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
Applicant’s amendments and remarks filed on January 26, 2026 are acknowledged. Claims 1-16, 20-21, 25, 27-33, 35, 37, 39, and 41 have been canceled. Claims 17-19, 24, 36, and 38 were amended. Claims 17-19, 22-24, 26, 34, 36, 38, and 40 are pending and are examined on the merits herein.
Withdrawn Objections
In view of Applicant’s amendments and response, the objection to claim 38 is withdrawn.
Withdrawn Rejections
In view of Applicant’s amendments and response, the 35 U.S.C 101 rejection is withdrawn.
Specification
The disclosure is objected to because of the following informality:
Paragraph [0019] refers to “red circles” and “blue circles” in FIG. 7B; however, color drawings were not submitted with the application.
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.
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.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Nie et al. (CN105602935A; reference previously cited by the Examiner) in view of Ryan et al. (US 2016/0289675; reference cited by Applicant) and Lorenz et al. (Microscopy and Microanalysis 2011; reference previously cited by the Examiner). A machine translation of Nie et al. was previously supplied, and references to the various passages of Nie et al. in the rejection refer to the machine translation.
Regarding claim 17, Nie et al. teaches the transformation of the CRISPR/Cas9 system and efficient and precise editing of mitochondrial targeted genes comprising of a guide RNA entering mitochondria and Cas9 nuclease localized in mitochondria [page 2]. Further, Nie et al. teaches removing the Cas9 N-terminal nuclear localization signal and introducing MTS-Flag or MTS-NES-Flag structure [page 5]. Nie et al. teaches gRNA that enters the mitochondria includes two types: 1. RNA composed of RNA mitochondrial positioning guide sequence, targeting target sequence, and gRNA skeleton sequence; 2. RNA composed of RNA mitochondrial localization guide sequence, any mitochondrial-encoded tRNA sequence or other additional spacer sequences, targeting target sequence, and gRNA backbone sequence wherein the RNA mitochondrial localization guide sequence is an RP sequence [page 6]. Figure 3A shows that the mitochondrial-targeted Cas9 nuclease can be localized to the mitochondria by mitochondrial leader peptide (MTS) fusion to the N-terminus of the Cas9 nuclease.
However, Nie et al. does not teach a cyanine moiety wherein the moiety is attached at the 5’ of the gRNA. Nie et al. also does not teach that the gRNA comprises one or more 2’-modified nucleotides.
Ryan et al. teaches that an “end” modification such as cyanine fluorescent dye is incorporated into the guide RNA [0048]. Further, guide RNAs containing modifications at specific positions were tolerated by active Cas protein and gRNA:Cas protein complexes wherein the modifications included 2’-O-methylribonucleotide and 5-aminoallyluracil coupled to Cy5 fluorophore [0282]. Ryan et al. also teaches that chemically modified nucleotides were incorporated into guide RNAs to improve certain properties such as improved organelle localization [0284]. Ryan et al. teaches in certain embodiments, a nucleotide sugar modification incorporated into the guide RNA is selected from the group consisting of 2′-O—C1-4alkyl such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H), 2′-O—C1-3alkyl-O—C1-3alkyl such as 2′-methoxyethyl (“2′-MOE”), 2′-fluoro (“2′-F”), 2′-amino (“2′-NH2”), 2′-arabinosyl (“2′-arabino”) nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-locked nucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), and 4′-thioribosyl nucleotide [0045].
Lorenz et al. teaches that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria. Lorenz et al. demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide [abstract].
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the gRNA of Nie et al. by incorporating a cyanine fluorescent dye because Nie et al. and Ryan et al. both teach guide RNAs and their use in CRISPR/CRISPR-associated systems and Ryan et al. teaches that it is within the skill of the art to include an end modification of a cyanine fluorescent dye to the guide RNA. One would have made such a modification in order to achieve the predictable result of improving certain properties of the guide RNA. One would have been motivated to make such a modification to localize the gRNA as taught by Ryan et al. because Ryan et al. taught that incorporating chemically modified nucleotides into guide RNAs can improve properties such as organelle localization and Lorenz et al. taught that Cy5 can be loaded into cells by attaching a short oligonucleotide.
