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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3-3-26 has been entered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Applicant's arguments filed 3-3-26 have been fully considered but they are not persuasive.
Claims 1-29 remain pending.
Election/Restrictions
Applicants elected Group I, claims 1-17, without traverse in the reply filed on 8-28-24.
Claims 18-29 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Claims 1-17 remain under consideration.
Claim objections
The preamble of claim 1, the body of claim 1, and the final result of claim 1 should have a nexus. The preamble says “genome editing”, but no “genome” is edited in the cell in the body of the claim. The final step of claim 1 requires repairing the target DNA, but the body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
The concept of “repair” in the phrase “nucleic acid repair template” in claim 1 is an intended use and does not necessarily have to occur. The body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
The phrase “to facilitate homology directed DNA break repair” in claim 1 is an intended use and does not necessarily have to occur. The body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
Step b) of claim 1 refers to the nucleic acid repair template but should refer to “the oligonucleotide” as newly amended.
The result of claim 1 says “the target DNA is edited” which is a different scope/concept than “repair”. The concept of “editing” encompasses nucleic acid insertions, deletions, and substitutions that do not alter the coding sequence or the function of a protein encoded by the coding sequence. The concept of “repair” infers the cell has a mutant gene in need of repair. Therefore, the scope of “editing” and “repair” cannot be determined. If the scope of “editing” encompasses making nucleic acid insertions, deletions, and substitutions that do not alter the function of a protein encoded by the coding sequence, then much clarification is required.
The term “enzyme” in the phrase “Cas9 enzyme” throughout the claim set is redundant. Delete “enzyme”.
Claim 2 can be written more clearly as ---wherein the RNA encoding Cas9 and the RNA encoding the sgRNA have a ratio from 1:20 to 20:1---.
Claim 14 can be written more clearly as ---further comprising transfecting the cell obtained in step b) with…---.
The concept of “editing” and “repair” in claim 14 lacks a nexus for reasons set forth above in claim 1. The concept of “editing” the 2nd target DNA encompasses nucleic acid insertions, deletions, and substitutions that do not alter the coding sequence or the function of a protein encoded by the coding sequence. The concept of “repair” infers the cell has a mutant gene in need of repair. Therefore, the scope of “editing” and “repair” cannot be determined. If the scope of “editing” the 2nd target DNA encompasses making nucleic acid insertions, deletions, and substitutions that do not alter the function of a protein encoded by the coding sequence, then much clarification is required.
Claim Rejections - 35 USC § 112
Written Description
Claims 1-17 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.
Withdrawn rejections
The rejection regarding a Cas9 with a D10A and H840A mutation as required in claim 8 has been withdrawn. Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191) taught “The Cas9D10A variant with a mutation in the active site of the RuvC-like domain cleaves the DNA strand complementary to the sgRNAbinding sequence, while Cas9H840A with a mutation in the HNH-like domain cleaves the noncomplementary strand, and Cas9D10A/H840A is catalytically dead (Jinek et al., 2012).” The specification teaches a Cas9D10A/H840A on pg 47, para 78, and one was described by Vriend.
The rejection of claim 3 regarding the second transfection occurs 12-18 hours after the first transfection has been withdrawn in view of steps 8) and 9) in section h) of Protocol V which describe a “second round after transfection” (step 9) 12-18 hours after confirming the 1st transfection was successful (step 8) (pg 31).
The rejection regarding a Cas9 comprising a nickase that bears a D10A mutation encoded by SEQ ID NO: 2 or H840A mutation encoded by SEQ ID NO: 3 in claim 7 has been withdrawn in view of Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191).
Pending rejections
The other previous rejections have been withdrawn in favor of the following rejections:
A) The specification lacks written description for the method of claim 1 other than a) transfecting an isolated mammalian cell with mRNA encoding Cas9, mRNA encoding a sgRNA, and an ssODN, and b) transfecting the cells obtained in step a) with the mRNA encoding a sgRNA, and the ssODN such that the target sequence in the genome of the cell is genetically modified.
