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 .
DETAILED ACTION
The amended claims filed on January 2, 2026, have been acknowledged. Claims 13-14 were cancelled. Claims 1-2 and 4-5 were amended. Claim 19 is new. Claims 1-12 and 15-19 are pending and examined on the merits.
Withdrawn Claim Objections
The prior objection to claim 4 is withdrawn because of the following amendments:
In claim 4, line 1, “wherein Cas endonuclease” has been changed to “wherein a Cas endonuclease”.
Withdrawn Claim Rejections - 35 USC § 112(b)
The prior rejection of claims 1-12 and 15-18 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn in light of Applicant’s amendments to claims 1-2 to recite “a first guide RNA cuts at the insertion locus”.
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 1-3, 5, 7-12, and 15-19 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by World Intellectual Property Organization Patent Application No. 2017/049266 (Bier; cited in ISR), as evidenced by Wu et al. (Quant Biol. 2: 59–70. 2014) and Oberhofer et al. (PNAS 116: 6250-6259. 2019. Published March 2019). This is a new rejection substantially similar to a previous rejection made in response to Applicant’s amendments to claims 1-2. Applicant’s traversal has been fully considered but is moot in response to the new rejection.
Regarding claims 1 and 17, a cleavage resistant preferred allele is interpreted to refer to an allele that is not targeted by a gRNA, and therefore cannot be cleaved by endonuclease activity at that target site. Furthermore, the preferred allele is considered to be the entire DNA sequence between the gRNA cut sites. Additionally, in regards to the allelic drive element and cleavage resistant preferred allele, these are interpreted to either be on separate nucleic acids or on the same nucleic acid (as contemplated by instant claim 18).
Bier teaches a method of introducing a preferred nucleotide sequence into a genome comprising:
An NCR construct can be transfected (i.e. contacting a cell) as a DNA plasmid (a nucleic acid encoding an allelic drive element) together with a plasmid source of Cas9 (a nucleic acid encoding a Cas endonuclease) protein into the germline of an organism to obtain transgenic organisms carrying this insertion. An NCR construct comprises 1) two guide polynucleotides (e.g., guide RNAs), and 2) homology arms flanking the NCR cassette that directly abut the endonuclease cut sites, as can be seen in Figures 4A-4F (shown below). The NCR element carries a correcting cassette (e.g., coding region of gene or cis-regulatory element) that has been recoded at the original guide-RNA cleavage sites to be immune to cleavage (i.e. a cleavage resistant preferred allele).
PNG
media_image1.png
538
509
media_image1.png
Greyscale
Figure 4A of WO2017/049266
PNG
media_image2.png
261
467
media_image2.png
Greyscale
PNG
media_image3.png
215
465
media_image3.png
Greyscale
Figure 4B of WO2017/049266 Figure 4C of WO2017/049266
As can be seen in Figures 4A-4C, a Cas-gRNA-2 complex and a Cas-gRNA-3 complex cuts at the insertion locus on a chromosome for the allelic drive element to be inserted into the genome.
PNG
media_image4.png
342
462
media_image4.png
Greyscale
PNG
media_image5.png
249
459
media_image5.png
Greyscale
Figure 4D of WO2017/049266 Figure 4E of WO2017/049266
PNG
media_image6.png
190
459
media_image6.png
Greyscale
Figure 4F of WO2017/049266
As can be seen in Figures 4D-4F, a Cas-gRNA-2 complex generates a first double strand break to allow insertion of the allelic drive element into the genome.
As can be seen in Figures 4D-4F, a Cas-gRNA-3 complex generates a second double strand break to allow insertion of the allelic drive element into the genome.
As can be seen in Figures 4E-F, the allelic drive element is inserted into the double stranded break created by the gRNA-2 by homology-directed repair.
As can be seen in Figures 4D-4F, the cleavage resistant sequence and the preferred allele is copied from the first chromosome to the second chromosome by homology directed repair (preferred allele and allelic drive element are on the same allele) (Figure 4 and paragraphs 0159-0163). It is important to note that that the endonuclease resistance is through mutations in the gRNA cleavage sites (i.e. the gRNA binding sites). As such, steps c and d of claim 1 are occurring at the same time as the allelic drive element and preferred allele are together on the same allele.
