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 .
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Status of Claims
Claims 184-204 are pending and under examination.
Claim Objection
Claim 195 should recite "the composition of 184 further comprising a donor template nucleic acid, wherein the donor template comprises a nucleic acid comprising at least a portion of a PTBP 1 gene …" to place the claim in proper form. Appropriate correction is required.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
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11,976,277
Claims 184, 186-190, 195 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 11,976,277 (hereinafter "Fernandes") in view of Roberson (BMC Genomics 20, 528 (2019); hereinafter Roberson, See PTO-892).
Although the claims at issue are not identical, they are not patentably distinct from each other because of the reasons stated below.
Regarding claim 184, 186-188, 190, 195: Claim 1 of Fernandes is directed to a composition comprising a CRISPR nuclease and a CRISPR guide RNA. Claim 7 of Fernandes was directed to a CasX comprising a SEQ ID NO: 196, which is 100% identical to instant SEQ ID NO: 133 (See alignment below). Claim 11 of Fernandes taught that the CRISPR RNA comprises a scaffold sequence. It is noted that claims of Fernandes did not teach or suggest a gRNA comprising a targeting sequence that is complementary sequence to a PTBP1 gene target nucleic acid sequence. However, it would have been obvious for a person of ordinary skill in the art to arrive at a gRNA for CasX using routine experimentation. For example Roberson taught that CasX guide sites are relatively common in all their tested genomes (Roberson, p.4, col. 2, para 2). Roberson also taught a method to arrive at suitable guide sequences for CasX. For example Roberson “performed the annotations on the Washington University Center for High Performance Computing cluster. The motif location output included the location of the hit in the genome (contig, start, end) and the associated sequences.” Roberson “calculated the size of the reference genomes from their FASTA index files, determined the uniqueness of guide RNA sites amongst all editing sites, counted PAM usage, and annotated any overlaps with the exons of known genes. It is worth noting that uniqueness of a guide was determined only among editing sites. Presumably another site that matches the guide exactly, but does not have the PAM site would not be cleaved. A guide that overlaps an exon can likely be used to knockout the gene or knock-in coding variants at the exon.” (See Roberson, p. 2, para 2, as required by claims 186, 190). As such Roberson determined that arriving at a suitable gRNA for CasX is routine and systematic.
One of ordinary skill in the art, given the teachings of Roberson would have readily designed a gRNA complementary to PTBP1 for use with the CasX protein taught by Fernandes, as design of such RNA is routine, predictable and well documented by prior art, as shown by Roberson.
Query 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN
Sbjct 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
Query 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG
Sbjct 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
Query 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE
Sbjct 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
Query 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF
Sbjct 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
Query 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR
Sbjct 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
Query 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE
Sbjct 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
Query 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
Sbjct 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
Query 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE
Sbjct 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
Query 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK
Sbjct 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
Query 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND
Sbjct 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
Query 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR
Sbjct 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
Query 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS
Sbjct 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
Query 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME
Sbjct 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
Query 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI
Sbjct 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
Query 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
Sbjct 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
Query 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
Sbjct 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
Query 961 TWQSFYRKKLKEVWKPAV 978
TWQSFYRKKLKEVWKPAV
Sbjct 961 TWQSFYRKKLKEVWKPAV 978
Regarding claim 189: Roberson taught that the CasX comprises a 4-basepair PAM: TTCN (See Roberson, p.1, col.2, para 2).
Claims 191-192 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 11,976,277 (hereinafter "Fernandes") in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and Ousterout et al (Nat Commun. 2015 Feb 18; See PTO-892; hereinafter “Ousterout”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a second gRNA comprising a scaffold and a targeting sequence as required by the claim. Ousterout taught that multiplex gRNA targeting to use two guide RNAs targeting the same exon or different genes are common practices in CRISPR technologies: “The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons.” (See Ousterout Abstract).
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Ousterout would have readily designed multiple gRNAs for editing same exon complementary to PTBP1 for use with the CasX protein taught by Fernandes, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Ousterout.
Claims 185, 195-196 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 11,976,277 (hereinafter "Fernandes") in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and WO2019084148A1 (Published May 2, 2019; See PTO-892; hereinafter “Nagy”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a donor template comprising a scaffold and a targeting sequence as required by the claim. Nagy taught the use of donor template to introduce a nucleic acid into a target site. For example claim 12 of Nagy taught a method of modifying a target site using CasX, a gRNA and a donor template. As such one of ordinary skill in the art would have bene motivated to generate a composition comprising a CasX comprising the claimed sequence and a gRNA targeting PTBP1 and a template in view of the cited prior art. It is also noted that Nagy taught that the CasX nuclease further comprises a nuclear localization signal as required by claim 185.
