CTNF 18/689,676 CTNF 101357 Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia 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 This action is in response to papers filed March 6, 2024. Claims 1-14, 16, 18, 21, 23, 24 and 26 are currently pending. It is noted that claim 1 is an independent claim. Therefore, claims 1-14, 16, 18, 21, 23, 24 and 26 are under examination to which the following grounds of rejection are applicable. Priority This application claims priority to International Application PCT/US2022/043004, filed on September 9, 2022. The International Application claims priority to US Provisional 63/243,260 September 13, 2021. Therefore, the earliest possible priority is September 13, 2021. Specification 06-49-06 AIA The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered. Claim Objections 07-29-01 Claims 1, 8 and 24 objected to because of the following informalities listed below. Appropriate correction is required. Regarding claim 1, line 2, the acronym “gRNA” is recited, but not defined in the claim. An acronym should be defined the first time it appears in an independent claim. For the purposes of examination, “gRNA” is interpreted to mean “guide RNA” . Regarding claim 8, line 2, the acronym “ssODNs” is recited, but not defined in the claim. An acronym should be defined the first time it appears in an independent claim or in the group of claims under an independent claim. For the purposes of examination, “ssODN” is interpreted to mean “Single-Stranded Oligodeoxynucleotide” . Regarding claim 24, it is objected to because the claims fail to further limit the subject matter of the claim from which it depends. For the sake of compact prosecution, it will be interpreted that claim 24 depends on claim 1. Claim Rejections - 35 USC § 112 07-30-02 AIA 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. 07-34-01 Claims 1-14, 16, 18, 21, 23, 24 and 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. Regarding claim 1 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. It is indefinite in its recitation of the term “designing and using multiple gRNAs” as it lacks essential steps of how gRNAs are being used and designed. The claim only requires the active step of designing and using multiple gRNAs. It is not apparent as to under what structural or functional parameters the designing and using multiple gRNAs is indicative to the preamble of the claim (e.g., a method of improving genome editing). It is noted that on pages 56-57, and 70-71 detail plasmid construction containing gRNAs, as well as generating transgenic lines containing the plasmid constructs. Regarding claims 1, 2, 7, 10, 12, 14 and 24 are indefinite in the phrase “improves” or “improve” . The term “improves” is a relative term which renders the claim indefinite. The term "improve" or “improves” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Moreover, it is unclear how these aspects are being improved. Appropriate action is required. Regarding claim 3, it is indefinite in its recitation of the term “high frequencies”. The term “high frequencies” in claim 3 is a relative term which renders the claim indefinite. The term “high frequencies” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear what is considered a “high” frequency and such terms must be defined in the Specification. Regarding claims 4 and 5, they are indefinite in the term “desired” as its metes and bounds are unclear. Claim scope cannot depend solely on the unrestrained, subjective opinion of a particular individual purported to be practicing the invention. Datamize LLC v. Plumtree Software, Inc., 417 F.3d 1342, 1350, 75 USPQ2d 1801, 1807 (Fed. Cir. 2005)); see also Interval Licensing LLC v. AOL, Inc., 766 F.3d 1364, 1373, 112 USPQ2d 1188 (Fed. Cir. 2014) (holding the claim phrase “unobtrusive manner” indefinite because the specification did not “provide a reasonably clear and exclusive definition, leaving the facially subjective claim language without an objective boundary”). Claims 1, 5 and 6 are indefinite in their recitation of “secondary or tertiary gRNAs” as it unclear whether primary, secondary or tertiary gRNAs share the same or different structure , they are used in sequential order or other. As such the metes and bounds of the claims are indefinite. Claim 10 is indefinite in its recitation of “genome editing efficiency”, as it is unclear what the term “efficiency” is drawn to and fails to provide a specific objective standard for its measurement (i.e. indel percentage, protein expression). As such, the metes and bounds of the claims are indefinite. Claim 12 is indefinite in its recitation of the phrase “precision genome editing rates”, as it is unclear what the term is drawn to, and fails to provide a specific objective standard for its measurement, or the degree of “precision” is intended. As such, the metes and bounds of the claims are indefinite. 07-34-03 Claims 12 and 13 recite the term “decreases”. The term “decreases” in claims 12 and 13 is a relative term which renders the claim indefinite. The term “decreases” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Claim 10-12 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph, as being incomplete for omitting essential elements, such omission amounting to a gap between the elements. See MPEP § 2172.01. It is unclear which “existing genome editing method” are the claims drawn to. 07-34-12 Claim 11 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. It is unclear how mutations are being blocked. Regarding claim 13, it is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. It is indefinite in its recitation of the term “to enhance HDR-mediated precision genome editing”, as it is unclear how RNP delivery enhances genome editing. 