Claims 18, 24, 26, 34, 36, 38, and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Nie et al. (CN105602935A; reference previously cited by the Examiner) in view of Ryan et al. (US 2016/0289675; reference cited by Applicant) and Lorenz et al. (Microscopy and Microanalysis 2011; reference previously cited by the Examiner). A machine translation of Nie et al. was previously supplied, and references to the various passages of Nie et al. in the rejection refer to the machine translation.
Regarding claims 18, 24, 26, 34, 36, 38, and 40, Nie et al. teaches the transformation of the CRISPR/Cas9 system and efficient and precise editing of mitochondrial targeted genes comprising of a guide RNA entering mitochondria and Cas9 nuclease localized in mitochondria [page 2]. Further, Nie et al. teaches removing the Cas9 N-terminal nuclear localization signal and introducing MTS-Flag or MTS-NES-Flag structure [page 5]. Nie et al. teaches gRNA that enters the mitochondria includes two types: 1. RNA composed of RNA mitochondrial positioning guide sequence, targeting target sequence, and gRNA skeleton sequence; 2. RNA composed of RNA mitochondrial localization guide sequence, any mitochondrial-encoded tRNA sequence or other additional spacer sequences, targeting target sequence, and gRNA backbone sequence wherein the RNA mitochondrial localization guide sequence is an RP sequence [page 6]. Figure 3A shows that the mitochondrial-targeted Cas9 nuclease can be localized to the mitochondria by mitochondrial leader peptide (MTS) fusion to the N-terminus of the Cas9 nuclease.
However, Nie et al. does not teach a cyanine moiety wherein the moiety is attached at the 5’ of the gRNA. Nie et al. also does not teach that the gRNA comprises one or more 2’-modified nucleotides. In addition, Nie et al. does not teach direct delivery of a guide RNA into mitochondria of a cell.
Ryan et al. teaches methods for genomic editing to modify a target polynucleotide in a mammalian cell [0240] comprising introducing or delivering the guide RNA into the cell [0245]. In certain embodiments, the guide RNA is introduced or delivered into cells [0223]. Ryan et al. teaches that an “end” modification such as cyanine fluorescent dye is incorporated into the guide RNA [0048]. Further, guide RNAs containing modifications at specific positions were tolerated by active Cas protein and gRNA:Cas protein complexes wherein the modifications included 2’-O-methylribonucleotide and 5-aminoallyluracil coupled to Cy5 fluorophore [0282]. Ryan et al. also teaches that chemically modified nucleotides were incorporated into guide RNAs to improve certain properties such as improved organelle localization [0284]. Ryan et al. teaches in certain embodiments, a nucleotide sugar modification incorporated into the guide RNA is selected from the group consisting of 2′-O—C1-4alkyl such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H), 2′-O—C1-3alkyl-O—C1-3alkyl such as 2′-methoxyethyl (“2′-MOE”), 2′-fluoro (“2′-F”), 2′-amino (“2′-NH2”), 2′-arabinosyl (“2′-arabino”) nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-locked nucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), and 4′-thioribosyl nucleotide [0045]. Ryan et al. also teaches that the total length of the two RNA pieces or the single guide RNA can be about 50-220 nucleotides in length [0169].
Lorenz et al. teaches that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria. Lorenz et al. demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide [abstract].
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the gRNA of Nie et al. by incorporating a cyanine fluorescent dye because Nie et al. and Ryan et al. both teach guide RNAs and their use in CRISPR/CRISPR-associated systems and Ryan et al. teaches that it is within the skill of the art to include an end modification of a cyanine fluorescent dye to the guide RNA. One would have made such a modification in order to achieve the predictable result of improving certain properties of the guide RNA. One would have been motivated to make such a modification to localize the gRNA as taught by Ryan et al. because Ryan et al. taught that incorporating chemically modified nucleotides into guide RNAs can improve properties such as organelle localization and Lorenz et al. taught that Cy5 can be loaded into cells by attaching a short oligonucleotide.
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to deliver the cyanine modified gRNA of Nie et al. and Ryan et al. into mitochondria of a cell because Nie et al. and Ryan et al. both teach guide RNAs and their use in CRISPR/CRISPR-associated systems and genomic editing. One would have been motivated to deliver the cyanine modified gRNA into the mitochondria of a cell for the purpose of genomic editing in the mitochondria.