Regarding the 10 kb distance between the first and second double strand breaks, Bier teaches that a CopyCat element is similar to an NCR in that the CopyCat element often carries one or two gRNAs and not Cas9. The CopyCat element often differs from an NCR in that the gRNAs are directed (via homology arms flanking the gRNA cut sites) to a locus in the genome other than an MCR. CopyCats carrying two gRNAs targeting nearby sequences in a region of the genome would delete the region between those cut sites and insert themselves into the gap (paragraph 00205). As can be seen in Figures 4A-4B, the plasmid comprising the gRNAs is a copycat element that is used to insert the gRNAs into an allele. Bier also teaches that CopyCat elements can carry two gRNAs cutting at some distance from each other, in which case the CopyCat element will generate a deletion between the two sites and then insert itself into that gap (e.g., 10kb gap) (paragraph 00351). As Bier identifies that CopyCat gRNAs can target sites 10 kb apart for insertion (i.e. Figures 4A-4C), these target sites would also cut 10 kb apart when targeting the MCR (Cas9 encoding nucleotide sequence) element in the chromosome for removal (i.e. Figures 4D-4F). Therefore, Bier identifies that the gRNAs can target sites 10 kb apart which falls within at the at least 10 kb limitation of claim 1.
Regarding claims 2 and 17, a cleavage resistant preferred allele is interpreted to refer to an allele that is not targeted by a gRNA, and therefore cannot be cleaved by endonuclease activity at that target site. Furthermore, the preferred allele is considered to be the entire DNA sequence between the gRNA cut sites. Additionally, in regards to the allelic drive element and cleavage resistant preferred allele, these are interpreted to either be on separate nucleic acids or on the same nucleic acid (as contemplated by instant claim 18).
Bier teaches a method of introducing a preferred nucleotide sequence into a genome comprising:
An NCR construct can be transfected (i.e. contacting a cell) as a DNA plasmid (a nucleic acid encoding an allelic drive element) together with a plasmid source of Cas9 (a nucleic acid encoding a Cas endonuclease) protein into the germline of an organism to obtain transgenic organisms carrying this insertion. An NCR construct comprises 1) two guide polynucleotides (e.g., guide RNAs), and 2) homology arms flanking the NCR cassette that directly abut the endonuclease cut sites, as can be seen in Figures 4A-4F (shown below). The NCR element carries a correcting cassette (e.g., coding region of gene or cis-regulatory element) that has been recorded at the original guide-RNA cleavage sites to be immune to cleavage (i.e. a cleavage resistant preferred allele).
PNG
media_image7.png
636
602
media_image7.png
Greyscale
Figure 4A of WO2017/049266
PNG
media_image2.png
261
467
media_image2.png
Greyscale
PNG
media_image3.png
215
465
media_image3.png
Greyscale
Figure 4B of WO2017/049266 Figure 4C of WO2017/049266
As can be seen in Figures 4A-4C, a Cas-gRNA-2 complex and a Cas-gRNA-3 complex cuts at the insertion locus on the first chromosome for the allelic drive element to be inserted into the genome.
PNG
media_image4.png
342
462
media_image4.png
Greyscale
PNG
media_image5.png
249
459
media_image5.png
Greyscale
Figure 4D of WO2017/049266 Figure 4E of WO2017/049266
PNG
media_image6.png
190
459
media_image6.png
Greyscale
Figure 4F of WO2017/049266
As can be seen in Figures 4D-4F, a Cas-gRNA-2 complex cuts the second chromosome to allow insertion of the allelic drive element into the genome.
As can be seen in Figures 4D-4F, a Cas-gRNA-3 complex cuts the second chromosome to allow insertion of the allelic drive element into the genome.
Bier is silent as to whether the gRNA target site is within 100 nucleotides of the non-preferred allele.
However, Wu evidences that the Cas9/sgRNA complex binds double-stranded DNA sequences that contain a sequence match to the first 17-20 nucleotides of the sgRNA if the target sequence is followed by a protospacer adjacent motif (PAM) (Figure 1). Once bound, two independent nuclease domains in Cas9 will each cleave one of the DNA strands 3 bases upstream of the PAM, leaving a blunt end DNA double stranded break (DSB) (page 1, paragraph 1).
