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 .
Claim Status
Applicant’s submission filed 05/18/2026 has been received and entered. Claims 66 and 85 have been cancelled. Claims 62, 70, 73-74, 79-81, 87 and 89-90 have been amended. Claims 67 and 89-90 were previously withdrawn as being directed to non-elected inventions. Accordingly, claims 62-65, 68-76, 79-81, 83-84, 86-87 and 91-98 are pending and under current examination.
Status of Prior Rejections/Response to Arguments
The objection to Specification is withdrawn:
Applicant’s amendment to the specification is effective to obviate the objection on record. The objection is withdrawn.
The objection to claim 70 is withdrawn:
Applicant’s amendment to claim 70 is effective to obviate the objection on record. The objection is withdrawn.
The rejection to claims 79-81 and 87 is withdrawn:
Applicant’s amendment to claims 79-81 and 87 replacing “RNA” with “polynucleotide” obviates the rejection on record. The rejection is withdrawn.
The rejection to claims 62-65, 68-76, 79-81, 83-85, 87, 91-94 and 96-98 under 35 USC §102(a)(1) over Yin et al., as evidenced by Nowak et al. and NCBI Reference Sequence: WP_306789577.1 is maintained:
The cancellation of claims 85 renders the rejection thereto moot.
Regarding claims 73-74, Yin et al. do not teach a modified tracr sequence with the special sets of unmodified nucleotides as recited in claims 73-74. Therefore the rejection to claims 73-73 is withdrawn. However, Yin et al.’s teaching still renders obviousness to the limitations set forth in the amended claims 73-74. Specifically, Yin et al. teach nucleotides at the tetraloop and the second stem-loop can be chemically modified by 2’OMe (see note in Supplementary figure 5), Yin et al. also teach the principle of this type of chemical modification: the tetraloop and the second stem-loop of sgRNAs extend outside of the Cas9 protein. It is hypothesized that these two loops could tolerate modifications in all nucleotides, as long as those modifications did not inhibit loop formation. Indeed, the sgRNA carrying the 2′ OMe (2′OMe, Loops) modification at these two loops was functional (see p1180, right column). This teaching indicates that the nucleotides at the tetraloop and the second stem-loop including position 53 tolerate modifications (that is, the nucleotides at these positions can be modified but not have to be modified) without decrease the functionality. As taught or suggested by Yin et al.’s teaching, PHOSITA would have substituted Yin et al.’s modification of all nucleotides in the tetraloop and the second stem-loop of sgRNAs, and leave some nucleotides (i.e., the nucleotide at position of 53 corresponding SEQ ID NO:61) unmodified according their research interest or preference. The new rejection is set forth in the 103 rejection part of current rejection.
Regarding claims 62-65, 68-72, 75-76, 79-81, 83-84, 87, 91-94 and 96-98, Applicant has amended claim 62 and traversed the rejection, asserting that that although Yin discloses a tracr sequence having a nucleotide base sequence that is 100% identical to SEQ ID NO: 61, none of Yin's tracr sequences comprises the unique set of chemically modified and unmodified nucleotides in the tracr sequence as specified in amended claim 62 (Remarks, p9).
Applicant’s argument is fully considered but not found persuasive. Specifically, the amended claim 62 limits “wherein the tracr sequence comprises chemically modified nucleotides at any one of positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47, 50-52, 54-60, 63-67, 69, or 72-76” in lines 9-11, and “wherein the tracr sequence comprises unmodified nucleotides at any one of positions 13, 35, 36, 40, 53, 61, 68, 70, or 71” in lines 13-14, does not limit the specific set of chemically modified and unmodified nucleotides in the tracr sequence, but only limits ANY combination of ANY of the modified and unmodified nucleotides listed in the claim. In instant case, Yin et al. teach various modification at different nucleotide positions in tracer sequence and spacer sequence, as well as the combination of tracer sequence and spacer sequence with different modifications. For instance, Yin et al. teach the 2’OMe modification of sgRNA (2’OMe, Loops) has modification at positions 10-12, 14-15, 17-19 and 50-59 of the corresponding position of SEQ ID NO:61, and has unmodified positions at, i.e., the corresponding positions 13, 35, 36 and 40 of SEQ ID NO:61, which reads on the limitation of amended claim 62. The rejection is maintained in modified form to address amended limitation.