Claims 19, 22, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Nie et al. (CN105602935A; reference previously cited by the Examiner) in view of Ryan et al. (US 2016/0289675; reference cited by Applicant) and Lorenz et al. (Microscopy and Microanalysis 2011; reference previously cited by the Examiner). A machine translation of Nie et al. was previously supplied, and references to the various passages of Nie et al. in the rejection refer to the machine translation.
Regarding claims 19, 22, and 23, Nie et al. teaches the transformation of the CRISPR/Cas9 system and efficient and precise editing of mitochondrial targeted genes comprising of a guide RNA entering mitochondria and Cas9 nuclease localized in mitochondria [page 2]. Further, Nie et al. teaches removing the Cas9 N-terminal nuclear localization signal and introducing MTS-Flag or MTS-NES-Flag structure [page 5]. Nie et al. teaches gRNA that enters the mitochondria includes two types: 1. RNA composed of RNA mitochondrial positioning guide sequence, targeting target sequence, and gRNA skeleton sequence; 2. RNA composed of RNA mitochondrial localization guide sequence, any mitochondrial-encoded tRNA sequence or other additional spacer sequences, targeting target sequence, and gRNA backbone sequence wherein the RNA mitochondrial localization guide sequence is an RP sequence [page 6]. Figure 3A shows that the mitochondrial-targeted Cas9 nuclease can be localized to the mitochondria by mitochondrial leader peptide (MTS) fusion to the N-terminus of the Cas9 nuclease.
However, Nie et al. does not teach a cyanine moiety wherein the moiety is attached at the 5’ of the gRNA. Nie et al. also does not teach that the gRNA comprises one or more 2’-modified nucleotides.
Ryan et al. teaches that an “end” modification such as cyanine fluorescent dye is incorporated into the guide RNA [0048]. Further, guide RNAs containing modifications at specific positions were tolerated by active Cas protein and gRNA:Cas protein complexes wherein the modifications included 2’-O-methylribonucleotide and 5-aminoallyluracil coupled to Cy5 fluorophore [0282]. Ryan et al. also teaches that chemically modified nucleotides were incorporated into guide RNAs to improve certain properties such as improved organelle localization [0284]. Ryan et al. teaches in certain embodiments, a nucleotide sugar modification incorporated into the guide RNA is selected from the group consisting of 2′-O—C1-4alkyl such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H), 2′-O—C1-3alkyl-O—C1-3alkyl such as 2′-methoxyethyl (“2′-MOE”), 2′-fluoro (“2′-F”), 2′-amino (“2′-NH2”), 2′-arabinosyl (“2′-arabino”) nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-locked nucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), and 4′-thioribosyl nucleotide [0045].
Lorenz et al. teaches that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria. Lorenz et al. demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide [abstract].
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the gRNA of Nie et al. by incorporating a cyanine fluorescent dye because Nie et al. and Ryan et al. both teach guide RNAs and their use in CRISPR/CRISPR-associated systems and Ryan et al. teaches that it is within the skill of the art to include an end modification of a cyanine fluorescent dye to the guide RNA. One would have made such a modification in order to achieve the predictable result of improving certain properties of the guide RNA. One would have been motivated to make such a modification to localize the gRNA as taught by Ryan et al. because Ryan et al. taught that incorporating chemically modified nucleotides into guide RNAs can improve properties such as organelle localization and Lorenz et al. taught that Cy5 can be loaded into cells by attaching a short oligonucleotide.
Response to Arguments
Applicant's arguments filed January 26, 2026 have been fully considered but they are not persuasive.
Applicant asserts the following:
PNG
media_image1.png
338
796
media_image1.png
Greyscale
PNG
media_image2.png
94
786
media_image2.png
Greyscale
Applicant asserts that paragraphs [0046] and [0048] of Ryan et al. disclose a laundry list of nucleobase modifications that may be used for guide RNA modifications and exemplary end modifications envisioned to be incorporated into the guide RNA for nuclear delivery. Applicant further asserts that there is no guidance in Ryan et al. as to which modifications or combination of modifications could result in successful delivery of a guide RNA into mitochondria. In addition, Ryan et al. does not teach or suggest positioning of specific modifications within the guide RNA to achieve mitochondrial localization. Applicant points to paragraph [0273] asserting that the modifications incorporated into the guide RNA are intended to improve nuclear localization with no mention of the modifications envisioned to improve mitochondrial localization. Applicant further asserts that it would be impossible to determine which of the modifications from paragraphs [0046] or [0048] of Ryan et al. to select and where to attach the modification on a guide RNA to achieve mitochondrial localization. Thus, Applicant asserts that Nie et al., Ryan et al., and Lorentz et al. do not provide a person having ordinary skill in the art a reason to include Ryan et al.’s modifications into Nie et al.’s guide RNA to deliver them into mitochondria.
