DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
A request for continued examination under 37 CFR 1.114 was filed in this application after appeal to the Patent Trial and Appeal Board, but prior to a decision on the appeal. 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examination under 37 CFR 1.114 and the fee set forth in 37 CFR 1.17(e) has been timely paid, the appeal has been withdrawn pursuant to 37 CFR 1.114 and prosecution in this application has been reopened pursuant to 37 CFR 1.114. Applicant’s submission filed on 09/12/2025 has been entered.
Amended claims 1, 10, 17, 47-61 and new claim 62 are pending in the present application, and they are examined on the merits herein.
Priority
The present application is a CON of US application with the Serial Number 15/388,248, filed on 12/22/2016, now Abandoned; which is a CON of US application with the Serial Number 14/604,641, filed on 01/23/2015, now Abandoned; which is a CIP of PCT/US2013/51418, filed on 07/21/2013; which claims benefit of the provisional application 61/675,778, filed on 07/25/2012; the provisional application 61/721,283, filed on 11/01/2012; the provisional application 61/736,465, filed on 12/12/2012; the provisional application 61/794,458, filed on 03/15/2013; and the provisional application 61/835,973, filed on 06/17/2013.
Upon review of the specifications of the above US applications and the above provisional application, it is determined that examined claims are only entitled to the effective filing date of 12/12/2012 of the provisional application 61/736,465. This is because there is no written support in any of the provisional applications 61/675,778 and 61/721,283 for a CRISPR-Cas9 system.
Terminal Disclaimer
The terminal disclaimers filed on 09/12/2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of any patent granted on any of Applications 16/906,580 and 16/938,110 have been reviewed and are accepted. The terminal disclaimer has been recorded.
Response to Amendment
1. The provisional nonstatutory double patenting rejection over claims 30-48 of copending Application No. 16/906,580; or claims 60-78 of copending Application No. 16/938,110 (reference application) was withdrawn in light of the Terminal Disclaimer filed on 09/12/2025.
2. The nonstatutory double patenting rejection based on claims of US Patent 10,876,100; 10,494,621; or 10,696,986 was also withdrawn in light of in light of the Terminal Disclaimer filed on 03/08/2024.
3. The nonstatutory double patenting rejection based on claims of U.S. Patent No. 10,377,998; 10,851,357; or 11,578,312 was also withdrawn because each of these US Patents has a later priority date than that of the present application; and importantly claims of each of these US Patents recite “features” that are not supported by the present application.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of pre-AIA 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 –
(e) the invention was described in (1) an application for patent, published under section 122(b), by another filed in the United States before the invention by the applicant for patent or (2) a patent granted on an application for patent by another filed in the United States before the invention by the applicant for patent, except that an international application filed under the treaty defined in section 351(a) shall have the effects for purposes of this subsection of an application filed in the United States only if the international application designated the United States and was published under Article 21(2) of such treaty in the English language.
Claims 1, 10, 17, 47-55 and 57-60 are rejected under pre-AIA 35 U.S.C. 102(e) as being anticipated by Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256). This is a modified rejection.
The instant claims are directed to an engineered composition comprising: a Cas9 protein linked or fused to one or more nuclear localization signals (NLSs), or a polynucleotide encoding the Cas9 protein linked or fused to one or more NLSs, wherein the polynucleotide is codon-optimized for expression in a eukaryotic cell, and an RNA comprising a first sequence comprising an engineered guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a nucleus of the eukaryotic cell, linked to a tracr mate sequence, and a second sequence comprising a sequence capable of hybridizing to the tracr mate sequence to form a hybridized RNA.
Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Accordingly, the teachings of Chen et al meet every limitation of the instant claims. Therefore, the reference anticipates the instant claims.
Claims 1, 47-53, 55, 57 and 59 are rejected under pre-AIA 35 U.S.C. 102(e) as being anticipated by Kim et al (US 2023/0374524 with an effective filing date of 10/23/2012 for the provisional application 61/717,324). This is a modified rejection.