07-34-03 Claim 14 recite the term “improve”. The term “improve” in claim 14 is a relative term which renders the claim indefinite. The term “Improve” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Claims 14 is indefinite in its recitation of “a disease-relevant model system” as it is unclear what the system is intended to be encompassed by the quoted phrase. It is unclear what denotes a model system to be “disease-relevant”, rendering the metes and bounds of this claim indefinite. Claim 21 is recites the term “and/or” in line 2. It is unclear what the metes and bounds of this term, as “and” could be interpreted to include only human cells, mammalian cells, or all of the cells, or, “or” would imply that the cell types are in the alternative. Regarding claim 24, it is indefinite in its recitation of the term “the gene drive efficiency ” as it lacks proper antecedent basis. There is no recitation of the gene drive anywhere else in the claims. Moreover, claim 24 is indefinite in its recitation of the phrase “The method of claim,” as it is unclear which claim this is dependent on. As such, the phrase “the method” also lacks proper antecedent basis. Regarding claim 26, it is indefinite in its recitation of the term, “boost genome efficiency”, as “boost” is a relative term of the degree for which the specification fails to provide an objective standard. More specifically, the term “boost” fails to provide a starting baseline or magnitude of increase rendering the metes and bounds of this claim indefinite. Claim Rejections - 35 USC § 112: Scope of Enablement 07-30-01 AIA The following is a quotation of the first paragraph of 35 U.S.C. 112(a): (a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention. The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention. 07-31-03 AIA Claim s 1-14, 16, 18, 21, 23, 24 and 26 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA), first paragraph, because the specification, while being enabling for limited in vitro and insect models (i.e. Example 1 and Example 2 of Specification), it does not reasonably provide enablement for other disease-model systems (e.g. in vivo models, improving gene editing within human, live animal models etc.). The disclosure provides, at most, working examples utilizing the “double tap” method in limited in vitro mammalian cell-line experiments and in one specific Drosophilia insect model. However, the claims are more broadly drawn to essentially any model ( in vivo , fungi, plant, human models) that can perform homology-directed repair, and it is unclear if the disclosed “double-tap” method can be performed on different models and/or disease-models . The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims. The criteria for enablement set out In re Wands, MPEP 2162.01(a), considers the following factors: Breadth of the Claims The claims are directed broadly to essentially any model ( in vivo , fungi, plant, human models) that can perform homology-directed repair. Moreover, the method is used to improve gene editing in any human disease. As such, the breadth of the claims is great. State of Prior Art Overbeek et al. (Molecular Cell, 2016; 63, 633-646) discusses that despite “the critical role that repair outcomes play in defining the nature of a genome edit, the specific insertion/deletion (indel) mutations that result from Cas9-initiated DSB repair have not been well characterized. There remains an outstanding need, therefore, for detailed characterization of repair outcomes at Cas9-initiated DSBs to anticipate functional outcomes” (pp. 633, col 1-2). Moreover, O’Brien et al . (Nat Biotechnol. 2016 Mar;34(3):339-44) teaches that HDR efficiency varies across cell type, genomic locus and other experimental conditions (“Different groups have recently demonstrated CRISPR base editing to achieve efficiencies of 44% to 100% (median = 82%) in mice, rabbits, rats and human embryos. The efficiency depends on factors including the surrounding sequence composition, and the position of the point mutation relative to the protospacer adjacent motif (PAM) at the target site. However, base editing is only efficient within a tight parameter space, e.g. only ~5 bases at each CRISPR-Cas binding site can be targeted with high efficiency”) These teachings establish that genome editing outcomes are highly dependent on the genomic sequence and the biological system, making the concept of improving genome efficiency highly unpredictable in the art. It is unclear if the “double-tap” method discussed in the Specification can be applied to fungi, human, bacteria, models. Amount of Direction Provided by the Inventor The specification provides insufficient direction and guidance to enable a person of ordinary skill in the art to practice the full scope of the claimed invention. Although the claims broadly encompass a method of improving genome editing in any organism that can perform HDR, the Specification only provides working examples for sickle-cell and Tay-Sachs disease models for genome editing (para 0103, “It was also demonstrated the utility of the double tap method by installing two disease-relevant mutations (an A to T