Claim 1 is drawn to a
A method for genome editing which comprises
a) transfecting an isolated mammalian cell with
i) a ribonucleic acid (RNA) that encodes Cas9 enzyme and has a 5' diguanosine cap and a 3' polyA tail,
ii) an RNA that encodes a single guide RNA (sgRNA) that has a 5' diguanosine cap, and
iii) an oligonucleotide that encodes a nucleic acid repair template to facilitate homology directed DNA break repair, and
b) transfecting the cells obtained in step a) with
i) the sgRNA and ii) the nucleic acid repair template [but] not the RNA that encodes Cas9 enzyme,
wherein the sgRNA hybridizes with a target DNA in the cell, the Cas9 enzyme cleaves the target DNA, and the nucleic acid repair template repairs the target DNA, whereby the target DNA is edited.
Claim 1 encompasses transfecting a cell with RNA encoding Cas9, RNA encoding an sgRNA, and a repair template followed by transfecting the same cell with the sgRNA and repair template but not the RNA encoding Cas9.
Example 6 (pg 13-42) is limited to generating a single base pair mutation (pg 13, end of para 44). Pg 7, para 23 and 26, and pg 42, last paragraph (Fig. 9), confirm a single base change was achieved.
The specification teaches using one or two sgRNAs that target the same gene (pg 7, para 21; pg 10, para 34).
Pg 16, para 60, describes doing so using mRNA encoding Cas9, mRNA encoding an sgRNA, and a ssODN (which must be DNA) followed by a second transfection using the mRNA encoding the sgRNA and the ssODN.
Protocol V (paragraph 71, pg 24-32), contains section h) (pg 29-32). Step 3 (pg 30) combines mRNA encoding Cas9, mRNA encoding sgRNA, and ssODN. Step 5 (pg 30) is the first transfection using the mixture. Step 9 (pg 31) is the second transfection using mRNA encoding sgRNA and ssODN but not mRNA encoding Cas9.
Messenger RNA (mRNA) is the only type of RNA that encodes anything, so the RNAs encoding Cas9 and sgRNA should be mRNA (pg 4, para 12). The specification does not teach any rRNA or tRNA encoding Cas9 or sgRNA as broadly encompassed by claim 1. Claim 1 should be limited to mRNA encoding Cas9 and mRNA encoding a sgRNA.
The specification and the art at the time of filing are limited to mRNA encoding Cas9 and sgRNA containing a 5 diguanosine cap and polyA tail (pg 4, para 12). The specification does not teach mRNA encoding an sgRNA without a polyA tail as broadly encompassed by claim 1.
The specification lacks written description for any oligonucleotide encoding a nucleic acid repair template as required in claim 1 other than a single stranded oligonucleotide (ssODN) (pg 13, para 47; et al.). Claim 1 encompasses double stranded oligonucleotides. The specification contemplates double stranded DNA (pg 5, para 15), but not double stranded oligonucleotides. The specification teaches single stranded oligos (pg 13, para 47, 48; pg 16, para 60; pg 30-32, Table 6-14; pg 40, d) 3)) but does not teach double stranded oligonucleotides as encompassed by claim 1.
The specification lacks written description for “repairing” any mutant mammalian gene as broadly encompassed by claim 1. The final step of claim 1 requires the “repair template repairs the target DNA” which encompasses “repairing” a mutant target DNA. The concepts of “editing” and “repairing” encompass nucleic acid insertions, deletions, and substitutions that alter a mutant coding sequence to a wild-type coding sequence. The concept of “repair” infers the cell has a mutant gene in need of repair. The specification does not teach any mutant genes, and “repair templates” the return a mutant gene to wild-type, or performing the method of claim 1 in a mammalian cell with a mutant gene. Example 6 (pg 13-42) is limited to generating a single base pair mutation (pg 13, end of para 44). Pg 7, para 23 and 26, and pg 42, last paragraph (Fig. 9), confirm a single base change was achieved. The specification does not correlate making the single base change to any “repair” as broadly encompassed by claim 1.