These arguments are not found persuasive. Nie et al. teaches the transformation of the CRISPR/Cas9 system and efficient and precise editing of mitochondrial targeted genes comprising of a guide RNA entering mitochondria and Cas9 nuclease localized in mitochondria [page 2]; however, Nie et al. does not teach a cyanine moiety wherein the moiety is attached at the 5’ of the gRNA and does not teach that the gRNA comprises one or more 2’-modified nucleotides. Therefore, the Ryan et al. reference was used in combination with the Nie et al. reference because Ryan et al. teaches incorporating an “end” modification such as a cyanine fluorescent dye into a guide RNA as well as incorporating chemically modified nucleotides into guide RNAs to improve properties such as organelle localization. There are a finite number of positions on a guide RNA and thus one of ordinary skill in the art would have been motivated to test the effects of a cyanine fluorescent dye and different chemical modifications at different positions to localize the gRNA of Nie et al. because Ryan et al. taught that incorporating chemically modified nucleotides into guide RNAs can improve properties such as organelle localization and Lorenz et al. taught that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria and demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide.
Applicant asserts that given the difference in chemical composition of RNA and DNA, it would be highly unpredictable what the effect of replacing the DNA oligo of Lorenz et al. with a guide RNA would be. Applicant also asserts that Applicant is the first to show that guide RNAs including 2’ modifications and a cyanine moiety attached to the 5’ end of the guide RNA are capable of stably translocating into mitochondria to edit mitochondrial genomes. Applicant refers to Figures 3A and 3B asserting that the figures show persistence of Cy3 crRNA in mitochondria. Applicant asserts surprisingly discovering that 3’ labeling with a cyanine moiety results in vesicular localization and does not co-localize with mitochondria (e.g., Figure 7B). Further, Applicant asserts unexpectedly discovering that the specific combination of modifications and attachment point of the cyanine moiety in the guide RNA results in the surprising effective mitochondrial delivery and genome editing.
These arguments are not found persuasive. Nie et al. taught the transformation of the CRISPR/Cas9 system and efficient and precise editing of mitochondrial targeted genes comprising of a guide RNA entering mitochondria and Cas9 nuclease localized in mitochondria. Figure 3A of Nie et al. shows that the mitochondrial-targeted Cas9 nuclease can be localized to the mitochondria by mitochondrial leader peptide (MTS) fusion to the N-terminus of the Cas9 nuclease. Ryan et al. taught incorporating “end” modifications such as cyanine fluorescent dye into the guide RNA to improve certain properties such as improved organelle localization. Further, guide RNAs containing modifications at specific positions were tolerated by active Cas protein and gRNA:Cas protein complexes wherein the modifications included 2’-O-methylribonucleotide and 5-aminoallyluracil coupled to Cy5 fluorophore [0282]. Lastly, Lorenz et al. taught that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria and demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide. Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the gRNA of Nie et al. by incorporating a cyanine fluorescent dye because Nie et al. and Ryan et al. both taught guide RNAs and their use in CRISPR/CRISPR-associated systems and Ryan et al. taught that it is within the skill of the art to include an end modification of a cyanine fluorescent dye to the guide RNA. One would have made such a modification in order to achieve the predictable result of improving certain properties of the guide RNA. One would have been motivated to make such a modification to localize the gRNA as taught by Ryan et al. because Ryan et al. taught that incorporating chemically modified nucleotides into guide RNAs can improve properties such as organelle localization and Lorenz et al. taught that labeling of organelles for microscopy is achieved generally by specific dyes that accumulate in a cellular compartment such as cyanine dyes in mitochondria and demonstrated that Cy5 can be loaded into cells by attaching a short oligonucleotide.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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 CHRISTINA TRAN whose telephone number is (571)270-0550. The examiner can normally be reached M-F 7:30 - 5:00pm.
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, Jennifer Dunston can be reached on (571) 272-2916. 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.
/C.T./
Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637