point mutation in the HBB gene that causes sickle cell disease, and a 4-bp insertion in the HEXA gene that causes Tay-Sachs disease)”). Moreover, Example 1 of the disclosure only utilizes specific gRNAs (HBB1, HBB3, HEXA2, HEXA4, and HEXA5), accounting for at minimum 40% of total repair outcomes for improving the sickle cell-relevant mutation and Tay-Sachs mutation in vitro (para 0090, “The sickle cell-relevant mutation E6V in hemoglobin, which is an A to T transversion mutation in the HBB gene, and the Tay-Sachs disease-relevant TATC 4-bp insertion in the HEXA gene were chosen as pegRNA-nicking gRNA combinations have already been optimized to introduce these mutations with prime editing…It was found that five out of the ten protospacers closely matched the inDelphi predictions; that is, these gRNAs (HBB1, HBB3, HEXA2, HEXA4, and HEXA5) generated the top three inDelphi predicted indels, and their collective introduction efficiencies represented >40% of all repair products”). Therefore, the improvements depended on specifically selected loci (HBB and HEXA gene) and gRNAs to generate favorable indel distributions, rather than broadly enabling all target genes, genomic loci or gRNA combinations encompassed by the claims. Moreover, the specification itself demonstrates the method is highly dependent on the genomic loci (para 0006, “In certain embodiments, the double tap method was tested in multiple human cell lines at 15 different genomic loci… larger improvements in HDR-mediated genome editing efficiencies were observed when targeting higher frequency indel sequences”). Further, in Example 2, the Specification discloses a specific gene-drive system that is only drawn to Drosophila (para 0033, “Double-tap trans-complementing gene drive (DT-tGD) experimental setup and inheritance analysis. FIG. 22 a . Schematic of the DT-tGD arrangement in which the Cas9 and gRNA elements are kept as two separate transgenic lines; gRNA-1 and gRNA-2 target the loci at which the Cas9 and gRNA elements are inserted, respectively… The y1-gRNA and w2-gRNA target the wildtype yellow and white loci, respectively. The y1b-gRNA and w2b-gRNA target a single base pair deletion of the most common indel generated at the yellow and white loci, respectively. ”), and does not enable the full scope of the claims to encompass other disease models or species. The Specification is silent on whether the y1b-gRNA and w2b-gRNA could be applied to other non-Drosophila models and also result in improved gene editing. Aside from the examples of gRNAs and indel sequences described in Examples 1 and 2, the Specification lacks step-by-step protocols, transfection conditions, specific sequences and processing conditions that would enable a skilled artisan to adapt the methods to other genomic sequences, indel sequences and disease models. Presence or Absence of Working Examples The specification provides working examples for improving gene editing in a sickle-cell disease model and Tay-Sachs disease in mammalian cells (para 0090, “It was found that five out of the ten protospacers closely matched the inDelphi predictions; that is, these gRNAs (HBB1, HBB3, HEXA2, HEXA4, and HEXA5) generated the top three inDelphi predicted indels, and their collective introduction efficiencies represented >40% of all repair products.”). Moreover, the specification details the addition of two gRNAs for circumventing indel sequences in Drosophila (para 0164, “To circumvent this phenomenon a CRISPR-based homing gene drive was supplemented with additional gRNAs targeting the most common resistance alleles generated by the drive process. This modification should provide a second opportunity for allelic conversion through HDR by allowing the drive element to also cut a subset of the resistance alleles, improving gene-drive inheritance. To do this, the “double-tap” trans-complementing gene drive (DT-tGD) was built, which contains two extra gRNAs”). The specification fails to teach how to identify indel sequences and how to design gRNAs to address the indel sequences. Moreover, the examples recited in the Specification are not representative of the full scope of the claims, which encompass ANY gRNA, ANY indel and ANY human disease. Quantity of Experimentation Necessary In view of the sequence-dependent behavior for improving genomic efficiency, a person of ordinary skill in the art would not reasonably expect the methods of using gRNAs listed in Examples 1 and 2, would result in the improved genomic editing in any disease model and any indel sequence. A person with ordinary skill in the art would have to perform undue, substantial, experimentation to full encompass the scope of the claims . Conclusion In light of the unpredictability surrounding the claimed subject matter and the lack of adequate guidance, one wishing to practice the presently claimed invention would be unable to do so without engaging in undue experimentation. It is especially noted that applicants provide no data, examples, figures, etc. demonstrating which gRNAs and indel sequences would successfully improve genome editing, and if this can be applied to the full scope of the claims. In the absence of such information, a person of ordinary skill in the art would reasonably require an undue quantity of experimentation. Claim Rejections - 35 USC § 112: Written Description 07-30-01 AIA The following is a quotation of the first paragraph of 35 U.S.C. 112(a): (a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention. The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention. 