The rejection to claims 62-65, 68-76, 79-81, 83-87, 91-94 and 96-98 under 35 USC §103 over Yin et al., as evidenced by Nowak et al. is maintained:
The rejection to claims 62-65, 68-76, 79-81, 83-85, 87 and 91-98 under 35 USC §103 over Yin et al., as evidenced by Nowak et al., in view of Ran et al. is maintained:
The cancellation of claims 85 renders the rejection thereto moot.
Applicant has amended claim 62 and traversed the rejection, asserting that independent claim 62 has been amended to recite a tracr sequence comprising a unique set of chemically modified and unmodified nucleotides, which is different from that of Yin's tracr sequences. While Yin discloses a set of chemical modifications "that can be modified while maintaining or enhancing genome-editing activity", it does not disclose or otherwise suggest the chemical modifications that are specified in the claims as amended (Remarks, p10).
Applicant’s argument is fully considered but not found persuasive. Specifically, as stated above, the amended claim 62 limits “wherein the tracr sequence comprises chemically modified nucleotides at any one of positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47, 50-52, 54-60, 63-67, 69, or 72-76” in lines 9-11, and “wherein the tracr sequence comprises unmodified nucleotides at any one of positions 13, 35, 36, 40, 53, 61, 68, 70, or 71” in lines 13-14, does not limit the specific set of chemically modified and unmodified nucleotides in the tracr sequence, but only limit ANY combination of ANY of the modified and unmodified nucleotides listed in the claim. In instant case, Yin et al. teach various modification at different nucleotide positions in tracer sequence and spacer sequence, as well as the combination of tracer sequence and spacer sequence with different modifications. For instance, Yin et al. teach the 2’OMe modification of sgRNA (2’OMe, Loops) has modification at positions 10-12, 14-15, 17-19 and 50-59 of the corresponding position of SEQ ID NO: 61, and has unmodified positions at, i.e., the corresponding positions 13, 35, 36 and 40 of SEQ ID NO: 61, which reads on the limitation of amended claims. Moreover, Yin et al. teach the principles of the modification (see results part, structure-guided chemical modification, especially p1180, right column, and figures 1-2). PHOSITA would have been taught and suggested to modify the nucleotides following the principle. The rejection is maintained in modified form to address amended limitation.
Modified Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 62-65, 68-72, 75-76, 79-81, 83-84, 87, 91-94 and 96-98 stand rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yin et al. (Nat Biotechnol. 2017 Dec;35(12):1179-1187, cited in IDS), as evidenced by Nowak et al. (Nucleic Acids Res. 2016 Nov 16;44(20):9555-9564) and NCBI Reference Sequence: WP_306789577.1 (available 2005). The rejection is modified as necessitated by Applicant’s amendment.
Yin et al. teach that guided by the structure of the Cas9–sgRNA complex, they identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and develop an optimal set of chemical modifications for in vivo applications (Abstract).
Regarding claim 62, Yin et al. teach targeting of the Cas9 complex is guided by sgRNAs, which combines CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). These sgRNAs are about 100 nucleotides (nt) long, with 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Yin et al. also teach the Cas9 they used to be spCas9 (see p1180, left column). CrRNA comprises a spacer sequence is evidenced by Nowak et al.. Nowak et al. teach Streptococcus pyogenes CRISPR-SpCas9 guide RNA and synthetic sgRNA anatomy (p9556, figure 1), and the 20 nt at the 5′ end hybridizing the complementary DNA sequence which referred in Yin et al. is the spacer sequence (orange color) in figure 1B. In addition, Nowak et al. teach SpCas9 is CRISPR type II-A protein (see p9555, right column), and SpCas9 interacts with the sgRNA in both sequence dependent and independent manners––the guide region is recognized in a sequence-independent mechanism, whereas SpCas9 recognition of the sgRNA repeat: anti-repeat duplex
involves sequence-dependent interactions (p9556, right column). This teaching reads on a single guide polynucleotide, comprising: (a) a spacer sequence and (b) a tracr sequence, wherein the tracr sequence serves as a binding scaffold for a Type II Cas protein, as recited in instant claim.
