DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 7-7-25.
Claims 1, 2, 11, 17-20, 29, 32, 35, 36, 51, 52, 61, 76, 77, 88, 96, 106, 107, 116, 127, 135, 137, , 139, 152, 157, 163, and 176 are pending in the instant application.
Election/Restrictions
Claims 29, 35, 61, , 76, 77, 88, 96, 106, 107, 116, 127, 135, 137 and 163 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention or species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7-7-25.
Applicant’s election without traverse of Group I, the species of the gRNA modification pattern comprising:
The crRNA portion modification pattern of crRNA 40: mN#mN#mN#dN#dN#dN#mNmNmNmNfNfNfNfNfNfNfNfNfNmNmGfUfUfUfufAmGm AmGmCmUmAmU#mG#mC#mU (SEQ ID NO: 6); and
The tracrRNA portion modification pattern of tracrRNA 92,
mA#mG#mC#mAmUmAmGmCmAmAmGrUrUmArAmAmArUmAmAmGmGrCrUmArG #1U#rC#mCrGrUrUmAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC#mU#mU#mU (SEQ ID NO: 98), wherein rN = RNA, mN = 2’-O-methyl RNA, fN = 2’-fluoro RNA, N#N = phosphorothioate linkage, and N = any nucleotide, claims 1, 2, 11, 17-20, 32, 36, 51, 52, 139, 152, 157, and 176, in the reply filed on 7-7-25 is acknowledged.
Claim Objections
Claims 2 and 18 are objected to because of the following informalities:
Appropriate correction is required.
In claim 2, line 4 from the bottom, “nucleotide” should perhaps be plural, not singular.
In claim 18, line 2, “selected any” should perhaps be changed to – selected from any --.
Claim Rejections - 35 USC § 112
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 2 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.
In claim 2, second line from the bottom, “at least about 50%” is vague and indefinite (e.g. perhaps deleting “about” would be remedial).
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.
Claims 1, 2, 11, 19, 20, 32, 51, 52, 139, 152, 157, and 176 are 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 breadth of the claims
The claims are broadly drawn to CRISPR genome editing systems comprising an RNA guided nuclease and chemically modified guide RNAs comprising (a) a crRNA portion comprising (1) a guide sequence capable of hybridizing to a target polynucleotide sequence, and (ii) a repeat sequence of any length; and (b) a tracrRNA portion comprising an anti-repeat nucleotide sequence that is complementary to the repeat sequence, which crRNA portion comprises at least 50% modified nucleotides; and which crRNA portion comprises between one and ten 2’-deoxy modified ribose groups, which modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof, optionally wherein each modification of the ribose group is independently selected from 2'-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(S)-constrained ethyl (S-cEt), a constrained MOE, or a 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNAN), or wherein at least 80%, at least 90%, or 100% of the ribose groups are chemically modified; each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification; each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N methyladenosine, pseudouridine, 2,6- diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups; or which guide RNA comprises at least 90% or 100% modified nucleotides; or which crRNA portion comprises between 1 and 20 phosphorothioate modifications; and/or the chemically modified guide RNA comprises at least about 50% activity relative to an un-modified guide RNA, or wherein at least one nucleotide of the crRNA portion comprises each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification, optionally wherein one or more of the nucleotides at positions 4, 5, 6, 12, 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion comprise a 2’-deoxy chemical modification; the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification; or wherein the nucleotide at position 12 from the 5’ end of the crRNA portion comprises each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification; or wherein the nucleotides at positions 15, 16, and 19 from the 5’ end of the crRNA portion comprise each of a 2’deoxy chemical modification and a phosphorothioate chemical modification; or the nucleotides at positions 22, 23, and 24 from the 5’ end of the crRNA portion comprise each of a 2’deoxy chemical modification and a phosphorothioate chemical modification or wherein the tracrRNA portion modification pattern is optionally selected from any of tracrRNAs 2-116 of Table 2. The claims are also drawn to chemically modified guide RNA comprising (a) a crRNA portion comprising (1) a guide sequence capable of hybridizing to a target polynucleotide sequence, and (ii) a repeat sequence; and (b) a tracrRNA portion comprising an anti-repeat nucleotide sequence that is complementary to the repeat