07-31-01 Claims 1-14, 16, 18, 21-26 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. The claims broadly encompass methods for improving genome editing using multiple gRNAs targeting indel sequences across numerous organisms and disease models, which include in vivo models, human models, fungi, plants etc. Moreover, the claims are primarily defined by their functional language rather than by their distinct structural features; the Specification does not reasonably convey to a person of ordinary skill in the art that the inventor had possession of the full scope of the claims at the time of filing. The Specification itself demonstrates the method is highly dependent on the genomic loci and the number of indels within a sequence (para 0006, “In certain embodiments, the double tap method was tested in multiple human cell lines at 15 different genomic loci… larger improvements in HDR-mediated genome editing efficiencies were observed when targeting higher frequency indel sequences”). Although the disclosure reveals that the double tap method could be implemented in any animal, fungi, or plant (see para 0006), these statements are merely exemplary and fail to demonstrate full possession of the claims. It is noted that on page 56, of the Specification, Applicants’ discuss a “double-tap” trans-complementing gene drive that “contains two extra gRNAs…targeting one of the most prevalent resistance alleles formed at each locus by [their] original trans-complementing gene drive [tGD]” (pp. 56). Further, they discuss that the addition of two additional gRNAs targeting common resistance alleles can improve gene-drive inheritance (see pp. 56 para 2) and specific primers used to target specific indels in fruit flies (see Table 2.) PNG media_image1.png 281 645 media_image1.png Greyscale Moreover, Example 1 is directed to a method for improving gene editing in a sickle-cell disease model and Tay-Sachs disease in vitro (para 0069) experiments containing HEK293T cells, HeLa and K562 cells (para 0090, “It was found that five out of the ten protospacers closely matched the inDelphi predictions; that is, these gRNAs (HBB1, HBB3, HEXA2, HEXA4, and HEXA5) generated the top three inDelphi predicted indels, and their collective introduction efficiencies represented >40% of all repair products.”) Despite this, however, the claims require targeting specific “indel sequences”, are not known and highly dependent on the specific target gene and target loci. As shown, the Specification recites a limited number of examples drawn to in vivo cell cultures and Drosophila and does not claim full possession of the claimed genus. Indel sequences and gRNAs are high variable depending on the genomic sequence, so a skilled artisan would fail to recognize which gRNAs and which target indels would fall within the scope of the claimed invention. Overbeek et al. discusses that despite “the critical role that repair outcomes play in defining the nature of a genome edit, the specific insertion/deletion (indel) mutations that result from Cas9-initiated DSB repair have not been well characterized. There remains an outstanding need, therefore, for detailed characterization of repair outcomes at Cas9-initiated DSBs to anticipate functional outcomes”. Moreover, O’Brien et al . teaches that HDR efficiency varies across cell type, genomic locus and other experimental conditions (“Different groups have recently demonstrated CRISPR base editing to achieve efficiencies of 44% to 100% (median = 82%) in mice, rabbits, rats and human embryos. The efficiency depends on factors including the surrounding sequence composition, and the position of the point mutation relative to the protospacer adjacent motif (PAM) at the target site. However, base editing is only efficient within a tight parameter space, e.g. only ~5 bases at each CRISPR-Cas binding site can be targeted with high efficiency”) These teachings establish that genome editing outcomes are highly dependent on the genomic sequence and the biological system, making the concept of improving genome efficiency highly unpredictable in the art. As a result, a person of ordinary skill in the art would be required to determine the indel profile for each target locus and design multiple desired gRNAs to target such indels. Claim Rejections - 35 USC § 103 07-06 AIA 15-10-15 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. 07-20-aia AIA 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. 07-23-aia AIA 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. 07-20-02-aia AIA This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention. 