Yin et al. also teach native strand sgRNA without modifications (101 nt, see Supplementary Table 5), the 21-100 nucleotide sequence of the native strand sgRNA is 100% identical to SEQ ID NO:61 of instant claim. This teaching reads on a tracr sequence having a nucleotide base sequence, wherein the nucleotide base sequence is 100% identity to SEQ ID NO: 61.
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Yin et al. further teach various modifications of sgRNA (see i.e., Supplementary Table 5). For instance, Yin et al. teach native crRNA and 2’OMe_1-20 nt (Supplementary Table 5), shows the 1-20 nt of crRNA is the guide sequence (spacer sequence). Yin et al. teach 2’OMe_1-10 nt (Supplementary Table 5) has 1-10 nt of spacer sequence modified and 11-20 nt of spacer sequence unmodified. This teaching reads on “(a) the spacer sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s)” as recited in instant claim. Yin et al. teach the 2’OMe modification of sgRNA (2’OMe, Loops) has modification at positions 10-12, 14-15, 17-19 and 50-59 of the corresponding position of SEQ ID NO:61, and has unmodified positions at, i.e., the corresponding positions 13, 35, 36 and 40 of SEQ ID NO:61. This teaching reads on “(b) the tracr sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s)”; “the tracr sequence comprises chemically modified nucleotides at any one of positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47,50-52, 54-60, 63-67, 69, or 72-76 at corresponding position of SEQ ID NO: 61” and ”the tracr sequence comprises unmodified nucleotides at any one of positions 13, 35, 36, 40, 53, 61, 68, 70, or 71, as numbered in SEQ ID NO: 61” as recited in instant claim. Therefore Yin et al. anticipate instant claim.
Regarding claims 63 and 64, following the discussion above, Yin et al. teach comprehensive, heavily modified sgRNA (named as SG-2′OMe), in which 60 out of 81 nucleotides were modified with 2′OMe RNA, fully retained its activity in cells with a moderate increase in editing efficiency (p1180, right column), this modification is in the 21-100 nt of the sgRNA (corresponding to the whole sequence of SEQ ID NO:61 of instant claim, see detailed modification in Supplementary Table 5). This teaching indicates more than 60/81 (74%) of the nucleotides in the tracr sequence are chemically modified.
Regarding claim 65, following the discussion above, Yin et al. teach sequence of SG-2’OMe (Supplementary Table 5), which has chemical modification (2’OMe modification) at positions 1, 8-12, 14-22, 26, 28, 30, 32-37, 40,41, 46, 47, 50-52, 54-61 and 63-80 of corresponding positions of SEQ ID NO: 61.
Regarding claim 68, following the discussion above, Yin et al. teach generating a number of sgRNAs modified with 2′-deoxy-2′-fluoro-ribonucleotide (2'F RNA), 2′ O-methyl ribonucleotide (2'OMe RNA) and the phosphorothioate bond (PS) at different positions (p1180, left column), as well as an modification example named SG-2’OMe (see Supplementary Table 5), reads on the one or more chemical modification(s) in the tracr sequence is a 2'-OMe-ribose sugar on the modified nucleotides, as recited in instant claim.
Regarding claim 69, following the discussion above, Yin et al. teach 5’&3’-sgRNA, which is chemical modification of both the 5′ and 3′ ends (2′OMe and phosphorothioate bond (PS) modifications of 3 nt at the 5′ and 3′ end, respectively). The Supplemental Table 5 discloses the detailed sequence of 5’&3’-sgRNA, shows that the 3'- terminal region of the tracr sequence comprises a repeating phosphorothioate linkages (phosphorothioate bond) in a backbone of the tracr sequence.