sequence, wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise a 2’-fluoro chemical modification or a phosphorothioate chemical modification, or which chemically modified guide RNA comprises one or more additional chemical modifications selected from a modification of a ribose group, phosphate group, nucleobase, or a combination thereof, optionally wherein each modification of the ribose group is independently selected from the group consisting of 2'-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(S)-constrained ethyl (S-cEt), a constrained MOE, and a 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNAN*),or at least 80%, at least 90%, or 100% of the ribose groups are chemically modified, wherein each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification, each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N methyladenosine, pseudouridine, 2,6- diaminopurine, inosine, thymidine, 5-methylcytosine, 5substituted pyrimidine, isoguanine, isocytosine, and halogenated
aromatic groups, and/or the guide RNA comprises at least 90% or 100% modified nucleotides, or wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise a 2’-fluoro chemical modification, or which chemically modified guide RNA further comprises a 2’fluoro chemical modification at one or more of positions 15, 16, 19, 22, 23, or 24 from the 5’ end of the crRNA portion; the chemically modified guide RNA further comprises a 2’fluoro chemical modification at positions 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion, or wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise phosphorothioate chemical modifications, optionally wherein the chemically modified guide RNA further comprises a 2’fluoro chemical modification at one or more of positions 15, 16, 19, 22, 23, or 24 from the 5’ end of the crRNA portion, or the chemically modified guide RNA further comprises a 2’fluoro chemical modification at positions 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion, which RNA guided nuclease optionally comprises S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9), which Cas9 is a variant Cas9 with altered activity, optionally wherein the variant Cas9 is selected from the group consisting of a Cas9 nickase (nCas9), a catalytically dead Cas9 (dCas9), a hyper accurate Cas9 (HypaCas9), a high fidelity Cas9 (Cas9-HF), an enhanced specificity Cas9 (eCas9), and an expanded PAM Cas9 (xCas9), and wherein the Cas9 off-target activity is reduced relative to an unmodified guide RNA; and/or the Cas9 on-target activity is increased relative to an unmodified guide RNA
Teachings in the specification
The specification teaches the following on page 3:
…existing guide RNAs suffer from several limitations, which limit their utility in therapeutic applications. For example, existing guide RNAs may be subject to rapid degradation in circulation and within cells. Moreover, chemical modifications of guide RNAs may reduce stability and editing efficiency. Accordingly, there exists a need in the art for optimized guide RNAs that retain efficient genome editing activity in vivo and ex vivo when paired with a CRISPR nuclease, such as Cas9.
The specification teaches that a pMCSG7 vector was used to express Cas9 from Streptococcus pyogenes, and contained three nuclear localization signals. This enzyme was used for screening the constructs for target cleavage.
And on pages 134-138, the specification teaches the following:
[0246] Screening of New Chemical Modification Patterns [0247] Structure-guided and systematic approaches were used to introduce 2'-OMe-RNA, 2'-F-RNA, 2’-deoxy, and PS modifications throughout guide RNAs. These modifications were chosen because they have been shown to improve stability, efficacy, and immunotoxicity associated with RNA. The strategy described herein yielded active RNP complexes with both extensively and fully modified versions of crRNAs and tracrRNAs. Figure 4 and Figure 5 depict a screen of crRNA patterns C23-C44, targeting both the MCV lasite and the MCV 1b site. The crRNAs C29, C39 and C40 demonstrate efficacy similar to that of the previously developed crRNA, C20. The crRNAs C20, C29, and C39 are fully modified in the sense that every nucleotide that does not have a ribose modification has a phosphodiester linkage modification. However, C20 still contains six unmodified ribose residues, while the new crRNA C39 only has three unmodified riboses, and C29 has only one unmodified ribose C40 is the newly developed, fully modified crRNA with no unmodified riboses in its composition. C45 is also a fully modified molecule with no unmodified ribose moieties. Like C40, this composition is expected to be very stable in vivo, though its activity is diminished somewhat in comparison to crRNA C20.
