DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status
Applicant’s amendments filed July 8, 2026, amending claims 1, 24, 25, 83 and 84, canceling claims 26, 27, 29, 31, 40, 49-52, 61 and 72, and adding new claims 89-99 is acknowledged. Claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-99 are pending.
Applicant’s election without traverse of Group I encompassing guide RNAs having a chemically modified crRNAs, and species SEQ ID NOs 5 for the modified crRNA sequence in the response filed July 8, 2026 is acknowledged. Applicant canceled claims there were directed to non-elected invention. The election of a species for an exNA-containing tracrRNA is moot in view of Applicant’s election and cancelation of nonelected groups.
Claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-99, and species of SEQ ID NOs 5 are under examination.
The application claims priority to provisional application 63/355,704, filed June 27, 2022, which is the effective filing date of the claimed invention.
Drawings
The drawings are objected to because they were submitted in color, but there is no granted petition to accept color drawings. See 37 CFR 1.84(a)(2) (“The Office will accept color drawings in utility patent applications only after granting a petition filed under this paragraph explaining why the color drawings are necessary”). Applicants must either provide that explanation via a petition and comply with all requirements of 37 CFR 1.84(a)(2)(i)-(iii) OR submit replacement sheets in black and white and include a clear instruction to replace the color drawings with the replacement sheets. The examiner takes no position on whether color drawings are necessary as the only practical medium by which to disclose the subject matter sought to be patented in this utility patent application.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Interpretation
The claims recite an “extended nucleic acid (exNA) intersubunit linkage.” The Specification does not explicitly define the term. However, from the Specification exNA intersubunit linkage is interpreted to mean a linkage in the sugar-phosphate backbone of a nucleic acid comprises at least one extra methyl group either 1) between the phosphate group and the 5’ carbon (e.g., as in Formula IIa) or 2) between a 3’ carbon of one nucleotide and the phosphate group of the subsequent nucleotide (e.g., as in Formula IIIa). ExNAs are not interpreted as the additional methyl group(s) are directly bonded to the phosphorus atom of the phosphate group.
Claim Rejections - 35 USC § 112(b)
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.
Claim 25 is 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.
Claim 25 recites “any one of tracrRNA 1 to tracrRNA 116 of Table 2… and tracrRNA 103 (6F-M12/M18/3’T-ExU) of Table 4.” Reference to tables in claims renders the claim indefinite when there is no practical way to define the invention in words. See MPEP 2173.05(s). Here, the tables contain exclusively text, and the appropriate text could simply be imported into the claim. Each of the tracrRNAs are text based. Additionally, each of the tracrRNA sequences and modifications are identified with SEQ ID NOs in the tables, which could also simply be recited in the claims. To overcome this rejection, applicants must amend claim 25 such that it clearly sets forth a closed list of alternatives for tracrRNA sequences either by name, sequence and modification pattern, or SEQ ID NO.
Claim Rejections - 35 USC § 103 – Mir in view of Khvorova
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.
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.
Claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99 are rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Khvorova (WO 2021242883 A1, published December 2, 2021). This rejection also addresses the elected species: crRNA having SEQ ID NO 5.
The Khvorova reference was published within 1 year of the effective filing date of the claimed invention and also names at least one non-inventor of the current application. Therefore, it constitutes prior art under §102(a)(1).
Regarding claim 1, Mir teaches heavily and fully modified guide RNAs for SpCas9 (title). Mir teaches guide RNAs have (a) a crRNA portion comprising a 3’ end, 5’ end, a guide/spacer sequence the hybridizes to the target polynucleotide sequence and a repeat nucleotide sequence (Fig. 1). Mir teaches guide RNAs have (b) a tracrRNA portion comprising a 3’ end, a 5’ end and an anti-repeat sequence that is complementary to the crRNA repeat sequence (Fig 1a). Mir teaches Cas9 guide RNAs have been extensively chemically modified including with 2’-O’methyl and 2’-fluoro sugar modifications, and phosphorothioate linkages (page 2, ¶4). Mir teaches similar modifications had been previously made in siRNA and ASO drugs to prevent innate immune responses and have been used in clinical trials (page 3, ¶1). Mir teaches applying the similar siRNAs and ASOs modification patterns to chemical modifications in crRNAs and tracrRNAs (page 3, ¶1). Regarding claim 4, Mir teaches the sequence of the crRNA is NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCU (Fig 1). Regarding claims 15, 24, 89-91 Mir teaches crRNA C20 having a crRNA sugar modification pattern of guide: mmmmmmmmmmffffrrffrm and repeat: mrrrffmmmmmmmmmm, wherein m = 2’-O-methyl, r = RNA, and f = 2’-fluoro, which is the sugar modification pattern and the phosphorothioate pattern of all the crRNAs in claim 24 and wherein 90% of the ribose groups are chemically modified (Table 1). Regarding claim 25, Mir teaches a tracrRNA sequence T1 having the same sequence and modification pattern as tracrRNA 1 (SEQ ID NO 171) in table 2 of the Specification (Table 2). Regarding claims 83-84, Mir teaches combining the C20 crRNA and tracrRNA of T1 with S. pyogenes Cas9 in a ribonucleoprotein (i.e., a CRISPR genome editing system) (Title, Fig 3). Regarding claims 93, Mir teaches the modified guide RNAs also comprised a phosphorothioate (PS) linkage (i.e., comprising at least one modification of a phosphate group) (Table 1).