Regarding claim 70, following the discussion above, Yin et al. teach sequence of SG-2’OMe (Supplementary Table 5), which has chemical modification (2’-OMe-ribose sugar modification) at positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47, 50-52, 54-60, 63-67, 69 and 72-80 of corresponding positions of SEQ ID NO: 61.
Regarding claim 71-72, following the discussion above, Yin et al. teach the 2’OMe modification of sgRNA (SG-2’OMe) has modification at positions 21-22, 32-37, 41 and 61 of corresponding position of SEQ ID NO:61.
Regarding claims 75, following the discussion above, Yin et al. teach the 2’OMe modification of sgRNA (SG-2’OMe) comprises unmodified nucleotides at positions 6, 7, 25, 27, 29, 45, 48 and 49 (supplementary Table 5), reads on the unmodified nucleotides at (i).
Regarding claim 76, following the discussion above, Yin et al. teach generating a number of sgRNAs modified with 2'F RNA, 2'OMe RNA and the phosphorothioate bond (PS) at different positions (p1180, left column), reads on the chemical modification comprises a 2'-OMe modification and phosphorothioate linkage as recited in instant claim.
Regarding claims 79-81, Yin et al. teach e-sgRNA (see supplementary Table 5), which has chemical modification in tracr sequence (i.e., at position 21-22 of corresponding positions of SEQ ID NO: 61), also comprises three contiguous phosphorothioate linkages (phosphorothioate bond) at 5’ end.
Regarding claim 83, following the discussion above, Yin et al. teach use of a cell reporter system to test the editing efficiency of modified sgRNAs. HEK293 cells were engineered to stably express GFP and spCas9 (p1180, left column). spCas9 the most commonly used form of Cas9 (p11179, left column). Moreover, Nowak et al. provide evidence that SpCas9 is CRISPR type II-A protein (see p9555, right column). This teaching reads on the Cas9 protein as recited in instant claim.
Regarding claims 87, 94, 97, following the discussion above, Yin et al. teach using lipid nanoparticle formulations of the enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9 (Abstract), and Nowak et al. provide evidence that SpCas9 is CRISPR type II-A protein (p9555, right column), and the e-sgRNA comprises chemical modification at i.e. nucleotides at positions 21-22 of corresponding position of SEQ ID NO:61 (see supplementary Table 5), therefore the teaching anticipates the composition as recited in instant claims.
Regarding claims 84 and 96, Yin et al. teach using spCas9, do not teach the amino acid sequence of spCas9. However, it is evidenced by NCBI Reference Sequence: WP_306789577.1. WP_306789577.1 discloses type II CRISPR RNA-guided endonuclease Cas9 [Staphylococcus aureus], wherein SEQ ID NO: 40 in instant claim is 100% identical to the NCBI Reference Sequence: WP_306789577.1 (sequence alignment is provided).
Regarding claim 91, following the discussion above, Yin et al. teach 5’&3’-sgRNA, which has chemical modification of both the 5′ and 3′ ends (2′OMe and PS modifications of 3 nt at the 5′ and 3′ end, respectively). The Supplemental Table 5 discloses the detailed sequence of 5’&3’-sgRNA, shows that the single guide RNA comprises three contiguous phosphorothioate bond at the 3’ end (first two phosphorothioate bonds are at corresponding positions 79-80 of SEQ ID NO:61), and does not have phosphonothioate linkages at corresponding positions 1-76 of SEQ ID NO: 61.
Regarding claim 92, Yin et al. teach 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Combining with the figure 1 of Nowak et al. shows the spacer sequence, the teachings read on “the spacer sequence hybridizes with a target polynucleotide sequence when contacted with the target polynucleotide sequence” as recited in instant claim.
Regarding claim 93, Yin et al. teach sgRNAs, which combines CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). These sgRNAs are about 100 nucleotides (nt) long, with 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Figure 1a (p1181) shows the connection for 5’-3’ of sgRNA. The spacer sequence (1-20 nt) is covalently linked to the tracr sequence.