[0248] New tracrRNA chemical modification patterns were also developed. Figure 6 depicts a screen of previously described tracrRNA patterns T2, T9, T12, T17, and T18, compared to new patterns T38, T39, and T41. The different tracrRNAs were paired with C21, C39, C40, or C45. The new crRNAs C39, C40, and C45 displayed higher editing efficiencies when paired with all tracrRNAs compared to the older C21 pattern. 20 [0249] Several new tracrRNAs are more heavily modified than the previous tracrRNA T2. TracrRNA T41, T12 and T17 show higher activity than T2. TracrRNAsT9, T18, T37, T38 and T92 display similar efficiencies as T2, while T49 and T95 display slightly diminished activity than T2 (Figure 7 and Figure 8).
[0250] The loss in efficacy seen in human cells with the fully modified crRNA C45 and heavily modified tracrRNAs T49 and T95 compared to the previously developed crRNA C20 or tracrRNA T2 may be offset by higher in vivo stability. All of the newly developed RNAs are functional in multiple combinations when tested in human cells
Example 3 — Chemically modified crRNA:tracrRNA pairs with and without conjugates targeting endogenous human genes [0251] To verify that the crRNA and tracrRNA designs of the disclosure are compatible with different guide sequences, including those targeting endogenous human genes, the designs C29, C30, C40, C42, and C45 were tested by targeting the PCSK9 gene (Figure 9). The crRNAs were paired with tracrRNA T2 or T6, and T2 was further used in a non-conjugate or GalNAc-conjugate form. C29, C39, C40, and C42 were also tested in a non-conjugate or GalNAc-conjugate form. The RNA designs were tested by electroporation of Cas9 RNP in the mouse Hepa 1-6 cell line. The graphs show indel percentages based on Inference of CRISPR Edits (ICE) analysis of PCR and Sanger sequencing data of the locus. The data represent the means from three independent biological replicates and error bars represent s.e.m. [0252] These results demonstrate that the modified crRNA and tracrRNA designs are also applicable to endogenous target sites and function with conjugates on both the crRNA and tracrRNA.
Example 4 — Chemically modified crRNAs with varied phosphorothioate content [0253] Additional chemically modified crRNAs were designed, synthesized, and tested for genome editing efficiency. crRNAs C52-C93 were tested in the TLR assay with the MCV 1a target site. Each crRNA was paired with the T41 tracrRNA. 2 pmol of an RNP containing Cas9 with the various crRNAs and the tracrRNA were transfected into the TLR-MCV1 line described above and the % mCherry expression was detected as a proxy for genome editing efficiency. The crRNAs C52-C93 contained the same chemical modification pattern as C40, except with respect to phosphorothioate placement. The crRNA sequences are shown in Table 6. The screen revealed that crRNAs containing at least up to 20 phosphorothioate modifications are tolerated (Figure 10).
Example 5 — Chemically modified crRNAs containing 2’-amino RNA and/or 4’- thio RNA modifications [0254] Additional chemically modified crRNAs containing either 2’-amino RNA or 4’-thio RNA (2.e., sugar ring oxygen in ribose sugar is replaced with sulfur modifications were designed, synthesized, and tested for gene editing efficiency. crRNAs C114-C134 were tested in the TLR assay with the MCV 1a target site or MCV 1b target site, or in the mTmG reporter system, each of which is described above.
As shown in Figure 11A, crRNAs C116-C118 and C122-C134 was paired with the T2 tracrRNA. 5 pmol of an RNP containing Cas9 with the various crRNAs and the tracrRNA were transfected into the TLR-MCV1 line described above and the % mCherry expression was detected. As shown in Figure 11B, crRNAs C116-C118 and C122-C134 were used in a modified TLR-MCV1 assay in which an unmodified tracrRNA and SpCas9 were stably expressed as well. 100 pmol of each crRNA was transfected into the cell line and the % mCherry expression was detected.
Finally, as shown in Figure 11C, crRNAs C114-C127 were tested in the mT'mG reporter assay described above with 5 pmol of an RNP containing Cas9 with the various crRNAs and the T2 tracrRNA. The crRNA sequences are shown in Table 6. Each crRNA tested either had one or more 2’-amino ribose modifications or one or more 4’-thio RNA modifications. The screen revealed that crRNAs containing one or more 2’-amino ribose modifications or one or more 4’-thio RNA modifications maintain effective gene editing activity, while possessing additional chemical modifications that can improve stability.