07-21-aia AIA Claim (s) 1-10, 12-14, 16, 18, 21, and 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ahflors et al. (Published 2017. WIPO Publication: WO 2017216771 A2) in view of Sehn et al (Published: 2015. Clinical Genomics, 2015, pp. 129–150) . Regarding claim 1, Ahlfors teaches a method for improving genome editing (para [0014]; “In some embodiments, we provide herein HDR repair templates having an improved design that can improve stability of the single-stranded oligodeoxynucleotide (ssODN) HDR donor template and/or allow genetic modifications to be made more efficiently at a target site of interest.”; para [0092], “ HDR mediated gene-editing strategy that requires multiple transfections of the HDR- plasmid and corrective HDR donor, targeting the disease-causing mutation, in this case cystic fibrosis caused by a W1282X mutation, an example of a non-sense or premature termination mutation.”; para [0248], “To overcome this difference in efficiency, we have developed an HDR-mediated gene-editing strategy with multiple transfections of HDR-plasmid and a corrective HDR donor in order to target disease-causing mutations or for gene enhancement.”), comprising designing and using multiple gRNAs (para [0025], “ where there are multiple gRNAs, the multiple gRNAs may be the same or different. For example, the first gRNA and the second gRNA may be the same or different from each other, and each may be the same or different from the third gRNA”) using primary gRNAs targeting a wild-type genomic sequence (para 0021, “In some embodiments, only one gRNA (e.g., only the first gRNA, the second gRNA, or the third gRNA) can bind to its target DNA sequence in a "normal" or non-disease causing TGR [target genome region]; in this case, the CRISPR/Cas9 system will only create a single nick in one strand of the double helix in the TGR.”; para 0240, “Selective repair of mutant mitochondrial DNA without affecting normal or wild type mitochondrial DNA is of great importance.”; para 0240, “To selectively repair the mutant DNA and increase the proportion of wild type mitochondrial DNA for complete recovery from the disease phenotype, an HDR donor ssODN that is linked with either PNA-MSP or PNA-TPP along with Cas9 enzyme and gRNA (Cas9+gRNA)-expressing plasmid can be used.”) and tertiary gRNA is targeting mutations (para 0021, “In some embodiments therefore, the CRISPR/Cas9 system can only bind and/or modify the TGR (e.g., the third target DNA sequence) in the mammalian cell of a patient suffering from a disease or in a mammalian cell where the TGR includes a disease- causing mutation.”; para 0079, “Moreover, the repeated attempts of HDR repair can repair the Indel mutations too.”; para [00240], “The gRNA is designed so that it exclusively binds to mutated mitochondrial DNA, but not to wild type DNA. For efficient mitochondrial DNA editing, either co-transfection of donor ssODN and Cas9+gRNA expressing plasmid, or transfection of donor ssODN 24 hours after transfection of Cas9+gRNA-expressing plasmid, can be used”) However, Ahlfors fails to explicitly teach designing secondary or tertiary gRNAs that specifically target indel sequences, as they only mention targeting mutations. Sehn teaches that indels are another form of mutations that can be caused by incorrect DNA repair (pp. 130, “Insertion–deletion mutations (indels) refer to insertion and/or deletion of nucleotides into genomic DNA and include events less than 1 kb in length.”; pp. 130 “Some types of DNA alterations, including insertions/deletions (indels), are not necessarily the direct result of DNA damage, per se . Instead, indels can originate from DNA polymerase errors or incorrect DNA repair following a genetic insult. As a result, indels may be complex (e.g., include both inserted and deleted bases) and often involve areas with repetitive sequences, factors that can make alignment, identification, and annotation difficult.”). Moreover, Sehn teaches that indel mutations play a role in diagnoses of diseases (pp. 130, “Accurate and reproducible identification of indels is critical in clinical testing, as indels are commonly implicated in constitutional (hereditary) and somatic (acquired, including cancer) diseases and may be important for diagnosis (e.g., trinucleotide repeat expansion diseases), patient management and counseling”) A person with ordinary skill in the art would have recognized that indel sequences discussed in Sehn generated during genome editing are a type of mutation, and that the application of Ahlfor’s genome editing technique would allow for the addition of multiple gRNAs to target and correct indel mutations. Such an application would be obvious to try in order to improve gene editing outcomes with the motivation of reducing the number of indel mutations and decrease risk of disease progression. Moreover, a skill in the art would have reasonable success in applying Ahlfor’s technique to target indel sequences, as they demonstrate that homology-directed repair (HDR) has been shown to repair indel mutations as well (see para 0079). Regarding claim 2, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches the method improves homology-directed repair (HDR)-meditates precision genome editing efficiencies (para 0013, “reduced off-site or non-specific modification (i.e., higher specificity of genomic modification); a higher efficiency of homology- directed repair (HDR); improved stability of a single- stranded DNA oligonucleotide (ssODN) HDR repair template; and/or other advantages as will become apparent herein”; para 0014, “In some embodiments, we provide herein HDR repair templates having an improved design that can improve stability of the ssODN HDR donor template and/or allow genetic modifications to be made more efficiently at a target site of interest…Thus, stability, efficiency, and/or traceability of the repair template, e.g., a ssODN, may be improved.” para 0079, “Moreover, the repeated attempts of HDR repair can repair the Indel mutations too.”). Regarding claim 3, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches that the indel sequences with high frequencies are targeted by end joining pathways, (para 