Mir does not teach the guide RNA comprises an intersubunit linkage with an exNA.
Khvorova teaches exNAs having an extra methyl group between the phosphate and the 5’ carbon (5’-exNA) or an extra alkyl group between the phosphate and the 3’ carbon (3’-exNA) (i.e., having Formula Ia) (FIG. 1). Khvorova teaches incorporating exNAs into oligonucleotides increases their stability (FIGs 17-19). Khvorova tested exNA linkages at different positions in siRNAs also having 2’-methyl and 2’-fluoro substituted ribose nucleotides (Tables 4 and 5). Khvorova teaches incorporating a single exNA in modified siRNAs increases their silencing efficiency over modified siRNAs not having an exNA linkage (FIG. 20B). Khvorova teaches that additional backbone modifications that provide higher metabolic stabilization are needed in the field of RNA therapeutics ([005]). Khvorova teaches the synthesis of exNA-containing, heavily modified RNA oligonucleotides and that exNAs are compatible with currently validated chemical modifications (Examples 1-10; [006]).
Regarding claims 1-2, 4, 6, 15, 25, 83-84, 89-91, 93 and 95, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have incorporated one exNA linkage into the highly modified C20 crRNA of Mir. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that an exNA could be incorporated into Mir’s C20 crRNA because Khvorova teaches exNAs can be added to heavily or fully modified short RNAs such as siRNAs and provides the protocols for producing them. The skilled artisan would have been motivated to have done so because 1) Mir teaches that nucleotide modifications first developed for siRNA technology can be applied to improving CRISPR RNAs and 2) Khvorova teaches that incorporating exNAs into RNA oligonucleotides increases their stability and enhances their effectiveness.
Regarding claim 24, it would have been obvious to include the exNA linkage specifically at the 3’ end of Mir’s C20 crRNA to arrive at crRNA Ex20-1 (36C-ExU) because Khvorova teaches including at single exNA linkage at the 3’ end of the RNA oligonucleotides increases its stability and effectiveness in vivo.
Claim 92 depends from claim 15 which recites “wherein the crRNA portion and/or the tracrRNA portion further comprises at least one modified nucleotide selected from a modification of the ribose group…” Claim 92 then recites “wherein 100% of the ribose groups are chemically modified”. Claim 92 is broadly but reasonably interpreted as either the crRNA or the tracrRNA or both portions have fully modified ribose groups. Mir teaches tracrRNA T8 having 100% modified ribose groups (Table 2).
Regarding claim 96, the combination of Mir’s tracrRNA T8 and the exNA-modified C20 crRNA rendered obvious above would have comprise 100% modified nucleotides since the RNA nucleotides of crRNA C20 have phosphorothioate linkages (Tables 1-2).
Regarding claim 99, Mir teaches that modified ASOs and siRNA have been shown to have reduced off-target effects (page 3, ¶1). Mir teaches that some of the modified crRNA/tracrRNA pairs had improved on-target and reduced off-target activity than unmodified guides (Supp Fig 8).
The skilled artisan would have predicted that incorporating a single exNA nucleotide into a modified crRNA/tracRNA would also provide increased on target activity and/or decreased off-target activity compared to an unmodified guide because Khvorova teaches exNA linkages in the context of heavily modified RNA oligonucleotides have increased stability and would therefore likely bind the intended target at a higher rate.
Claim 94 is rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Khvorova (WO 2021242883 A1, published December 2, 2021) as applied to claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99 above, and further in view of Hoy (Hoy et al., Bio-orthogonal chemistry-based conjugation strategy facilitates investigation of impacts of s2U, s4U, m1A and m6A guide RNA modifications on CRISPR activity. bioRx, doi: https://doi.org/10.1101/2022.06.09.495561, posted June 9, 2022).