Example 6 — Chemically modified tracrRNAs containing 4’-thio RNA modifications [0255] Additional chemically modified tracrRNAs containing 4’-thio RNA modifications were designed, synthesized, and tested for gene editing efficiency. tracrRNAs T107-T116 were tested in the TLR assay or in the mTmG reporter system, each of which is described above. Each of T107-T116 had the same chemical modification pattern as T2, except a 4’-thio RNA modification was introduced at one or more of the unmodified residues. 5 pmol of an RNP containing Cas9 with the various tracrRNAs and the C20 crRNA were transfected into the TLR-MCV1 line or mTmG line and the fluorescence was detected. The tracrRNA sequences are shown in Table 2. As shown in Figure 12, all of the tracrRNAs tested retained effective gene editing activity. The inclusion of 4°-thio RNA modifications at previously unmodified positions provides tracRNAs that are closer to being 100% chemically modified. T107 for example, has a modification at all but 5 nucleotides.
Example 7 — In vivo gene editing [0256] The various chemically modified guide RNAs have displayed substantial gene editing activity in vitro while possessing enhanced stability (e.g.,serum stability). The in vivo activity of select chemically modified guide RNAs was next determined in the mTmG transgenic mouse. RNPs made up of select crRNAs and tracrRNAs, along with Cas9, were intrastriatally (IS) injected into the mouse at a dose of 150-200 pmol. Six days following injection, mouse brain tissue was stained to detect 15 GFP expression. The guide RNA crRNA/ tracrRNA pairs were used: C20 / T2, C29 / T2, C20 / T41, and C29 / T41.
As shown in Figure 13, GFP was expressed in brain tissue from mice receiving a C20 / T2 containing RNP. As shown in Figure 14, GFP was expressed in brain tissue from mice receiving a C20 / T41 containing RNP. The data shows that the chemically modified guide RNAs are capable of gene editing activity in vivo.
[Emphases added}
The teachings in the specification are not representative of the expansive genus of molecules claimed. The specification fails to provide the requisite guidance for making and using the large genus of modulatory agents instantly claimed, and further whereby off target activity is reduced and/or on target activity is increased relative to an unmodified guide RNA.
Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus of modulators, and because the claimed genus is highly variant, the description provided is insufficient, one of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the myriad of modulatory agents instantly claimed.
Thus, Applicant was not in possession of the broadly claimed genus.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 2, 11, 17-20, 32, 36, 51, 52, 139, 152, 157, and 176 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Sontheimer et al (WO 2019/183000).
The applied reference has a common inventors with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Sontheimer et al (WO 2019/183000) teach CRISPR genome editing systems comprising chemically modified guide RNA and an RNA guided nuclease, which guide RNA comprises the modification patterns comprising:
The crRNA portion modification pattern of crRNA 40: mN#mN#mN#dN#dN#dN#mNmNmNmNfNfNfNfNfNfNfNfNfNmNmGfUfUfUfufAmGm AmGmCmUmAmU#mG#mC#mU (SEQ ID NO: 6); and
The tracrRNA portion modification pattern of tracrRNA 92,
mA#mG#mC#mAmUmAmGmCmAmAmGrUrUmArAmAmArUmAmAmGmGrCrUmArG #1U#rC#mCrGrUrUmAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC#mU#mU#mU (SEQ ID NO: 98), wherein rN = RNA, mN = 2’-O-methyl RNA, fN = 2’-fluoro RNA, N#N = phosphorothioate linkage, and N = any nucleotide.
Sontheimer teaches modified guide RNA optionally further comprising targeting ligands, linkers and branched lipids (see entire document, esp. the attached sequence alignments and page 32).
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.
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(s) 1, 2, 11, 19, 20, 32, 51, 152, 157, and 176 is/are rejected under 35 U.S.C. 103 as being unpatentable over Stoner et al (WO 2019/222545).