Regarding claim 98, instant claim is directed to a GC% content of “a portion of” the mRNA that encodes the protein is at least 60%. Without a definition in the specification about the length of “a portion” the sequence, it is interpreted under broadest reasonable interpretation as two or more contiguous RNA sequences, therefore any RNA fragment “GC”, or a RNA fragment comprises three RNAs , wherein two out of the three RNAs are “G” and “C” in Yin et al.’s Cas9 mRNA have a GC% content more than 60%. Therefore Yin et al. anticipates instant claim.
Modified 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.
Claims 62-65, 68-72, 75-76, 79-81, 83-84, 87, 91-94 and 96-98 stand rejected and claims 73-74 newly rejected under 35 U.S.C. 103 as being unpatentable over Yin et al. (Nat Biotechnol. 2017 Dec;35(12):1179-1187, cited in IDS), as evidenced by Nowak et al. (Nucleic Acids Res. 2016 Nov 16;44(20):9555-9564) and NCBI Reference Sequence: WP_306789577.1 (available 2005). The rejection is modified as necessitated by Applicant’s amendment.
Yin et al. teach that guided by the structure of the Cas9–sgRNA complex, they identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and develop an optimal set of chemical modifications for in vivo applications (Abstract).
Regarding claim 62, Yin et al. teach targeting of the Cas9 complex is guided by sgRNAs, which combines CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). These sgRNAs are about 100 nucleotides (nt) long, with 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Yin et al. also teach the Cas9 they used to be spCas9 (see p1180, left column). CrRNA comprises a spacer sequence is evidenced by Nowak et al.. Nowak et al. teach Streptococcus pyogenes CRISPR-SpCas9 guide RNA and synthetic sgRNA anatomy (p9556, figure 1), and the 20 nt at the 5′ end hybridizing the complementary DNA sequence which referred in Yin et al. is the spacer sequence (orange color) in figure 1B. In addition, Nowak et al. teach SpCas9 is CRISPR type II-A protein (see p9555, right column), and SpCas9 interacts with the sgRNA in both sequence dependent and independent manners––the guide region is recognized in a sequence-independent mechanism, whereas SpCas9 recognition of the sgRNA repeat: anti-repeat duplex
involves sequence-dependent interactions (p9556, right column). This teaching reads on a single guide polynucleotide, comprising: (a) a spacer sequence and (b) a tracr sequence, wherein the tracr sequence serves as a binding scaffold for a Type II Cas protein, as recited in instant claim.
Yin et al. also teach native strand sgRNA without modifications (101 nt, see Supplementary Table 5), the 21-100 nucleotide sequence of the native strand sgRNA is 100% identical to SEQ ID NO:61 of instant claim. This teaching reads on a tracr sequence having a nucleotide base sequence, wherein the nucleotide base sequence is 100% identity to SEQ ID NO: 61.
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Yin et al. further teach various modifications of sgRNA (see i.e., Supplementary Table 5). For instance, Yin et al. teach native crRNA and 2’OMe_1-20 nt (Supplementary Table 5), shows the 1-20 nt of crRNA is the guide sequence (spacer sequence). Yin et al. teach 2’OMe_1-10 nt (Supplementary Table 5) has 1-10 nt of spacer sequence modified and 11-20 nt of spacer sequence unmodified. This teaching reads on “(a) the spacer sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s)” as recited in instant claim. Yin et al. teach the 2’OMe modification of sgRNA (2’OMe, Loops) has modification at positions 10-12, 14-15, 17-19 and 50-59 of the corresponding position of SEQ ID NO:61, and has unmodified positions at, i.e., the corresponding positions 13, 35, 36 and 40 of SEQ ID NO:61. This teaching reads on “(b) the tracr sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s)”; “the tracr sequence comprises chemically modified nucleotides at any one of positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47,50-52, 54-60, 63-67, 69, or 72-76 at corresponding position of SEQ ID NO: 61” and ”the tracr sequence comprises unmodified nucleotides at any one of positions 13, 35, 36, 40, 53, 61, 68, 70, or 71, as numbered in SEQ ID NO: 61” as recited in instant claim. Therefore Yin et al. reads on instant claim.