Accordingly, the concept lacks written description other than other than a) transfecting an isolated mammalian cell with mRNA encoding Cas9, mRNA encoding a sgRNA, and an ssODN, and b) transfecting the cells obtained in step a) with the mRNA encoding a sgRNA, and the ssODN such that the target sequence in the genome of the cell is genetically modified.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
B) The specification lacks written description for double stranded oligonucleotide encoding a nucleic acid repair template as required in claim 4. Claim 4 is limited to a double stranded oligonucleotides. The specification contemplates double stranded DNA (pg 5, para 15), but not double stranded oligonucleotides as required in claim 4. The specification teaches single stranded oligos (pg 13, para 47, 48; pg 16, para 60; pg 30-32, Table 6-14; pg 40, d) 3)) but does not teach double stranded oligonucleotides as required in claim 4.
C) The specification lacks written description for a repair template that is RNA capable of targeting, repairing, and integrating DNA as required in claim 6. The specification does not teach the structures of any RNA template capable of targeting, repairing, and integrating DNA as required in claim 6. Accordingly, the concept lacks written description.
D) The specification lacks written description for using Cas9 with D10A and H840 mutations as required in claim 8 because Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191) taught Cas9 with a D10A and H840A mutation did not have enzymatic function. Vriend taught “The Cas9D10A variant with a mutation in the active site of the RuvC-like domain cleaves the DNA strand complementary to the sgRNAbinding sequence, while Cas9H840A with a mutation in the HNH-like domain cleaves the noncomplementary strand, and Cas9D10A/H840A is catalytically dead (Jinek et al., 2012).” The specification teaches a Cas9D10A/H840A on pg 47, para 78, and one was described by Vriend, but it didn’t function to cleave or nick double stranded DNA. Without evidence the art or applicants figured out how to make a Cas9D10A/H841A mutant function as a nickase after the publication of Vriend, the concept in claim 8 lacks written description.
E) The specification lacks written description for the reagents and “repair” in claim 14 for reasons set forth above in rejection A).
F) The specification lacks written description for fusing the nucleic acid repair template and the RNA encoding the sgRNA on the same nucleic acid as required in claim 15. The concept cannot be found in the specification or the art at the time of filing. In particular, the ssODN described by applicants is the only oligo that is a “repair template” described by applicants (see above), and ssODNs MUST be DNA. Single stranded DNA can never be in the same nucleic acid as RNA as claimed. Accordingly, the concept in claim 15 lacks written description.
Response to arguments
Applicants argue it was readily apparent sgRNA and repair templates could be joined in para 37. Applicants’ argument is not persuasive for reasons set forth above. In no way do the teachings in para 37 have a nexus with the RNA encoding sgRNA in claim 1 that MUST be mRNA as discussed above. In no way do the teachings in para 37 have a nexus with the oligonucleotide encoding the repair template in claim 1 which MUST be DNA. The oligo MUST be single stranded DNA for reasons cited above which MUST be DNA. It cannot be RNA as required in claim 15.
Applicants point to WO 2017180711 which describes RNA repair templates. Applicants’ argument is not persuasive. In no way do the teachings in WO 2017180711 have a nexus with the oligonucleotide encoding the repair template in claim 1 which MUST be DNA. The oligo MUST be single stranded DNA for reasons cited above which MUST be DNA. It cannot be RNA as required in claim 15.
G) The specification lacks written description for an oligo comprising the repair template further comprising an aptamer as required in claim 16. The specification contemplates a repair template having an aptamer that binds Cas9 (pg 11, para 38), but claim 16 makes no such requirement. The specification does not correlate an aptamer that binds Cas9 to any aptamer as broadly claimed. However, the specification does not teach any ssODN or other oligo that is a repair template further comprising an aptamer that binds Cas9 or has any function as broadly encompassed by claim 16. Accordingly, the concept lacks written description.
Response to arguments
Applicants argue it was readily apparent that an aptamer could be used as described in para 38. Applicants’ argument is not persuasive for reasons set forth above.