As Bier teaches that the homology arms flanking the cassette directly abut the endonuclease cut sites (i.e. gRNA target sites) and Wu evidences that the endonuclease cut site occurs 3 bases upstream of the PAM sequence (i.e. the start of the preferred allele), the cleavage resistant sequence of Bier would be within ~20 nucleotides of the non-preferred allele.
As can be seen in Figures 4E-F, the allelic drive element is inserted into the double stranded break created by the gRNA-2 by homology-directed repair.
As can be seen in Figures 4D-4F, the cleavage resistant sequence and the preferred allele is copied from the first chromosome to the second chromosome by homology directed repair (preferred allele and allelic drive element are on the same allele) (Figure 4 and paragraphs 0159-0163). It is important to note that that the endonuclease resistance is through mutations in the gRNA cleavage sites (i.e. the gRNA binding sites). As such, steps c and d of claim 2 are occurring at the same time as the allelic drive element and preferred allele are together on the same allele.
Regarding the 10 kb distance between the first and second double strand breaks, Bier teaches that a CopyCat element is similar to an NCR in that the CopyCat element often carries one or two gRNAs and not Cas9. The CopyCat element often differs from an NCR in that the gRNAs are directed (via homology arms flanking the gRNA cut sites) to a locus in the genome other than an MCR. CopyCats carrying two gRNAs targeting nearby sequences in a region of the genome would delete the region between those cut sites and insert themselves into the gap (paragraph 00205). As can be seen in Figures 4A-4B, the plasmid comprising the gRNAs is a copycat element that is used to insert the gRNAs into an allele. Bier also teaches that CopyCat elements can carry two gRNAs cutting at some distance from each other, in which case the CopyCat element will generate a deletion between the two sites and then insert itself into that gap (e.g., 10kb gap) (paragraph 00351). As Bier identifies that CopyCat gRNAs can target sites 10 kb apart for insertion (i.e. Figures 4A-4C), these target sites would also cut 10 kb apart when targeting the MCR (Cas9 encoding nucleotide sequence) element in the chromosome for removal (i.e. Figures 4D-4F). Therefore, Bier identifies that the gRNAs can target sites 10 kb apart which falls within at the at least 10 kb limitation of claim 1.
Regarding claim 3, Bier teaches that the endonuclease (i.e. Cas endonuclease) is in a separate plasmid or on a separate chromosome (Figure 4D) from the allelic drive element (i.e. not integrated into the allelic drive element) (Figure 4A).
Regarding claims 5-6, Bier teaches that allelic conversion of the NCR construct (also known as the ERACR construct) into a genome can occur at a rate of at least 99% (paragraph 00532). As such, allelic conversion to the preferred allele would be 99% in the second filial generation.
Regarding claim 7, Wu evidences that the Cas9/sgRNA complex binds double-stranded DNA sequences that contain a sequence match to the first 17-20 nucleotides of the sgRNA if the target sequence is followed by a protospacer adjacent motif (PAM) (Figure 1). Once bound, two independent nuclease domains in Cas9 will each cleave one of the DNA strands 3 bases upstream of the PAM, leaving a blunt end DNA double stranded break (DSB) (page 1, paragraph 1). As such, the second gRNA would necessarily bind to a sensitive portion (i.e. a gRNA binding site) that is within 20 nucleotides of the non-preferred allele.
Regarding claim 8, Bier teaches that their NCR construct can be transfected (i.e. contacting a cell) as a DNA plasmid (a nucleic acid encoding an allelic drive element) together with a plasmid source of Cas9 (a nucleic acid encoding a Cas endonuclease) protein into the germline of an organism to obtain transgenic organisms carrying this insertion. As such, the progeny survive in the presence of Cas9.
Regarding claim 9, Oberhofer evidences that maternal carryover of Cas9/gRNA complexes without a transgene encoding the Cas9 is known to happen (Figure 1 and page 6251, column 2, paragraph 4-page 6252, column 1, paragraph 1). As such, it would also occur in the method of Bier.