Regarding claims 63 and 64, following the discussion above, Yin et al. teach comprehensive, heavily modified sgRNA (named as SG-2′OMe), in which 60 out of 81 nucleotides were modified with 2′OMe RNA, fully retained its activity in cells with a moderate increase in editing efficiency (p1180, right column), this modification is in the 21-100 nt of the sgRNA (corresponding to the whole sequence of SEQ ID NO:61 of instant claim, see detailed modification in Supplementary Table 5). This teaching indicates more than 60/81 (74%) of the nucleotides in the tracr sequence are chemically modified.
Regarding claim 65, following the discussion above, Yin et al. teach sequence of SG-2’OMe (Supplementary Table 5), which has chemical modification (2’OMe modification) at positions 1, 8-12, 14-22, 26, 28, 30, 32-37, 40,41, 46, 47, 50-52, 54-61 and 63-80 of corresponding positions of SEQ ID NO: 61.
Regarding claim 68, following the discussion above, Yin et al. teach generating a number of sgRNAs modified with 2′-deoxy-2′-fluoro-ribonucleotide (2'F RNA), 2′ O-methyl ribonucleotide (2'OMe RNA) and the phosphorothioate bond (PS) at different positions (p1180, left column), as well as an modification example named SG-2’OMe (see Supplementary Table 5), reads on the one or more chemical modification(s) in the tracr sequence is a 2'-OMe-ribose sugar on the modified nucleotides, as recited in instant claim.
Regarding claim 69, following the discussion above, Yin et al. teach 5’&3’-sgRNA, which is chemical modification of both the 5′ and 3′ ends (2′OMe and phosphorothioate bond (PS) modifications of 3 nt at the 5′ and 3′ end, respectively). The Supplemental Table 5 discloses the detailed sequence of 5’&3’-sgRNA, shows that the 3'- terminal region of the tracr sequence comprises a repeating phosphorothioate linkages (phosphorothioate bond) in a backbone of the tracr sequence.
Regarding claim 70, following the discussion above, Yin et al. teach sequence of SG-2’OMe (Supplementary Table 5), which has chemical modification (2’-OMe-ribose sugar modification) at positions 1, 8-12, 14-22, 26, 28, 30, 32-34, 37, 41, 46, 47, 50-52, 54-60, 63-67, 69 and 72-80 of corresponding positions of SEQ ID NO: 61.
Regarding claim 71-72, following the discussion above, Yin et al. teach the 2’OMe modification of sgRNA (SG-2’OMe) has modification at positions 21-22, 32-37, 41 and 61 of corresponding position of SEQ ID NO:61.
Regarding claims 75, following the discussion above, Yin et al. teach the 2’OMe modification of sgRNA (SG-2’OMe) comprises unmodified nucleotides at positions 6, 7, 25, 27, 29, 45, 48 and 49 (supplementary Table 5), reads on the unmodified nucleotides at (i).
Regarding claim 76, following the discussion above, Yin et al. teach generating a number of sgRNAs modified with 2'F RNA, 2'OMe RNA and the phosphorothioate bond (PS) at different positions (p1180, left column), reads on the chemical modification comprises a 2'-OMe modification and phosphorothioate linkage as recited in instant claim.
Regarding claims 79-81, Yin et al. teach e-sgRNA (see supplementary Table 5), which has chemical modification in tracr sequence (i.e., at position 21-22 of corresponding positions of SEQ ID NO: 61), also comprises three contiguous phosphorothioate linkages (phosphorothioate bond) at 5’ end.