Kim et al already taught at least a composition for cleaving a target DNA in eukaryotic cells (e.g., human embryonic kidney (HEK) 293T cells) comprising a chimeric guide RNA (a fusion of crRNA and tracr-RNA) and a Cas9 derived from Streptoccoccus pyogenes, wherein the Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or chimeric guide RNA and the trinucleotide 5’-NGG-3’ protospacer adjacent motif (PAM) recognized by Cas9 itself (Abstract; paragraphs [0013], [0026], [0074], [0087], [0089]-[0090], [0094], [0112], [0129]; Fig. 1A; and claims 58-69). Kim et al also disclosed that the chimeric guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Fig. 1A); and the Cas9 has a peptide tag comprising the sequence NH2-GGSGPPKKKRKVYPYDVPDYA-COOH containing the HA epitope and a nuclear localization at the C-terminus of Cas9 (paragraph [0164]).
Accordingly, the teachings of Kim et al meet every limitation of the instant claims. Therefore, the reference anticipates the instant claims.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 1 and 56 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256).
Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Although Chen et al did not teach explicitly that the Cas9 protein is flanked on each end by one or more NLSs linked or fused to the N-terminus and one or more NLSs linked or fused to C-terminus of the Cas9 protein, it would have been obvious for an ordinary skilled artisan to also incorporate at least one NLS at both the N-terminus and C-terminus of the Cas9 protein to facilitate or enhance transport of the Cas9 protein into the nuclei of eukaryotic cells with a reasonable expectation of success. This is because since Chen et al already taught explicitly that the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells, including the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or an internal location of the endonuclease; and this teaching indicates or suggests that it is unimportant whether the at least one NLS is located at the N-terminus or C-terminus of the Cas9 protein to retain the functionality of the at least one NLS to promote entry of Cas9 into the nuclei of eukaryotic cells.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 1 and 61-62 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,251) in view of Doudna et al (US 2014/0068797; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008).
Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Chen et al did not teach specifically the use of Streptococcus thermophilus LMD-9 CRISPR1 Cas9 along with its recognition PAM comprising the sequence NNAGAAW.
At about the effective filing date of the present application (12/2/2012), Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus (e.g., LMD-9, YP_820832.1); L. innocua, Campylobacter jejuni and N. meningitidis can be used (at least paragraphs [0595], [0596]; and Fig. 8).
Additionally, Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus, including Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease (at least Abstract; page 1410, right column, first full paragraph; and Figs. 5 and 7).
Accordingly, at the effective filing date of the present application it would have been obvious and within the scope of skill for an ordinary artisan to modify the teachings of Chen et al by also utilizing Streptococcus thermophilus LMD-9 CRISPR1 Cas9 along with its recognition PAM comprising the sequence NNAGAAW for targeted genome modification of a eukaryotic cell; in light of the teachings of Doudna et al and Horvath et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modification because: (i) Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus such as LMD-9 and YP_820832.1 can also be used; and (ii) Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus, including Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease.
An ordinary skilled artisan would have a reasonable expectation of success to carry out the above modification in light of the teachings of Chen et al, Doudna et al and Horvath et al; coupled with the level of skill for an ordinary skilled artisan in the relevant art.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 1, 10, 17, 54 and 56-62 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Kim et al (US 2023/0374524 with an effective filing date of 10/23/2012 for the provisional application 61/717,324) in view of Inouye et al (US 2014/0294801), Doudna et al (US 2014/0068797 with an effective filing date of 05/25/2012; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008).
Kim et al already taught at least a composition for cleaving a target DNA in eukaryotic cells (e.g., human embryonic kidney (HEK) 293T cells) comprising a chimeric guide RNA (a fusion of crRNA and tracr-RNA) and a Cas9 derived from Streptoccoccus pyogenes, wherein the Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or chimeric guide RNA and the trinucleotide 5’-NGG-3’ protospacer adjacent motif (PAM) recognized by Cas9 itself (Abstract; paragraphs [0013], [0026], [0074], [0087], [0089]-[0090], [0094], [0112], [0129]; Fig. 1A; and claims 58-69). Kim et al also disclosed that the chimeric guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Fig. 1A); and the Cas9 has a peptide tag comprising the sequence NH2-GGSGPPKKKRKVYPYDVPDYA-COOH containing the HA epitope and a nuclear localization at the C-terminus of Cas9 (paragraph [0164]).