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Nagy would have readily arrived at a composition comprising a gRNA targeting PTBP1 for use with the CasX protein taught by Fernandes. The person would have been motivated to further modify the composition with a donor template to insert a sequence at the target site for further modifying the target site, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Nagy.
U.S. Application No. 18/266,076
Claim 184, 186-190, 195 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 176-203 of copending Application No. 18/266,076 (reference application) in view of Roberson (BMC Genomics 20, 528 (2019); hereinafter Roberson, See PTO-892).
Although the claims at issue are not identical, they are not patentably distinct from each other because of the reasons stated below. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claim 184, 186-188, 190, 195: Claim 1 of reference application is directed to a composition comprising a CRISPR nuclease and a CRISPR guide RNA. Claim 185 of reference application was directed to a CasX comprising a SEQ ID NO: 145, which is 100% identical to instant SEQ ID NO: 133 (See alignment below). Claim 194 of reference application taught that the CRISPR RNA comprises a scaffold sequence. It is noted that claims of reference application did not teach or suggest a gRNA comprising a targeting sequence that is complementary sequence to a PTBP1 gene target nucleic acid sequence. However, it would have been obvious for a person of ordinary skill in the art to arrive at a gRNA for CasX using routine experimentation. For example Roberson taught that CasX guide sites are relatively common in all their tested genomes (Roberson, p.4, col. 2, para 2). Roberson also taught a method to arrive at suitable guide sequences for CasX. For example Roberson “performed the annotations on the Washington University Center for High Performance Computing cluster. The motif location output included the location of the hit in the genome (contig, start, end) and the associated sequences.” Roberson “calculated the size of the reference genomes from their FASTA index files, determined the uniqueness of guide RNA sites amongst all editing sites, counted PAM usage, and annotated any overlaps with the exons of known genes. It is worth noting that uniqueness of a guide was determined only among editing sites. Presumably another site that matches the guide exactly, but does not have the PAM site would not be cleaved. A guide that overlaps an exon can likely be used to knockout the gene or knock-in coding variants at the exon.” (See Roberson, p. 2, para 2, as required by claims 186, 190). As such Roberson determined that arriving at a suitable gRNA for CasX is routine and systematic.
One of ordinary skill in the art, given the teachings of Roberson would have readily designed a gRNA complementary to PTBP1 for use with the CasX protein taught by reference application, as design of such RNA is routine, predictable and well documented by prior art, as shown by Roberson.
Query 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN
Sbjct 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
Query 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG
Sbjct 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
Query 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE
Sbjct 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
Query 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF
Sbjct 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
Query 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR
Sbjct 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
Query 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE
Sbjct 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
Query 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
Sbjct 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
Query 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE
Sbjct 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
Query 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK
Sbjct 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
Query 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND
Sbjct 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
Query 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR
Sbjct 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
Query 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS
Sbjct 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
Query 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME
Sbjct 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
Query 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI
Sbjct 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
Query 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
Sbjct 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
Query 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
Sbjct 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
Query 961 TWQSFYRKKLKEVWKPAV 978
TWQSFYRKKLKEVWKPAV
Sbjct 961 TWQSFYRKKLKEVWKPAV 978
Regarding claim 189: Roberson taught that the CasX comprises a 4-basepair PAM: TTCN (See Roberson, p.1, col.2, para 2).
Claims 184-204 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 176-203 of copending Application No. 18/266,076 (reference application) in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and Ousterout et al (Nat Commun. 2015 Feb 18; See PTO-892; hereinafter “Ousterout”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a second gRNA comprising a scaffold and a targeting sequence as required by the claim. Ousterout taught that multiplex gRNA targeting to use two guide RNAs targeting the same exon or different genes are common practices in CRISPR technologies: “The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons.” (See Ousterout Abstract).
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Ousterout would have readily designed multiple gRNAs for editing same exon complementary to PTBP1 for use with the CasX protein taught by Fernandes, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Ousterout.
Claims 185, 195-196 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 176-203 of copending Application No. 18/266,076 (reference application) in view of in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and WO2019084148A1 (Published May 2, 2019; See PTO-892; hereinafter “Nagy”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a donor template comprising a scaffold and a targeting sequence as required by the claim. Nagy taught the use of donor template to introduce a nucleic acid into a target site. For example claim 12 of Nagy taught a method of modifying a target site using CasX, a gRNA and a donor template. As such one of ordinary skill in the art would have bene motivated to generate a composition comprising a CasX comprising the claimed sequence and a gRNA targeting PTBP1 and a template in view of the cited prior art. It is also noted that Nagy taught that the CasX nuclease further comprises a nuclear localization signal as required by claim 185.