Regarding claim 10, Bier teaches that an NCR (also referred to as an ERACR sequence) targeting one or more genes, for example those required for female fertility or survival, may reduce the damage caused by many of these pests (paragraphs 00159 and 00563). As the NCR resistant allele of Bier comprises a recoded gene or cis-regulatory element that restores a genetic function (paragraph 00159), it would be inherent that the recoded gene that restores a genetic function would be viable in a homozygous state.
Regarding claim 11, the phrase “in-frame fusion” is interpreted to mean that the cleavage sites mutations for resistance to endonuclease cleavage are in-frame.
Bier teaches that an NCR construct comprises a recoded gene or cis-regulatory element that restores a genetic function that cannot be cut by the guide polynucleotide(s) (e.g., guide RNA(s)). For example, sequences encoding this gene would directly abut the left homology arm (based on an orientation in which transcription of the gene locus is from left to right; i.e. the 5’ end) so that it is in frame with the undisturbed portion of the gene and carries 3' UTR sequences necessary for producing a functional and stable coding mRNA product (paragraph 00159).
Regarding claim 12, Bier teaches that the feasibility of a gene drive strategy in mosquitoes was tested by generating an MCR that carries one of several well-studied effector gene cassettes capable of blocking transmission of the malarial parasite Plasmodium falciparum (FIGURE 29B). This kh-MCR targets insertion into an eye pigmentation locus (kynurenine hydroxylase =kh = cinnabar in Drosophila) in the Asian vector, Anopheles stephensi. The blood-meal inducible gene cassette carried by the - 17kb kh-MCR expresses two single-chain antibodies that block different steps of the parasite life cycle and are 100% effective in preventing propagation of P. falciparum in mosquitoes. It can also be possible to make use of a combination of MCRs, ERACRs (Bier teaches that NCR can also be a ERACR [Elements to Reverse the Autocatalytic Chain Reaction] (paragraph 00159)), and transposons to broadly disseminate multiple copies of effector gene cassettes. For example, in the exemplary scheme depicted in FIGURE 29D, an MCR carries a copy of a transposase gene (e.g., P-transposase ~2-3) while a matched ERACR carries a desired effector cassette flanked by corresponding transposon ends. The MCR can first be released and allowed to spread broadly throughout the population. These animals do not express the effector genes. Subsequently animals carrying the ERACR, which allows the expression of the effector gene, can be released. When an ERACR encounters an MCR, the transposase encoded by the MCR can mobilize transposition of the effector cassette carried between the transposon ends. Because the ERACR also deletes the MCR, transposition can take place for one generation, thereby creating a singular burst of transposon mobilization peaking at the point where the frequencies of the ERACR and MCR are equal (paragraphs 0058, 00493, and 00496). As such, the NCR of Figure 4 could be used in this example.
Regarding claim 15, Bier teaches that their methods are used for multiplex engineering of large chromosome segments encompassing drought resistance (paragraph 00371).
Regarding claim 16, Bier teaches that pests or weeds that are resistant to pesticides or herbicides (e.g., glyphosate), respectively, may also be targeted by MCRs and/or NCRs. For example, MCRs may replace resistant alleles to restore susceptibility to a pesticide or herbicide (paragraph 00378).
Regarding claim 18, as stated supra, Bier teaches that the NCR element carries a correcting cassette (e.g., coding region of gene or cis-regulatory element) that has been recorded at the original guide-RNA cleavage sites to be immune to cleavage (i.e. a portion of a cleavage resistant preferred allele) (Figure 4 and paragraphs 0159-0163).
Regarding claims 1 and 19, the “a Cas endonuclease” used as part of the contacting a cell with a nucleic acid encoding for a Cas endonuclease is broadly considered to encompass a separate endonuclease than what is used in sub-claims a) and b). A cleavage resistant preferred allele is interpreted to refer to an allele that is not targeted by a gRNA, and therefore cannot be cleaved by endonuclease activity at that target site. Furthermore, the preferred allele is considered to be the entire DNA sequence between the gRNA cut sites. Additionally, in regards to the allelic drive element and cleavage resistant preferred allele, these are interpreted to either be on separate nucleic acids or on the same nucleic acid (as contemplated by instant claim 18).