Enablement
Claims 1-17 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Withdrawn rejections
The rejection regarding a Cas9 with a D10A and H840A mutation as required in claim 8 has been withdrawn. Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191) taught “The Cas9D10A variant with a mutation in the active site of the RuvC-like domain cleaves the DNA strand complementary to the sgRNAbinding sequence, while Cas9H840A with a mutation in the HNH-like domain cleaves the noncomplementary strand, and Cas9D10A/H840A is catalytically dead (Jinek et al., 2012).” The specification teaches a Cas9D10A/H840A on pg 47, para 78, and one was described by Vriend.
The rejection of claim 3 regarding the second transfection occurs 12-18 hours after the first transfection has been withdrawn in view of steps 8) and 9) in section h) of Protocol V which describe a “second round after transfection” (step 9) 12-18 hours after confirming the 1st transfection was successful (step 8) (pg 31).
The rejection regarding a Cas9 comprising a nickase that bears a D10A mutation encoded by SEQ ID NO: 2 or H840A mutation encoded by SEQ ID NO: 3 in claim 7 has been withdrawn in view of Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191).
Pending rejections
The other previous rejections have been withdrawn in favor of the following rejections:
A) The specification does not enable using the method as broadly encompassed by claim 1 other than a) transfecting an isolated mammalian cell with mRNA encoding Cas9, mRNA encoding a sgRNA, and an ssODN, and b) transfecting the cells obtained in step a) with the mRNA encoding a sgRNA, and the ssODN such that the target sequence in the genome of the cell is genetically modified.
Claim 1 and its breadth are described above.
The teachings in the specification and examples are described above.
The specification teaches using one or two sgRNAs that target the same gene (pg 7, para 21; pg 10, para 34).
Example 6 (pg 13-42) is limited to generating a single base pair mutation (pg 13, end of para 44). Pg 7, para 23 and 26, and pg 42, last paragraph (Fig. 9), confirm a single base change was achieved.
Messenger RNA (mRNA) is the only type of RNA that encodes anything, so the RNAs encoding Cas9 and sgRNA should be mRNA (pg 4, para 12). The specification does not teach any rRNA or tRNA encoding Cas9 or sgRNA as broadly encompassed by claim 1. Claim 1 should be limited to mRNA encoding Cas9 and mRNA encoding a sgRNA.
The specification and the art at the time of filing are limited to mRNA encoding Cas9 and sgRNA containing a 5 diguanosine cap and polyA tail (pg 4, para 12). The specification does not teach mRNA encoding an sgRNA without a polyA tail as broadly encompassed by claim 1.
The specification does not enable making/using any oligonucleotide encoding a nucleic acid repair template as required in claim 1 other than a single stranded oligonucleotide (ssODN) (pg 13, para 47; et al.). Claim 1 encompasses double stranded oligonucleotides. The specification contemplates double stranded DNA (pg 5, para 15), but not double stranded oligonucleotides. The specification teaches single stranded oligos (pg 13, para 47, 48; pg 16, para 60; pg 30-32, Table 6-14; pg 40, d) 3)) but does not teach double stranded oligonucleotides as encompassed by claim 1.
The specification lacks written description for “repairing” any mutant mammalian gene as broadly encompassed by claim 1. The final step of claim 1 requires the “repair template repairs the target DNA” which encompasses “repairing” a mutant target DNA. The concepts of “editing” and “repairing” encompass nucleic acid insertions, deletions, and substitutions that alter a mutant coding sequence to a wild-type coding sequence. The concept of “repair” infers the cell has a mutant gene in need of repair. The specification does not teach any mutant genes, and “repair templates” the return a mutant gene to wild-type, or performing the method of claim 1 in a mammalian cell with a mutant gene. Example 6 (pg 13-42) is limited to generating a single base pair mutation (pg 13, end of para 44). Pg 7, para 23 and 26, and pg 42, last paragraph (Fig. 9), confirm a single base change was achieved. The specification does not correlate making the single base change to any “repair” as broadly encompassed by claim 1.