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Nagy would have readily arrived at a composition comprising a gRNA targeting PTBP1 for use with the CasX protein taught by Fernandes. The person would have been motivated to further modify the composition with a donor template to insert a sequence at the target site for further modifying the target site, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Nagy.
U.S. Application No. 18/255,172
Claim 184, 186-190, 195 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 209-246 of copending Application No. 18/255,172 (reference application) in view of Roberson (BMC Genomics 20, 528 (2019); hereinafter Roberson, See PTO-892).
Although the claims at issue are not identical, they are not patentably distinct from each other because of the reasons stated below. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claim 184, 186-188, 190, 195: Claim 1 of reference application is directed to a composition comprising a CRISPR nuclease and a CRISPR guide RNA. Claim 222 of reference application was directed to a CasX comprising a SEQ ID NO: 133, which is 100% identical to instant SEQ ID NO: 133 (See alignment below). Claim 219 of reference application taught that the CRISPR RNA comprises a scaffold sequence. It is noted that claims of reference application did not teach or suggest a gRNA comprising a targeting sequence that is complementary sequence to a PTBP1 gene target nucleic acid sequence. However, it would have been obvious for a person of ordinary skill in the art to arrive at a gRNA for CasX using routine experimentation. For example Roberson taught that CasX guide sites are relatively common in all their tested genomes (Roberson, p.4, col. 2, para 2). Roberson also taught a method to arrive at suitable guide sequences for CasX. For example Roberson “performed the annotations on the Washington University Center for High Performance Computing cluster. The motif location output included the location of the hit in the genome (contig, start, end) and the associated sequences.” Roberson “calculated the size of the reference genomes from their FASTA index files, determined the uniqueness of guide RNA sites amongst all editing sites, counted PAM usage, and annotated any overlaps with the exons of known genes. It is worth noting that uniqueness of a guide was determined only among editing sites. Presumably another site that matches the guide exactly, but does not have the PAM site would not be cleaved. A guide that overlaps an exon can likely be used to knockout the gene or knock-in coding variants at the exon.” (See Roberson, p. 2, para 2, as required by claims 186, 190). As such Roberson determined that arriving at a suitable gRNA for CasX is routine and systematic.
One of ordinary skill in the art, given the teachings of Roberson would have readily designed a gRNA complementary to PTBP1 for use with the CasX protein taught by reference application, as design of such RNA is routine, predictable and well documented by prior art, as shown by Roberson.
Query 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN
Sbjct 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
Query 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG
Sbjct 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
Query 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE
Sbjct 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
Query 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF
Sbjct 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
Query 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR
Sbjct 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
Query 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE
Sbjct 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
Query 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
Sbjct 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
Query 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE
Sbjct 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
Query 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK
Sbjct 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
Query 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND
Sbjct 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
Query 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR
Sbjct 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
Query 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS
Sbjct 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
Query 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME
Sbjct 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
Query 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI
Sbjct 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
Query 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
Sbjct 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
Query 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
Sbjct 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
Query 961 TWQSFYRKKLKEVWKPAV 978
TWQSFYRKKLKEVWKPAV
Sbjct 961 TWQSFYRKKLKEVWKPAV 978
Regarding claim 189: Roberson taught that the CasX comprises a 4-basepair PAM: TTCN (See Roberson, p.1, col.2, para 2).
Claims 184-204 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 209-246 of copending Application No. 18/255,172 (reference application) in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and Ousterout et al (Nat Commun. 2015 Feb 18; See PTO-892; hereinafter “Ousterout”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a second gRNA comprising a scaffold and a targeting sequence as required by the claim. Ousterout taught that multiplex gRNA targeting to use two guide RNAs targeting the same exon or different genes are common practices in CRISPR technologies: “The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons.” (See Ousterout Abstract).
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Ousterout would have readily designed multiple gRNAs for editing same exon complementary to PTBP1 for use with the CasX protein taught by Fernandes, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Ousterout.
Claims 185, 195-196 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 209-246 of copending Application No. 18/255,172 (reference application) in view of in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and WO2019084148A1 (Published May 2, 2019; See PTO-892; hereinafter “Nagy”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a donor template comprising a scaffold and a targeting sequence as required by the claim. Nagy taught the use of donor template to introduce a nucleic acid into a target site. For example claim 12 of Nagy taught a method of modifying a target site using CasX, a gRNA and a donor template. As such one of ordinary skill in the art would have bene motivated to generate a composition comprising a CasX comprising the claimed sequence and a gRNA targeting PTBP1 and a template in view of the cited prior art. It is also noted that Nagy taught that the CasX nuclease further comprises a nuclear localization signal as required by claim 185.