The claims are drawn to CRISPR genome editing systems comprising an RNA guided nuclease and chemically modified guide RNAs comprising (a) a crRNA portion comprising (1) a guide sequence capable of hybridizing to a target polynucleotide sequence, and (ii) a repeat sequence of any length; and (b) a tracrRNA portion comprising an anti-repeat nucleotide sequence that is complementary to the repeat sequence, which crRNA portion comprises at least 50% modified nucleotides; and which crRNA portion comprises between one and ten 2’-deoxy modified ribose groups, which modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof, optionally wherein each modification of the ribose group is independently selected from 2'-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(S)-constrained ethyl (S-cEt), a constrained MOE, or a 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNAN), or wherein at least 80%, at least 90%, or 100% of the ribose groups are chemically modified; each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification; each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N methyladenosine, pseudouridine, 2,6- diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups; or which guide RNA comprises at least 90% or 100% modified nucleotides; or which crRNA portion comprises between 1 and 20 phosphorothioate modifications; and/or the chemically modified guide RNA comprises at least about 50% activity relative to an un-modified guide RNA, or wherein at least one nucleotide of the crRNA portion comprises each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification, optionally wherein one or more of the nucleotides at positions 4, 5, 6, 12, 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion comprise a 2’-deoxy chemical modification; the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification; or wherein the nucleotide at position 12 from the 5’ end of the crRNA portion comprises each of a 2’-deoxy chemical modification and a phosphorothioate chemical modification; or wherein the nucleotides at positions 15, 16, and 19 from the 5’ end of the crRNA portion comprise each of a 2’deoxy chemical modification and a phosphorothioate chemical modification; or the nucleotides at positions 22, 23, and 24 from the 5’ end of the crRNA portion comprise each of a 2’deoxy chemical modification and a phosphorothioate chemical modification or wherein the tracrRNA portion modification pattern is optionally selected from any of tracrRNAs 2-116 of Table 2. The claims are also drawn to chemically modified guide RNA comprising (a) a crRNA portion comprising (1) a guide sequence capable of hybridizing to a target polynucleotide sequence, and (ii) a repeat sequence; and (b) a tracrRNA portion comprising an anti-repeat nucleotide sequence that is complementary to the repeat sequence, wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise a 2’-fluoro chemical modification or a phosphorothioate chemical modification, or which chemically modified guide RNA comprises one or more additional chemical modifications selected from a modification of a ribose group, phosphate group, nucleobase, or a combination thereof, optionally wherein each modification of the ribose group is independently selected from the group consisting of 2'-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(S)-constrained ethyl (S-cEt), a constrained MOE, and a 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNAN*),or at least 80%, at least 90%, or 100% of the ribose groups are chemically modified, wherein each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification, each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N methyladenosine, pseudouridine, 2,6- diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated
aromatic groups, and/or the guide RNA comprises at least 90% or 100% modified nucleotides, or wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise a 2’-fluoro chemical modification, or which chemically modified guide RNA further comprises a 2’fluoro chemical modification at one or more of positions 15, 16, 19, 22, 23, or 24 from the 5’ end of the crRNA portion; the chemically modified guide RNA further comprises a 2’fluoro chemical modification at positions 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion, or wherein the nucleotides at positions 4, 5, and 6 from the 5’ end of the crRNA portion comprise phosphorothioate chemical modifications, optionally wherein the chemically modified guide RNA further comprises a 2’fluoro chemical modification at one or more of positions 15, 16, 19, 22, 23, or 24 from the 5’ end of the crRNA portion, or the chemically modified guide RNA further comprises a 2’fluoro chemical modification at positions 15, 16, 19, 22, 23, and 24 from the 5’ end of the crRNA portion, which RNA guided nuclease optionally comprises S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).
Stoner et al (WO 2019/222545) teach CRISPR genome editing systems comprising chemically modified guide RNA and an RNA guided nuclease optionally comprising Cas9, which modified guide strands comprise the nucleotide, internucleotide linkage and sugar modifications instantly claimed. Stoner also teaches modified guide strands optionally further comprising linkers and conjugated moieties (see entire document, esp. para 0002-00013, 0019, 0028, 0073-0074, 0088-0090, 0104-0108, 0185, claims 1-24, 94-97).
It would have been obvious to design and optimize modifications on the guide strands of CRISPR genome editing systems because all of the modifications were well known in the art. The insertion and optimal placing of modifications, and testing target cleavage upon insertion of modified moieties into the guide strands involved routine experimentation at the time of the instant invention.
For these reasons, the instant invention would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free).
Jane Zara
10-10-25
/JANE J ZARA/Primary Examiner, Art Unit 1637