Bier teaches a method of introducing a preferred nucleotide sequence into a genome comprising:
a trans-complementing MCR consisting of two components: 1) a nucleotide sequence encoding a Cas9 endonuclease under control of regulatory sequences that direct its expression in cells, which is flanked by genomic sequences acting as homology arms precisely abutting the site at which a first gRNA-1 directs cutting in the host genome, (denoted here as "<cas9>" where the symbols"<>" represent the homology arms flanking gRNA-1 cut site; and 2) a nucleotide sequence encoding two gRNA genes each under the control promoters regulating their expression, one of which (gRNA-1) cuts at the previously mentioned site of Cas9 insertion in the genome, while the other (gRNA-2) cuts at the site of insertion of the two-gRNA gene cassette. The two-gRNA gene cassette can be precisely abutted by host homology arms flanking the gRNA-2 cut site in the genome and can be denoted as <gRNAl; gRNA2>. Each of these constructs can be inserted into the genome independently (i.e., by co-injecting a plasmid containing the gRNA construct described in point (2) with a plasmid encoding cas9) (i.e. contacting a cell containing the genome with a nucleic acid coding for a Cas endonuclease and a nucleic acid coding for an allelic-drive element that comprises first and second guide RNAs).
Figure 20 shows an illustrative scheme for a trans-complementing mutagenic chain reaction (MCR). Two separate trans-complementing elements <cas9> and a <gRNA> shown inserted on two different chromosomes together create a drive system that results in each element being copied to the sister chromosome. Such a dual element arrangement is functionally equivalent to that of a single-unit coupled <cas9; gRNA> MCR element. In this scheme, gRNA1 cleaves at the Cas9 insertion site while gRNA2 cleaves at the <gRNA1,2> insertion site.
PNG
media_image8.png
453
1308
media_image8.png
Greyscale
Figure 4A of WO2017/049266
As can be seen in Figure 20,
a Cas endonuclease is guided by the first guide RNA (gRNA2) cuts at an insertion locus for the allelic-drive element (gRNA1 and gRNA2), generating a first double stranded DNA break and the Cas9 transgene inserted at gRNA cut-site 1 can copy themselves from one chromosome to the sister chromosome through homologous directed repair
a Cas endonuclease guided by the second guide RNA (gRNA1) cuts the non-preferred allele (the non-Cas9 encoding allele), but not the preferred allele (the Cas9 encoding allele), generating a second double stranded DNA break, wherein the second double stranded DNA break is on a different, non-homologous chromosome and the gRNA cassette inserted at gRNA cut-site 2, can copy themselves from one chromosome to the sister chromosome through homologous directed repair (paragraphs 00131, 00250, and 00412-00419 and Figure 20).
Bier teaches that a recoded wild-type allele of the locus into which the gene encoding an endonuclease or the MCR element has integrated can be provided wherein the guide polynucleotide cleavage sites are mutated to be resistant to endonuclease cleavage at those sites (paragraph 0058).
Claim Rejections - 35 USC § 103
Claims 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over World Intellectual Property Organization Patent Application No. 2017/049266 (Bier). This rejection is repeated with regards to the rejection in the Non-final Office action mailed on October 1, 2025. Applicant’s traversal has been addressed above.
The teachings of Bier are as discussed above.
Figure 4 of Bier does not include a Cas endonuclease integrated into the allelic drive element.
However, figure 31 of Bier teaches that the Cas9 can be on the same allele as the allelic drive element (the gRNA) and this leads to exponential spread (i.e. super mendelian). When the gRNA does not have the Cas9 on the same allele, this leads to linear spread. As such, it would have been obvious to integrate the Cas9 to get exponential spread instead of just linear spread. Furthermore, Bier teaches that NCR constructs can include a gene encoding a Cas9 (paragraph 00159).
Conclusion
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 KEENAN A BATES whose telephone number is (571)270-0727. The examiner can normally be reached M-F 7:30-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Doug Schultz can be reached on (571) 272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KEENAN A BATES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631