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to perform the method as broadly encompassed by claim 1 other than other than a) transfecting an isolated mammalian cell with mRNA encoding Cas9, mRNA encoding a sgRNA, and an ssODN, and b) transfecting the cells obtained in step a) with the mRNA encoding a sgRNA, and the ssODN such that the target sequence in the genome of the cell is genetically modified.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
B) The specification does not enable making/using a double stranded oligonucleotide encoding a nucleic acid repair template as required in claim 4. Claim 4 is limited to a double stranded oligonucleotides. The specification contemplates double stranded DNA (pg 5, para 15), but not double stranded oligonucleotides as required in claim 4. The specification teaches single stranded oligos (pg 13, para 47, 48; pg 16, para 60; pg 30-32, Table 6-14; pg 40, d) 3)) but does not teach double stranded oligonucleotides as required in claim 4.
C) The specification does not enable making/using a repair template that is RNA capable of targeting, repairing, and integrating DNA as required in claim 6. The specification does not teach the structures of any RNA template capable of targeting, repairing, and integrating DNA as required in claim 6.
D) The specification does not enable using Cas9 with D10A and H840 mutations as required in claim 8 because Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191) taught Cas9 with a D10A and H840A mutation did not have enzymatic function. Vriend taught “The Cas9D10A variant with a mutation in the active site of the RuvC-like domain cleaves the DNA strand complementary to the sgRNAbinding sequence, while Cas9H840A with a mutation in the HNH-like domain cleaves the noncomplementary strand, and Cas9D10A/H840A is catalytically dead (Jinek et al., 2012).” The specification teaches a Cas9D10A/H840A on pg 47, para 78, and one was described by Vriend, but it didn’t function to cleave or nick double stranded DNA. Without evidence the art or applicants figured out how to make a Cas9D10A/H841A mutant function as a nickase after the publication of Vriend, the concept in claim 8 is not enabled.
E) The specification does not enable the reagents and “repair” in claim 14 for reasons set forth above in rejection A).
F) The specification does not enable fusing the nucleic acid repair template and the RNA encoding the sgRNA on the same nucleic acid as required in claim 15. The concept cannot be found in the specification or the art at the time of filing. In particular, the ssODN described by applicants is the only oligo that is a “repair template” described by applicants (see above), and ssODNs MUST be DNA. Single stranded DNA can never be in the same nucleic acid as RNA as claimed.
Response to arguments
Applicants argue it was readily apparent sgRNA and repair templates could be joined in para 37. Applicants’ argument is not persuasive for reasons set forth above. In no way do the teachings in para 37 have a nexus with the RNA encoding sgRNA in claim 1 that MUST be mRNA as discussed above. In no way do the teachings in para 37 have a nexus with the oligonucleotide encoding the repair template in claim 1 which MUST be DNA. The oligo MUST be single stranded DNA for reasons cited above which MUST be DNA. It cannot be RNA as required in claim 15.
Applicants point to WO 2017180711 which describes RNA repair templates. Applicants’ argument is not persuasive. In no way do the teachings in WO 2017180711 have a nexus with the oligonucleotide encoding the repair template in claim 1 which MUST be DNA. The oligo MUST be single stranded DNA for reasons cited above which MUST be DNA. It cannot be RNA as required in claim 15.
G) The specification does not enable making/using an oligo comprising the repair template further comprising an aptamer as required in claim 16. The specification contemplates a repair template having an aptamer that binds Cas9 (pg 11, para 38), but claim 16 makes no such requirement. The specification does not correlate an aptamer that binds Cas9 to any aptamer as broadly claimed. However, the specification does not teach any ssODN or other oligo that is a repair template further comprising an aptamer that binds Cas9 or has any function as broadly encompassed by claim 16. Accordingly, the concept lacks written description.
Response to arguments
Applicants argue it was readily apparent that an aptamer could be used as described in para 38. Applicants’ argument is not persuasive for reasons set forth above.