0242, “Correcting mutations via high fidelity HDR pathways with a donor template is generally less efficient than non-homologus end joining (NHEJ), which can repair a DSB by directly rejoining the two ends in a process that does not require any homology repair template.”) Regarding claim 4, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches wherein the indel sequence(s) are re-targeted for double-stand break (DSB) at a desired genomic locus be processed by HDR using a same donor template (para 0248, “To overcome this difference in efficiency, we have developed an HDR-mediated gene-editing strategy with multiple transfections of HDR-plasmid and a corrective HDR donor in order to target disease-causing mutations or for gene enhancement.”; Example 8: Repeated correction of non-functional genes to increase the incidence of HDR.) Regarding claim 5, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches the secondary or tertiary gRNAs decreases unwanted indel products and increases desired precision genome editing outcome (para 0079, “During the HDR repair process, it is possible that the cellular machinery may introduce intrinsic corrections to restore the histidine codon (H) and it also possible that indels may be formed by NHEJ.”; para 0092, “several indels may occur in some cells along with HDR-mediated repair and gene correction in other cells, while some cells will not receive the HDR-plasmid and/or corrective HDR donor due to less than 100% efficiency of introduction into all cells at each round…Thus every attempt to introduce the HDR-plasmid and corrective HDR template to the cell population (in vitro or in vivo) significantly increases the number of corrected cells, without further harming the remaining cells.”; para 0226, “Once a large cell population is successfully obtained with a reasonable indel rate or HDR-mediated correction or HDR-mediated knock-in”). Regarding claim 6, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches that wherein the secondary or tertiary gRNAs does not introduce off-target DSBs at a level that impacts cell viability (para 0048, “In other embodiments, methods provided herein may be used to modify one or more gene in a cancer cell, such as one or more gene that results in death of the cell, termination or reduction in proliferation and/or growth of the cell, and/or confers dependence on the presence or introduction (or the lack of presence) of a substance for continued survival of the cell.”; para 0062, “ This approach can be fine-tuned to deliver the Cas9/Cas9n protein at the optimum concentration to limit off-target cleavage by utilizing a cell's own endogenous degradation machinery to rapidly degrade the Cas9 or Cas9n protein, once it has completed its activity.”; para 0079, “The strategy attempts to introduce HDR plasmid through several rounds of transfections along with corrective HDR donor template to repair the H46R mutation in the majority of cells") Regarding claim 7, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches wherein the method improved HDR-mediated precision genome editing efficiency for installation of point mutations, small, insertions, deletions or gene knock in, (para 0060, “ In some embodiments, this method is particularly useful where the disease-causing mutation is a heterozygous mutation, e.g., a point mutation, e.g., a gain of function mutation, and only one chromosome requires repair”; para 0026, “ Once a large cell population is successfully obtained with a reasonable indel rate or HDR-mediated correction or HDR-mediated knock-in”). Regarding claim 8, the combined teachings of Sehn and Ahlfors render obvious claims 1 and 7. Moreover, Ahlfors teaches that the deletions are done with ssODNs, (para 0047, “the repair template is a ssODN having the sequence set forth in SEQ ID NO: 6 or 7; and/or one or more gRNA comprises the sequence set forth in SEQ ID NO: 4 or 5.”). Regarding claim 9, the combined teachings of Sehn and Ahlfors render obvious claims 1 and 7. Moreover, Ahlfors discloses wherein gene knock-in is using dsDNA donor templates (para 0222, “…for large modifications [>100 bp insertion/deletion], using a double stranded DNA donor (dsDNA) is generally more efficient than a ssODN; however, a ssODN provides a more effective HOR than a dsDNA template when small modifications [<50 bp] in the target sequence are needed”) Regarding claim 10, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches the use of the method of claim 1 in combination with an existing genome editing method to improve genome editing efficiency without perturbing gene expression levels or cell cycles (para 132, “CRISPR gRNAs are delivered in combination with Cas9 or Cas9n as episomal or non-episomal plasmids”; para 0008, “However, HDR typically occurs less frequently and is substantially more variable in frequency than NHEJ.”; para 0009, “A potential limitation of CRISPR nuclease systems is that they can cause off-target mutagenesis. To minimize off-target activity, several mutant forms of Cas9 protein have been developed to maximize introduction of DSBs at the desired target site.”). Regarding claim 12, the combined teachings of Sehn and Ahlfors render obvious claims 1 and 10. Moreover, Ahlfors teaches wherein the existing genome editing method is an additional HDR-enhancing method to further improve precision genome editing rates and decreases unwanted indel rates (para 0226, “Once a large cell population is successfully