The teachings of Mir and Khvorova are recited above and applied as for claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99. Khvorova also teaches the exNA-modified oligonucleotides can comprise modified nucleobases such as N6-methyladenosine, 2-thiouridine and 4-thiouridine (FIG. 11, [0420]). Khvorova teaches that using N6-methyladenosine blocks the activity of adenosine deaminase ([0420]).
Mir and Khvorova do not teach guide RNAs having modified nucleobases.
Hoy teaches guide RNAs having at least one N6-methyladenosine, 2-thiouridine or 4-thiouridine modified nucleobase (Figure 4). Hoy teaches the guide RNA having N6-methyladenosine modifications (sgRNA 8) increased the activity of Cas9 cleavage of the target guide RNA over unmodified guide RNAs at 1 hour and 16 hours (Figure 5).
It would have been obvious to one skilled in the before the effective filing date of the claimed invention to have additionally modified the exNA-crRNA 20 rendered obvious above by incorporating an N6-methyladenosine. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that the N6-methyladenosine modification taught in both Khvorova and Hoy could be incorporated into Mir’s guide RNA because both references either teach or suggest its incorporation into highly modified RNA oligonucleotides. Additionally, Hoy provides the protocols for the synthesis of N6-methyladenosine-containing guide RNAs. The skilled artisan would have been motivated to have done so because 1) Khvorova suggests using N6-methyladenosine in high modified RNA oligonucleotides for preventing deamination by adenosine deaminase and 2) Hoy demonstrates increased Cas9-mediated cleavage upon N6-methyladenosine incorporation.
Claims 97-98 are rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Khvorova (WO 2021242883 A1, published December 2, 2021) as applied to claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99 above, and further in view of Doudna (US 20140068797 A1).
The teachings of Mir and Khvorova are recited above and applied as for claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99. Mir also teaches that Cas9-gRNA-DNA complex formation activates the His–Asn–His (HNH) and RuvC-like endonuclease domains that cleave the target strand and the non-target strand of the DNA, respectively, resulting in a double-strand break (DSB) (page 2, ¶2).
Mir and Khvorova does not teach modified guide RNAs with a variant Cas9 having altered activity.
Doudna teaches variant Cas9 polypeptides having reduced or no catalytic activity ([0449]). Doudna teaches dCas9 proteins can be fused to various protein domains to functionalize dCas9 for labeling, target gene transcriptional control or chromatin modification ([0456]). Doudna teaches that the D10A and H840A mutations render Cas9 catalytically inactive (FIG. 11A), but do not affect guide RNA and target DNA binding (FIG. 11B0. Doudna teaches guide RNAs having a variety of backbone modifications ([0194]) and modified sugars ([0205]).
It would have been obvious to one skilled in the before the effective filing date of the claimed invention to have combined the exNA-crRNA 20 rendered obvious with a catalytically inactive dCas9. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that dCas9 could be used with the obvious exNA-crRNA because Doudna teaches the only changes in the protein are to the catalytic residues, which does not block guide RNA or DNA binding. The skilled artisan would have been motivated to have done so to expand the use of the Cas9/exNA-C20 crRNA system for labeling and gene regulation as taught in Doudna.
Claim Rejections - 35 USC § 103 – Mir in view of Haly
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.
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.
Claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93 and 95-96 are rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Haly (Haly et al., Nucleosides and Nucleotides (1996), 15: 1383-1395). This rejection also addresses the elected species: crRNA having SEQ ID NO 5.
The teachings of Mir regarding the guide RNAs comprising heavy and fully modified crRNA and tracrRNAs that read on limitations recited in claims 1, 4, 15, 24-25, 83-84, 89-91 and 93 are recited above in paragraph 16 and incorporated here.
Mir does not teach the guide RNA comprises an intersubunit linkage with an exNA.
Haly teaches antisense therapeutics for inhibition of gene expression (page 1383, ¶1). Haly teaches antisense oligonucleotides (ASOs) need to be chemically modified to be nuclease resistant (page 1383, ¶1). Haly teaches oligonucleotides having a “stretched” backbone (page 1384, ¶1). Haly teaches erythro (alpha) linked oligonucleotides (i.e., 3’-exNA intersubunit linkages; i.e., having formula Ia) (Figure 2, structure 13). Haly teaches the synthesis of exNA-containing oligonucleotides (Scheme I).
Regarding claims 1-2, 4, 6, 15, 25, 83-84, 89-91, 93 and 95, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have incorporated one exNA linkage into the highly modified C20 crRNA of Mir. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that an exNA could be incorporated into Mir’s C20 crRNA because Haly teaches exNA-containing oligonucleotides can be synthesized and provides the protocols for synthesizing them. The skilled artisan would have been motivated to have done so because 1) Mir teaches that nucleotide modifications first developed for antisense technology can be applied to improving CRISPR RNAs and 2) Haly teaches that incorporating exNAs provides nuclease resistance in RNA oligonucleotides such that it increases their stability.