Indefiniteness
The rejections of claims 1-17 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential elements and having gaps between the elements has been withdrawn in view of the amendment.
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.
Claims 1-17 are 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.
A) The preamble of claim 1, the body of claim 1, and the final result of claim 1 do not have a nexus. This makes the metes and bounds of claim 1 indefinite. The preamble says “genome editing”, but no “genome” is edited in the cell in the body of the claim. The final step of claim 1 requires repairing the target DNA, but the body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
The concept of “repair” in the phrase “nucleic acid repair template” in claim 1 is an intended use and does not necessarily have to occur. The body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
The phrase “to facilitate homology directed DNA break repair” in claim 1 is an intended use and does not necessarily have to occur. The body of the claim doesn’t require the genome of the cell has a gene that needs repaired.
Step b) of claim 1 refers to the nucleic acid repair template but should refer to “the oligonucleotide” as newly amended.
The result of claim 1 says “the target DNA is edited” which is a different scope/concept than “repair”. The concept of “editing” encompasses nucleic acid insertions, deletions, and substitutions that do not alter the coding sequence or the function of a protein encoded by the coding sequence. The concept of “repair” infers the cell has a mutant gene in need of repair. Therefore, the scope of “editing” and “repair” cannot be determined. If the scope of “editing” encompasses making nucleic acid insertions, deletions, and substitutions that do not alter the function of a protein encoded by the coding sequence, then much clarification is required.
Overall, those of skill would not be able to determine when they had achieved the desire result of claim 1.
B) Claim 14 is indefinite for reasons recited above in claim 1.
Claim Rejections - 35 USC § 102
Withdrawn rejection
The rejection of claims 1, 5, 9, 13, 15 under 35 U.S.C. 102a1 as being anticipated by Qin (Genetics, 2015, Vol. 200, No. 2, pg 423-430) has been withdrawn. Qin genetically modified isolated mammalian cells using mRNA encoding Cas9, sgRNA and a repair template (“To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide” abstract) which is equivalent to claim 1. Qin did not teach transfecting the cells obtained with sgRNA and a cDNA template as newly required in claim 1 as amended.
Pending rejection
Claims 1, 2, 4, 5, 9-16 remain rejected under 35 U.S.C. 102a1 as being anticipated by Hoge (20160367702).
Hoge transfected an isolated mammalian cell with mRNA encoding Cas9 (para 14; Fig. 3; para 17; Table 7), a single guide RNA (sgRNA) that cleaves a target DNA in the cell (abstract, paragraph 2), and a cDNA template (para 181, 183, 192-196, 522, 523578, 662, 680) which is a “oligonucleotide [ ] repair template” as required in claim 1. The mRNA encoding Cas9 has a 5’ diguanosine cap (para 140) and polyA tail (para 44, 135; 167-174) as required in claim 1. The mRNA encoding the sgRNA inherently MUST have a 5’ diguanosine cap and polyA tail as required in claim 1 because it is functionally expressed. The transfection can be repeated twice, at least 3 times, at least 4 times… (para 622) which is equivalent to claim 1.
The ratios of nucleic acids taught by Hoge in para 726 are equivalent to those in claim 2.
Hoge taught using strategies of Mali (para 3, 6, 176, 709), Cong (para 9, 709), Gilbert (para 8, 9, 14, 15), Trilink (para 14), Maeder (para 15, 718) which used single or double strand DNA templates as required in claims 4 and 5.
Hoge taught “FLAG tagged Cas9 mRNA constructs containing Cong Cas9”, “untagged Cas9 mRNA construct containing Mali Cas9”, a “HA tagged dCas9 construct containing Gilbert dCas9”, a FLAG-tagged dCas9 construct containing Maeder dCas9” and a HA-tagged Trilink Cas9 construct” (pg 72, para 725), all of which are Cas9/FokI fusion proteins. FokI is “another enzyme that can alter epigenetic markers on either the DNA or chromatin proteins” as required in claim 9. In an alternative interpretation, Hoge taught Cas9 is a dCAS9 fusion protein (para 23) dCAS9-effector (activator or inhibitor) fusion protein (para 24, 27, 66, 98, 663, 709, 722), fused to VP64, p65AD, KRAB, Mxi1, et al. (para 7, 21, 33), fused to a HA-tag or “NLSs” (para 713), all of which are “another enzyme that can alter epigenetic markers on either the DNA or chromatin proteins” as required in claim 9.