obtained with a reasonable indel rate or HDR-mediated correction or HDR-mediated knock-in, edited cells will be purified using magnetic separation or other suitable methods known in the art such as, e.g., cell sorting”; para 0037, “In some embodiments, one or more cap may serve to increase stability of the repair template, increase efficiency of the HDR pathway, and/or allow monitoring of cellular uptake and/or distribution of the repair template in a cell.”) Regarding claim 13, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches RNP delivery to enhance HDR-mediated precision genome editing (para 0062, “Further, in some embodiments, gRNAs produced using in vitro transcription (also referred to as "IVT gRNAs") are pre-loaded onto a Cas9 protein. In some embodiments, cleavage specificity of the CRISPR/Cas9 system is further enhanced by direct introduction into a cell of a pre-complexed Cas9 or Cas9n protein with IVT gRNA to form a Cas9/gRNA ribonucleoprotein complex (Cas9/gRNA RNP)”). Regarding claim 14, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches that the method is used for disease modelling (para 0049, “In a sixth aspect, there is provided a method for treating or preventing ALS or Frontotemporal Dementia resulting from a mutated C90RF72 gene in a patient in need thereof, comprising use of the CRISPR/Cas9 systems provided herein.”; para 0228, “Here we use ALS as a genetic disease model to demonstrate gene-editing strategies for correcting genetic mutations associated with ALS.”). Regarding claim 16, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches that the method is applied in a subject or system that has HDR as a DNA repair mechanism and suffers from low HDR frequencies, and wherein the HDR frequencies are increased after applying said method (para 0008, “However, HDR typically occurs less frequently and is substantially more variable in frequency than NHEJ. The repair template can either be in the form of double-stranded DNA”; para 0014, “In general, HDR occurs at a much lower frequency and is therefore less efficient than NHEJ in CRISPR/Cas9 gene editing systems… In some embodiments, we provide herein HDR repair templates having an improved design that can improve stability of the ssODN HDR donor template and/or allow genetic modifications to be made more efficiently at a target site of interest.”) Regarding claim 18, the combined teachings of Sehn and Ahlfors render obvious claims 1 and 16. Moreover, Ahlfors teaches that the subject is an animal (para 0103, “ "Genomic DNA" refers to the DNA of a genome of an organism including, but not limited to, the DNA of the genome of a bacterium, fungus, archea, plant or animal, including mammals, including humans.”) Regarding claim 21, the combined teachings of Sehn and Ahlfors render obvious claims 1 and 16. Moreover, Ahlfors teaches that the system comprises mammalian cells (para 0017, “In a first broad aspect, there is provided a method for targeted genomic modification within a target genome region (TGR) in a mammalian cell, the method comprising providing a CRISPR/Cas9 system and contacting the mammalian cell with the CRISPR/Cas9 system”). Regarding claim 26, the combined teachings of Sehn and Ahlfors render obvious claim 1. Moreover, Ahlfors teaches that the method is used for treating a disease (para 0049, “In a sixth aspect, there is provided a method for treating or preventing ALS or Frontotemporal Dementia resulting from a mutated C90RF72 gene in a patient in need thereof, comprising use of the CRISPR/Cas9 systems provided herein.”; para 0228, “Here we use ALS as a genetic disease model to demonstrate gene-editing strategies for correcting genetic mutations associated with ALS.”). *** 07-21-aia AIA Claim (s) 1 and 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ahflors et al. (Published 2017. WIPO Publication: WO 2017216771 A2) in view of Sehn et al (Published: 2015. Clinical Genomics, 2015, pp. 129–150) and in further view of Paquet et al. (Published: 2016. Cite No. 24 on IDS filed 5/02/2025. Nature 533, 125–129 (2016)) With regard to claim 1, the combined teachings of Ahlfors and Sehn render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the combined teachings fail to disclose that the existing genome editing method is blocking mutations. Regarding claim 11, Paquet teaches that blocking mutations increased HDR accuracy (pp. 125 col 2 para 2, “Without blocking mutations, only 6 to 35% of reads that incorporated pathogenic mutations had accurate HDR, but presence of a CRISPR/Cas-blocking PAM mutation increased HDR accuracy in both iPS cells and HEK293 cells two- to tenfold, depending on locus and cell type, which may increase the probability of accurately editing both alleles in a cell up to 100-fold (assuming independent allele editing).”) It would have been obvious for a person with ordinary skill in the art to modify the invention based on the combined teaching of Sehn and Ahlfors to further comprise blocking mutations taught by Paquet for the sake of improving HDR accuracy. