3, Figure 1B).
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 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 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 claims 11, 12, 14 and 15, 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 claims 13, 16 and 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 the Cas9 protein 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 individual Cas9 and helitron components of Buerckstuemmer to include a Cas9-transposase complex as taught by Chen 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 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 amplifying genomic content in eukaryotic cells and targeted transposition to enable directed transposition in cell-free reactions 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 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 explicitly teach the fusion of the Cas9 and helitron.
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 specifically teaches a transposase–dCas9 fusion protein where there is a dCas9 protein fused to the N-terminus of the Hsmar1 with a second dCas9 protein fused to the C-terminus of the Hsmar1 to increase the rate of dimerization locally while minimizing off-target insertions with two guide RNAs (Page 377, Figure 1A and Page 393, 1st 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 individual Cas9 and helitron components of Buerckstuemmer to include a dCas9-transposase-dCas9 complex as taught by Chen 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 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 amplifying genomic content in eukaryotic cells and targeted transposition to enable directed transposition in cell-free reactions as taught by Buerckstuemmer and Chen.
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].
Buerckstuemmer does not explicitly teach the fusion of the Cas9 and helitron.
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).
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 individual Cas9 and helitron components of Buerckstuemmer to include a Cas9-transposase complex as taught by Chen 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 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 amplifying genomic content in eukaryotic cells and targeted transposition to enable directed transposition in cell-free reactions 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) 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).
The teachings of Buerckstuemmer and Chen are described above and applied as before.
Regarding claim 5, Buerckstuemmer and Chen do not teach the donor construct is a linear single-stranded (ssDNA) or double-stranded (dsDNA) molecule.
Jun teaches the codelivery system called “sgR-DNA” that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA (Page 1651, Abstract). Jun teaches successful recombineering with linear DNA constructs and observed moderate viability compared to the plasmid form, indicating continuous expression of markers and effective selection pressure (Page 1655, 2nd Column bridging Page 1656, 1st 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 and Chen to include the donor DNA as a linear construct as taught by Jun 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, 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 and Jun teaches codelivery system called “sgR-DNA” that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA.
One would have been motivated to make such a modification in order to receive the expected benefit of successful recombineering with linear DNA constructs and moderate viability compared to the plasmid form as taught by Jun.
Claims 21 is rejected 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)).
The teachings of Buerckstuemmer and Chen are described above and applied as before.
Regarding claim 21, Buerckstuemmer and Chen do 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 and Chen 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, 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 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.
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637