Regarding claim 24, it would have been obvious to include the exNA linkage specifically at the 3’ end of Mir’s C20 crRNA to arrive at crRNA Ex20-1 (36C-ExU) because Haly teaches the exNA provides resistance to exonuclease III, which cleaves from the ends of the oligonucleotides.
Claim 92 depends from claim 15 which recites “wherein the crRNA portion and/or the tracrRNA portion further comprises at least one modified nucleotide selected from a modification of the ribose group…” Claim 92 then recites “wherein 100% of the ribose groups are chemically modified”. Claim 92 is broadly but reasonably interpreted as either the crRNA or the tracrRNA or both portions have fully modified ribose groups. As indicated above, Mir teaches tracrRNA T8 having 100% modified ribose groups (Table 2).
Regarding claim 96, the combination of Mir’s tracrRNA T8 and the exNA-modified C20 crRNA rendered obvious above would have comprise 100% modified nucleotides since the RNA nucleotides of crRNA C20 have phosphorothioate linkages (Tables 1-2).
Claim 94 is rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Haly (Haly et al., Nucleosides and Nucleotides (1996), 15: 1383-1395) as applied to claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, and 95-96 above, and further in view of Hoy (Hoy et al., Bio-orthogonal chemistry-based conjugation strategy facilitates investigation of impacts of s2U, s4U, m1A and m6A guide RNA modifications on CRISPR activity. bioRx, doi: https://doi.org/10.1101/2022.06.09.495561, posted June 9, 2022).
The teachings of Mir and Haly are recited above and applied as for claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93 and 95-96.
Mir and Haly do not teach guide RNAs having modified nucleobases.
Hoy teaches guide RNAs having at least one N6-methyladenosine, 2-thiouridine or 4-thiouridine modified nucleobase (Figure 4). Hoy teaches the guide RNA having N6-methyladenosine modifications (sgRNA 8) increased the activity of Cas9 cleavage of the target guide RNA over unmodified guide RNAs at 1 hour and 16 hours (Figure 5).
It would have been obvious to one skilled in the before the effective filing date of the claimed invention to have additionally modified the exNA-crRNA 20 rendered obvious above by incorporating an N6-methyladenosine. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that the N6-methyladenosine modification taught in Hoy could be incorporated into Mir’s guide RNA because Hoy teaches its incorporation into highly modified RNA oligonucleotides and provides protocols for the synthesis of N6-methyladenosine-containing guide RNAs. The skilled artisan would have been motivated to have done so because Hoy demonstrates increased Cas9-mediated cleavage upon N6-methyladenosine incorporation.
Claims 97-98 are rejected under 35 U.S.C. 103 as being unpatentable over Mir (Mir et al., Nature Communications (2018), 9: 2641, pages 1-9) in view of Khvorova (WO 2021242883 A1, published December 2, 2021) as applied to claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93, 95-96 and 99 above, and further in view of Doudna (US 20140068797 A1).
The teachings of Mir and Haly are recited above and applied as for claims 1-2, 4, 6, 15, 24-25, 83-84 and 89-93 and 95-96. Mir also teaches that Cas9-gRNA-DNA complex formation activates the His–Asn–His (HNH) and RuvC-like endonuclease domains that cleave the target strand and the non-target strand of the DNA, respectively, resulting in a double-strand break (DSB) (page 2, ¶2).
Mir and Haly do not teach modified guide RNAs with a variant Cas9 having altered activity.
Doudna teaches variant Cas9 polypeptides having reduced or no catalytic activity ([0449]). Doudna teaches dCas9 proteins can be fused to various protein domains to functionalize dCas9 for labeling, target gene transcriptional control or chromatin modification ([0456]). Doudna teaches that the D10A and H840A mutations render Cas9 catalytically inactive (FIG. 11A), but do not affect guide RNA and target DNA binding (FIG. 11B0. Doudna teaches guide RNAs having a variety of backbone modifications ([0194]) and modified sugars ([0205]).
It would have been obvious to one skilled in the before the effective filing date of the claimed invention to have combined the exNA-crRNA 20 rendered obvious with a catalytically inactive dCas9. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that dCas9 could be used with the obvious exNA-crRNA because Doudna teaches the only changes in the protein are to the catalytic residues, which does not block guide RNA or DNA binding. The skilled artisan would have been motivated to have done so to expand the use of the Cas9/exNA-C20 crRNA system for labeling and gene regulation as taught in Doudna.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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