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results *** 07-21-aia AIA Claim s (s) 1 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ahflors et al. (Published 2017. WIPO Publication: WO 2017216771 A2) in view of Sehn et al (Published: 2015. Clinical Genomics, 2015, pp. 129–150) and in further view of Grunwald et al. (Published: 2019. Cited in IDS filed 05/02/2025. Nature;566(7742):105-109.) . With regard to claim 1, the combined teachings of Ahlfors and Sehn render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the combined teachings fail to explicitly disclose that the system comprises mouse germline cells. Grunwald teaches assessing genetic systems in mice (pp. 105, col 2, “To assess the feasibility of active genetic systems in mice, we designed a ‘CopyCat’ element that differs from the genetic element used initially in insects in that it cannot self-propagate, because it encodes a gRNA but not the Cas9 protein (Fig. 1a).”). Moreover, Grunwald taught different genetic tool strategies comprising a mouse embyo (pp. 105, col 2, “For this analysis, we assessed eight different genetic strategies that use existing tools to provide spatial and temporal control of Cas9 expression in the early embryo and in the male and female germlines.”). It would have been obvious for a person with ordinary skill in the art to apply the genome editing method of Ahlfors in a mouse embryo taught by Grunwald to determine the feasibility of gene editing tools in mice. Moreover, a person with ordinary skill in the art would have been motivated to apply the gene editing method of Ahlfors to the mouse embryo of Grunwald to assess gene editing efficiency in a biologically relevant system such as a mouse embryo. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results. *** 07-21-aia AIA Claim (s) 1 and 24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ahflors et al. (Published 2017. WIPO Publication: WO 2017216771 A2) in view of Sehn et al (Published: 2015. Clinical Genomics, 2015, pp. 129–150) and in further view of Champer et al. (Published: 2017. Cited in IDS filed 05/02/2025. PLOS Genetics, vol. 13, no. 7, 20 July 2017, p. e1006796) . With regard to claim 1, the combined teachings of Ahlfors and Sehn render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the combined teachings fail to teach the explicit use of additional gRNAs are encoded into the gene drive to target resistant alleles. Champer teaches that additional gRNAs are encoded to target commonly generated resistant alleles (pp. 2, “The design of such CRISPR gene drives has now also been demonstrated in mosquitoes, including approaches aimed for population suppression of A. gambiae [27] and for spreading a malaria resistance gene in Anopheles stephensi [28]. These approaches use a Cas9 endonuclease to cleave chromosomes at a specific location dictated by a guide RNA(gRNA), which can be engineered to target any unique nucleotide sequence.”; pp. 12, “Several additional strategies have been suggested to lower resistance potential. For example, a drive construct could contain multiple gRNAs targeting different sites, in which case resistance may need to evolve; independently at all sites in order to halt the drive”). Moreover, Champer teaches that targeting resistance alleles aids in gaining more control of Cas9 constructs (pp. 12, “As we have highlighted in this study, one prerequisite for lowering resistance potential is better control of Cas9 expression.”) It would have been obvious for a skilled artisan to modify the method of Ahlfors to comprise additional gRNAs targeting resistant alleles with the motivation of gaining better control of Cas9 constructs. Such modifications in Ahlfors’ method would allow for improved CRISPR/Cas9 gene drive efficiency and reduce overall resistance rates. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results. Conclusion No claims allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to Katriel B Kasayan whose telephone number is (571)272-1402. The examiner can normally be reached 10-4p. 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, Maria G Leavitt can be reached at (571) 272-1085. 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. /KATRIEL BARCELLANO KASAYAN/Examiner, Art Unit 1634 /MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634 Application/Control Number: 18/689,676 Page 2 Art Unit: 1634 Application/Control Number: 18/689,676 Page 3 Art Unit: 1634 Application/Control Number: 18/689,676 Page 4 Art Unit: 1634 Application/Control Number: 18/689,676 Page 5 Art Unit: 1634 Application/Control Number: 18/689,676 Page 6 Art Unit: 1634 Application/Control Number: 18/689,676 Page 7 Art Unit: 1634 Application/Control Number: 18/689,676 Page 8 Art Unit: 1634 Application/Control Number: 18/689,676 Page 9 Art Unit: 1634 Application/Control Number: 18/689,676 Page 10 Art Unit: 1634 Application/Control Number: 18/689,676 Page 11 Art Unit: 1634 Application/Control Number: 18/689,676 Page 12 Art Unit: 1634 Application/Control Number: 18/689,676 Page 13 Art Unit: 1634 Application/Control Number: 18/689,676 Page 14 Art Unit: 1634 Application/Control Number: 18/689,676 Page 15 Art Unit: 1634 Application/Control Number: 18/689,676 Page 16 Art Unit: 1634 Application/Control Number: 18/689,676 Page 17 Art Unit: 1634 Application/Control Number: 18/689,676 Page 18 Art Unit: 1634 Application/Control Number: 18/689,676 Page 19 Art Unit: 1634 Application/Control Number: 18/689,676 Page 20 Art Unit: 1634 Application/Control Number: 18/689,676 Page 21 Art Unit: 1634 Application/Control Number: 18/689,676 Page 22 Art Unit: 1634 Application/Control Number: 18/689,676 Page 23 Art Unit: 1634 Application/Control Number: 18/689,676 Page 24 Art Unit: 1634 Application/Control Number: 18/689,676 Page 25 Art Unit: 1634
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