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Nagy would have readily arrived at a composition comprising a gRNA targeting PTBP1 for use with the CasX protein taught by Fernandes. The person would have been motivated to further modify the composition with a donor template to insert a sequence at the target site for further modifying the target site, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Nagy.
U.S. Application No. 17/791,130
Claim 184, 186-190, 195 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 215-237 of copending Application No. 17/791,130 (reference application) in view of Roberson (BMC Genomics 20, 528 (2019); hereinafter Roberson, See PTO-892).
Although the claims at issue are not identical, they are not patentably distinct from each other because of the reasons stated below. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claim 184, 186-188, 190, 195: Claim 1 of reference application is directed to a composition comprising a CRISPR nuclease and a CRISPR guide RNA. Claim 222 of reference application was directed to a CasX comprising a SEQ ID NO: 145, which is 100% identical to instant SEQ ID NO: 133 (See alignment below). Claim 226 of reference application taught that the CRISPR RNA comprises a scaffold sequence. It is noted that claims of reference application did not teach or suggest a gRNA comprising a targeting sequence that is complementary sequence to a PTBP1 gene target nucleic acid sequence. However, it would have been obvious for a person of ordinary skill in the art to arrive at a gRNA for CasX using routine experimentation. For example Roberson taught that CasX guide sites are relatively common in all their tested genomes (Roberson, p.4, col. 2, para 2). Roberson also taught a method to arrive at suitable guide sequences for CasX. For example Roberson “performed the annotations on the Washington University Center for High Performance Computing cluster. The motif location output included the location of the hit in the genome (contig, start, end) and the associated sequences.” Roberson “calculated the size of the reference genomes from their FASTA index files, determined the uniqueness of guide RNA sites amongst all editing sites, counted PAM usage, and annotated any overlaps with the exons of known genes. It is worth noting that uniqueness of a guide was determined only among editing sites. Presumably another site that matches the guide exactly, but does not have the PAM site would not be cleaved. A guide that overlaps an exon can likely be used to knockout the gene or knock-in coding variants at the exon.” (See Roberson, p. 2, para 2, as required by claims 186, 190). As such Roberson determined that arriving at a suitable gRNA for CasX is routine and systematic.
One of ordinary skill in the art, given the teachings of Roberson would have readily designed a gRNA complementary to PTBP1 for use with the CasX protein taught by reference application, as design of such RNA is routine, predictable and well documented by prior art, as shown by Roberson.
Query 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN
Sbjct 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
Query 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG
Sbjct 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
Query 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE
Sbjct 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
Query 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF
Sbjct 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
Query 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR
Sbjct 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
Query 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE
Sbjct 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
Query 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
Sbjct 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
Query 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE
Sbjct 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
Query 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK
Sbjct 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
Query 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND
Sbjct 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
Query 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR
Sbjct 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
Query 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS
Sbjct 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
Query 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME
Sbjct 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
Query 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI
Sbjct 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
Query 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
Sbjct 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
Query 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
Sbjct 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
Query 961 TWQSFYRKKLKEVWKPAV 978
TWQSFYRKKLKEVWKPAV
Sbjct 961 TWQSFYRKKLKEVWKPAV 978
Regarding claim 189: Roberson taught that the CasX comprises a 4-basepair PAM: TTCN (See Roberson, p.1, col.2, para 2).
Claims 184-204 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 215-237 of copending Application No. 17/791,130 (reference application) in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and Ousterout et al (Nat Commun. 2015 Feb 18; See PTO-892; hereinafter “Ousterout”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a second gRNA comprising a scaffold and a targeting sequence as required by the claim. Ousterout taught that multiplex gRNA targeting to use two guide RNAs targeting the same exon or different genes are common practices in CRISPR technologies: “The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons.” (See Ousterout Abstract).
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Ousterout would have readily designed multiple gRNAs for editing same exon complementary to PTBP1 for use with the CasX protein taught by Fernandes, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Ousterout.
Claims 185, 195-196 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 215-237 of copending Application No. 17/791,130 (reference application) in view of in view of Roberson (BMC Genomics (2019); hereinafter Roberson, See PTO-892) and WO2019084148A1 (Published May 2, 2019; See PTO-892; hereinafter “Nagy”).