Regarding claim 83, following the discussion above, Yin et al. teach use of a cell reporter system to test the editing efficiency of modified sgRNAs. HEK293 cells were engineered to stably express GFP and spCas9 (p1180, left column). spCas9 the most commonly used form of Cas9 (p11179, left column). Moreover, Nowak et al. provide evidence that SpCas9 is CRISPR type II-A protein (see p9555, right column). This teaching reads on the Cas9 protein as recited in instant claim.
Regarding claims 87, 94, 97, following the discussion above, Yin et al. teach using lipid nanoparticle formulations of the enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9 (Abstract), and Nowak et al. provide evidence that SpCas9 is CRISPR type II-A protein (p9555, right column), and the e-sgRNA comprises chemical modification at i.e. nucleotides at positions 21-22 of corresponding position of SEQ ID NO:61 (see supplementary Table 5), therefore the teaching anticipates the composition as recited in instant claims.
Regarding claims 84 and 96, Yin et al. teach using spCas9, do not teach the amino acid sequence of spCas9. However, it is evidenced by NCBI Reference Sequence: WP_306789577.1. WP_306789577.1 discloses type II CRISPR RNA-guided endonuclease Cas9 [Staphylococcus aureus], wherein SEQ ID NO: 40 in instant claim is 100% identical to the NCBI Reference Sequence: WP_306789577.1 (sequence alignment is provided).
Regarding claim 91, following the discussion above, Yin et al. teach 5’&3’-sgRNA, which has chemical modification of both the 5′ and 3′ ends (2′OMe and PS modifications of 3 nt at the 5′ and 3′ end, respectively). The Supplemental Table 5 discloses the detailed sequence of 5’&3’-sgRNA, shows that the single guide RNA comprises three contiguous phosphorothioate bond at the 3’ end (first two phosphorothioate bonds are at corresponding positions 79-80 of SEQ ID NO:61), and does not have phosphonothioate linkages at corresponding positions 1-76 of SEQ ID NO: 61.
Regarding claim 92, Yin et al. teach 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Combining with the figure 1 of Nowak et al. shows the spacer sequence, the teachings read on “the spacer sequence hybridizes with a target polynucleotide sequence when contacted with the target polynucleotide sequence” as recited in instant claim.
Regarding claim 93, Yin et al. teach sgRNAs, which combines CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). These sgRNAs are about 100 nucleotides (nt) long, with 20 nt at the 5′ end hybridizing the complementary DNA sequence and the remaining structure for Cas9 recognition and binding (p1180, left column). Figure 1a (p1181) shows the connection for 5’-3’ of sgRNA. The spacer sequence (1-20 nt) is covalently linked to the tracr sequence.
Regarding claim 98, instant claim is directed to a GC% content of “a portion of” the mRNA that encodes the protein is at least 60%. Without a definition in the specification about the length of “a portion” the sequence, it is interpreted under broadest reasonable interpretation as two or more contiguous RNA sequences, therefore any RNA fragment “GC”, or a RNA fragment comprises three RNAs , wherein two out of the three RNAs are “G” and “C” in Yin et al.’s Cas9 mRNA have a GC% content more than 60%. Therefore Yin et al. anticipates instant claim.