Hoge taught modifying nucleotides of the mRNA encoding Cas9 or the sgRNA (para 233, 235, 243) as required in claims 10 and 11, specifically metyl-cytodine (para 235), 2 thiouracil (para 243) or pseudoriacil (para 243) as required in claim 12.
The ratios of mRNA encoding Cas9 and sgRNA used by Hoge (para 726) are 1:1000 to 1000:1 as required in claim 13.
Hoge taught using strategies of Mali (para 3, 6, 176, 709), Cong (para 9, 709), Gilbert (para 8, 9, 14, 15), Trilink (para 14), Maeder (para 15, 718) which used “multiple sgRNA targeting” sites and Cas9 “from different species or bearing different mutations” as required in claim 14.
Claim 15 has been included because Hoge taught the “template” and sgRNA are on the same sequence (see above).
Claim 16 has been included because Hoge taught the sgRNA comprises an aptamer (para 58, 62, 227, 378, 381, 382, 383).
Response to arguments
Applicants argue Hoge did not teach two separate transfection steps. Applicants’ argument is not persuasive. Hoge taught the transfection can be repeated twice, at least 3 times, at least 4 times… (para 622) which is equivalent to two separate transfection steps as required in claim 1.
Claim Rejections - 35 USC § 103
A) Claims 1-6, 9-16 remain rejected under 35 U.S.C. 103 as being unpatentable over Hoge (20160367702) in view of Storici (Nature, 2007, Vol. 447, pg 338-341).
Hoge transfected an isolated mammalian cell with mRNA encoding Cas9 (para 14; Fig. 3; para 17; Table 7), a single guide RNA (sgRNA) that cleaves a target DNA in the cell (abstract, paragraph 2), and a cDNA template (para 181, 183, 192-196, 522, 523578, 662, 680) which is a “repair template” as required in claim 1. The mRNA encoding Cas9 has a 5’ diguanosine cap (para 140) and polyA tail (para 44, 135; 167-174) as required in claim 1. The transfection can be repeated twice, at least 3 times, at least 4 times… (para 622) which is also equivalent to claim 1.
Hoge did not teach the first and second transfections were 16-18 hours apart as required in claim 3.
However, the time between transfections was simply a matter of routine optimization by the ordinary artisan. Those of ordinary skill in the art at the time of filing would have been motivated to wait 16-18 hours until the 2nd transfection to ensure cleavage of the target gene and incorporation of the repair template.
Hoge did not teach the repair template was RNA as required in claim 6.
However, Storici taught RNA-templated DNA repair (title, abstract, Methods).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to perform the method of Hoge using an RNA repair template described by Storici. Those of ordinary skill in the art at the time of filing would have been motivated to use an RNA repair template because Storici taught “RNA as a homologous template in DNA repair may contribute to both genome integrity and evolution” (pg 340, col. 2, 3rd paragraph).
Claims 2, 4, 5, 9-16 have been included for reasons set forth above.
Response to arguments
Applicants argue Hoge does not teach the method of claim 1. Applicants’ argument is not persuasive for reasons set forth above.
B) Claims 1-5, 7, 9-16 remain rejected under 35 U.S.C. 103 as being unpatentable over Hoge (20160367702) in view of BCK79008 described by Chen (CN104611368).
Hoge taught genetically modifying a target gene in an isolated mammalian cell using mRNA encoding Cas9 (para 14; Fig. 3; para 17; Table 7), a single guide RNA (sgRNA) (abstract, paragraph 2), and a donor for reasons set forth above. The transfection can be repeated twice, at least 3 times, at least 4 times… (para 622) which is also equivalent to claim 1.