The teachings of Fernandes in view of Roberson are set forth above. Fernandes in view of Roberson did not teach a donor template comprising a scaffold and a targeting sequence as required by the claim. Nagy taught the use of donor template to introduce a nucleic acid into a target site. For example claim 12 of Nagy taught a method of modifying a target site using CasX, a gRNA and a donor template. As such one of ordinary skill in the art would have bene motivated to generate a composition comprising a CasX comprising the claimed sequence and a gRNA targeting PTBP1 and a template in view of the cited prior art. It is also noted that Nagy taught that the CasX nuclease further comprises a nuclear localization signal as required by claim 185.
One of ordinary skill in the art, given the teachings of Fernandes in view of Roberson and Nagy would have readily arrived at a composition comprising a gRNA targeting PTBP1 for use with the CasX protein taught by Fernandes. The person would have been motivated to further modify the composition with a donor template to insert a sequence at the target site for further modifying the target site, as design of such RNAs is routine, predictable and well documented by prior art, as shown by Roberson and Nagy.
U.S. Application No. 18/568,029
Claim 184, 186-190, 195 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-4, 13-14, 18-20, 23, 25-31, 35, 37-39, 41-43, 45 of copending Application No. 18/568,029 (reference application) in view of Roberson (BMC Genomics 20, 528 (2019); hereinafter Roberson, See PTO-892).
Although the claims at issue are not identical, they are not patentably distinct from each other because of the reasons stated below. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claim 184, 186-188, 190, 195: Claim 1 of reference application is directed to a composition comprising a CRISPR nuclease and a CRISPR guide RNA. Claim 39 of reference application was directed to a CasX comprising SEQ ID NOs: 635, 668, 892, 829, 871, 868, 828, 703, 878, 931, 928, 929, 834, which are > 90% identical to instant SEQ ID NO: 133 (See for alignment of SEQ ID NO: 635 with SEQ ID NO: 133 below). Claim 41 of reference application taught that the CRISPR RNA comprises a scaffold sequence. It is noted that claims of reference application did not teach or suggest a gRNA comprising a targeting sequence that is complementary sequence to a PTBP1 gene target nucleic acid sequence. However, it would have been obvious for a person of ordinary skill in the art to arrive at a gRNA for CasX using routine experimentation. For example Roberson taught that CasX guide sites are relatively common in all their tested genomes (Roberson, p.4, col. 2, para 2). Roberson also taught a method to arrive at suitable guide sequences for CasX. For example Roberson “performed the annotations on the Washington University Center for High Performance Computing cluster. The motif location output included the location of the hit in the genome (contig, start, end) and the associated sequences.” Roberson “calculated the size of the reference genomes from their FASTA index files, determined the uniqueness of guide RNA sites amongst all editing sites, counted PAM usage, and annotated any overlaps with the exons of known genes. It is worth noting that uniqueness of a guide was determined only among editing sites. Presumably another site that matches the guide exactly, but does not have the PAM site would not be cleaved. A guide that overlaps an exon can likely be used to knockout the gene or knock-in coding variants at the exon.” (See Roberson, p. 2, para 2, as required by claims 186, 190). As such Roberson determined that arriving at a suitable gRNA for CasX is routine and systematic.
One of ordinary skill in the art, given the teachings of Roberson would have readily designed a gRNA complementary to PTBP1 for use with the CasX protein taught by reference application, as design of such RNA is routine, predictable and well documented by prior art, as shown by Roberson.
Query 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN
Sbjct 1 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN 60
Query 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG
Sbjct 61 TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG 120
Query 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE
Sbjct 121 NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE 180
Query 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF
Sbjct 181 AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF 240
Query 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR
Sbjct 241 LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR 300
Query 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE
Sbjct 301 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE 360
Query 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
Sbjct 361 DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE 420
Query 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE
Sbjct 421 AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE 480
Query 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK
Sbjct 481 LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK 540
Query 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND
Sbjct 541 LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND 600
Query 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR
Sbjct 601 LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR 660
Query 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS
Sbjct 661 GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS 720
Query 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME
Sbjct 721 RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME 780
Query 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI
Sbjct 781 DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI 840
Query 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
Sbjct 841 NGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR 900
Query 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
Sbjct 901 FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE 960
Query 961 TWQSFYRKKLKEVWKPAV 978
TWQSFYRKKLKEVWKPAV
Sbjct 961 TWQSFYRKKLKEVWKPAV 978
Regarding claim 189: Roberson taught that the CasX comprises a 4-basepair PAM: TTCN (See Roberson, p.1, col.2, para 2).
Claims 184-204 are provisionally rejected o