Regarding claims 73 and 74, following the discussion above, Yin et al. teach the 2’OMe modification of sgRNA (2’OMe, Loops) has unmodified positions at the corresponding positions 2-7, 13, 23-25, 27, 29, 31, 35, 36, 38-40, 42-45, 48, 49, 61, 62, 68, 70 and 71 of SEQ ID NO:61. Instant claims differ from Yin et al.’s teaching that instant claims have the tracr sequence also have unmodified position at position 53. However, Yin et al. teach the principle of the modification of the tetraloop and the second stem-loop of sgRNAs: the tetraloop and the second stem-loop of sgRNAs extend outside of the Cas9 protein. It is hypothesized that these two loops could tolerate modifications in all nucleotides, as long as those modifications did not inhibit loop formation. Indeed, the sgRNA carrying the 2' OMe (2
'OMe, Loops) modification at these two loops was functional, and, in fact, it showed a small but significant increase of editing efficiency (p118, right column). This teaching indicates that the nucleotides at the tetraloop and the second stem-loop including position 53 tolerate modifications (that is, the nucleotides at these positions can be modified but not have to be modified) without decrease the functionality. Following Yin et al.’s principle of nucleotide modification, the nucleotide at position 53 can be modified (i.e., the modification would not reduce the sgRNA’s editing ability) or stay unmodified.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Yin et al.’s 2’OMe modification of sgRNA and keep the nucleotide of position 53 unmodified. The only difference between instant claim and Yin et al.’s 2’OMe modification of sgRNA is instant claims have one nucleotide (at position 53 corresponding SEQ ID NO: 61) stay unmodified. Given that Yin et al. teach nucleotides at the tetraloop and the second
stem-loop can tolerate chemical modification by 2’OMe (see note in Supplementary figure 5), one of ordinary skill in the art would have substituted Yin et al.’s 2’OMe modification of sgRNA having all the nucleotides at the tetraloop and the second stem-loop modified, and keep some nucleotides (i.e., nucleotide at position 53) unmodified according to their research interest or preference. This simple substitution is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 86, Yin et al. do not teach the exact sequence of the sgRNAs because of different spacer sequence (guide RNA) target to different gene of interest. However, Yin et al. teach the same principle of the modification. For instance, regarding gRNA ID GA004 in Table 1 of instant claim, there are 5’-PS-2’OMe modification on the 5’ end (see 5’&3’ sgRNA and e-sgRNA in figure 3, p1183), and modification of tracr sequence (nt 21-101 of SEQ ID NO: 255 in gRNA ID GA004) with same pattern as SG-2’OMe (Supplemental Table 5) of Yin et al.(yellow part shows the same modification of tracr sequence).
GA004:
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68
507
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Greyscale
SG-2’OMe:
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128
927
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Greyscale
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Yin et al.’s sgRNA, use different spacer sequence (guide sequence) in order to target to different DNA fragment, and use the same modification in tracr sequence. The skilled artisan would have been motivated to use the same modification in tracr sequence since Yin et al. teach this modification can enhance CRISPR genome- tracr sequence since Yin et al. teach the sequence of tracr sequence and the modification of tracr sequence (i.e., supplementary Table 5).
Claims 62-65, 68-72, 75-76, 79-81, 83-84, 87 and 91-98 stand rejected under 35 U.S.C. 103 as being unpatentable over Yin et al. (Nat Biotechnol. 2017 Dec;35(12):1179-1187, cited in IDS), as evidenced by Nowak et al. (Nucleic Acids Res. 2016 Nov 16;44(20):9555-9564) and NCBI Reference Sequence: WP_306789577.1 (available 2005), as applied to 62-65, 68-76, 79-81, 83-84, 87, 91-94 and 96-98 above, in view of Ran et al. (Cell. 2013 Sep 12;154(6):1380-9).
The teaching of Yin et al. is set forth above.
Regarding claim 95, Yin et al. do not teach the Cas9 protein comprises a Cas9 nickase (nCas9). However, this was disclosed by Ran et al. at the time of instant invention.
Ran et al. describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double- strand breaks (Abstract).
Regarding claim 95, Ran et al. teach to improve the specificity of Cas9-mediated genome editing, they developed a strategy that combines the D10A mutant nickase version of Cas9 (Cas9n) with a pair of offset sgRNAs complementary to opposite strands of the target site (p1381, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Yan et al.’s spCas9, and use the D10A mutant nickase version of Cas9 as taught by Ran et al.. The skilled artisan would have been motivated to use the D10A mutant nickase version of Cas9 since Ran et al. teach the Cas9 nickase involved double nicking maintains high on-target efficiencies while reducing off-target modifications to background levels (p1387, left column). There would be a reasonable expectation of success of using the D10A mutant nickase version of Cas9 since Ran et al. teach the method of providing a Cas9 nickase: mutations of the catalytic residues (D10A in RuvC and H840A in HNH) convert Cas9 into DNA nickases (p1382, left column).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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