Hoge did not teach the first and second transfections were 16-18 hours apart as required in claim 3.
However, the time between transfections was simply a matter of routine optimization by the ordinary artisan. Those of ordinary skill in the art at the time of filing would have been motivated to wait 16-18 hours until the 2nd transfection to ensure cleavage of the target gene and incorporation of the repair template.
Hoge did not teach the Cas9 had a D10A or an H840A mutation as required in claim 7.
However, Chen described BCK79008 which is 99.2% identical to applicants’ nucleic acid sequence of SEQ ID NO: 2 with 21 mismatches and has a D10A mutation.
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Dependent claims have been included for reasons set forth above.
Response to arguments
Applicants argue Hoge did not teach the invention of claim 1, but applicants fail to point to one specific element that is missing from Hoge. Applicants’ arguments is not persuasive for reasons set forth above.
C) Claims 1-5, 9-17 remain rejected under 35 U.S.C. 103 as being unpatentable over Hoge (20160367702) in view of Totary-Jain (10188750).
Hoge transfected an isolated mammalian cell with mRNA encoding Cas9 (para 14; Fig. 3; para 17; Table 7), a single guide RNA (sgRNA) that cleaves a target DNA in the cell (abstract, paragraph 2), and a cDNA template (para 181, 183, 192-196, 522, 523578, 662, 680) which is a “repair template” as required in claim 1. The mRNA encoding Cas9 has a 5’ diguanosine cap (para 140) and polyA tail (para 44, 135; 167-174) as required in claim 1. The first and second transfections in claim 1 can be at the same time which is equivalent to the teachings of Hoge. The transfection can be repeated twice, at least 3 times, at least 4 times… (para 622) which is also equivalent to claim 1.
Hoge did not teach the first and second transfections were 16-18 hours apart as required in claim 3.
However, the time between transfections was simply a matter of routine optimization by the ordinary artisan. Those of ordinary skill in the art at the time of filing would have been motivated to wait 16-18 hours until the 2nd transfection to ensure cleavage of the target gene and incorporation of the repair template.
Hoge did not teach the method included adding B18R as required in claim 17.
However, 10188750 taught using Cas9 in combination with B18R (para 38-39; Fig. 37-38).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to perform the method of Hoge using B18R described by 10188750. Those of ordinary skill in the art at the time of filing would have been motivated to add B18R because it caused a synergistic effect (para 38 and 39).
Claims 2, 4, 5, 9-16 have been included for reasons set forth above.
Response to arguments
Applicants argue Hoge does not teach the method of claim 1. Applicants’ argument is not persuasive for reasons set forth above.
Claim 8 is free of the prior art because Vriend (Methods in Enzymology, 2014, Vol. 546, pg 175-191) taught Cas9 with a D10A and H840A mutation did not have enzymatic function. Vriend taught “The Cas9D10A variant with a mutation in the active site of the RuvC-like domain cleaves the DNA strand complementary to the sgRNAbinding sequence, while Cas9H840A with a mutation in the HNH-like domain cleaves the noncomplementary strand, and Cas9D10A/H840A is catalytically dead (Jinek et al., 2012).” The specification teaches a Cas9D10A/H840A on pg 47, para 78, and one was described by Vriend, but it didn’t function to cleave or nick double stranded DNA. Without evidence the art or applicants figured out how to make a Cas9D10A/H841A mutant function as a nickase after the publication of Vriend, then the art at the time of filing did not reasonably teach or suggested the method of claim 8.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, Immunity, 2005, Vol. 23, No. 2, pg 165-175.
Ryan (10337001) taught mRNA encoding Cas9 and sgRNA with modified nucleotides.
10676499 taught 3’ and 5’ caps for mRNA encoding Cas9 and sgRNAs.
11027025 taught sgRNAs with modifications (Detailed Description paragraph 668).
11097012 taught mRNA encoding Cas9 and sgRNA with modifications (claims 12 and 18).
No claim is allowed.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
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