Kim et al did not teach that the Cas9 contains two or more peptide tags containing the PKKKRKV nuclear localization sequence at the C-terminus and/or at the N-terminus of Cas9; the use of a Streptococcus thermophilus LMD-9 Cas9 along with its recognition PAM having the sequence NNAGAAW; and/or the engineered composition comprising two or more RNAs, each with an engineered sequence capable of hybridizing to a different target sequence in the eukaryotic cell.
At about the effective filing date of the present application (12/12/2012), Inouye et al already taught an mRNA interferase polypeptide/protein having an activity of cleaving an RNA sequence having the target sequence of UUACUCA, and to facilitate the delivery of the polypeptide to a cell and/or through the cell membrane and into the cytosol or nucleus of the cell the polypeptide is fused to one or more of an NLS, CPP, and/or other domains (see at least Abstract; Summary of the Invention; particularly paragraphs [0005]-[0006], [0011] and [0029]).
Additionally, Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus (e.g., LMD-9, YP_820832.1); L. innocua, Campylobacter jejuni and N. meningitidis can be used (at least paragraphs [0595], [0596]; and Fig. 8). Doudna et al also taught that multiple DNA-targeting RNAs are used simultaneously in the same cell, wherein the DNA-targeting RNAs target at different locations on the same target DNA or on different target DNAs (paragraph [0444]).
Moreover, Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus, including Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease (at least Abstract; page 1410, right column, first full paragraph; and Figs. 5 and 7).
Accordingly, at the effective filing date of the present application it would have been obvious and within the scope of skill for an ordinary artisan to modify the teachings of Kim et al by also at least linking or fusing at least two or more peptide tags containing the PKKKRKV nuclear localization sequence at the C-terminus and/or at the T-terminus of Cas9 to facilitate or enhance its delivery to the nucleus of a eukaryotic cell for cleaving a target DNA, as well as utilizing a Streptococccus thermophilus LMD-9 Cas9 molecule with its recognition PAM comprising the sequence NNAGAAW, and two or more gRNAs targeting different target sequences for RNA-directed cleavage of a target DNA molecule in a eukaryotic cell; in light of the teachings of Inouye et al, Doudna et al and Horvath et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because: (i) Inouye et al already taught fusing one or more an NLS, CPP, and/or other domains at any location to a sequence-specific mRNA interferase polypeptide/protein to facilitate the delivery of the polypeptide to a cell and/or through the cell membrane and into the cytosol or nucleus of the cell; (ii) Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus such as LMD-9 and YP_820832.1 can also be used. Additionally, Doudna et al already taught that multiple DNA-targeting RNAs are used simultaneously in the same cell, wherein the DNA-targeting RNAs target at different locations on the same target DNA or on different target DNAs; and (iii) Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus, including Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease.
An ordinary skilled artisan would have a reasonable expectation of success to carry out the above modification in light of the teachings of Kim et al, Inouye et al, Doudna et al and Horvath et al; coupled with the level of skill for an ordinary skilled artisan in the relevant art.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments related to the above modified 102(e) and 103(a) rejections in the Amendment filed on 09/12/2025 (pages 6-7) have been fully considered, but they are respectfully not found persuasive for the reason discussed below.
Applicant argued that the Chen reference (US 2016/0017366) was abandoned in 2016 and so could not have been and cannot be subjected to an interference; whereas the Office issued the Kim reference (US 2015/0322457) into US Pat. No. 10,851,380 on December 01, 2020 which is after the Office declared Patent Interference No. 106,115 between the Broad Institute et al and the Regents of The University of California et al. Since the Office allowed the Kim reference to issue into the ‘380 patent, rather than having the Kim application become part of the ‘115 Interference, the Office effectively decided that the claims of the ‘380 patent are patentably distinct from the subject matter of the ‘115 Interference. Using this rationale, the Office should not subject the claims of the ‘380 patent and the claims of the present application to an interference proceeding. Thus, it is unnecessary for Applicants to suggest an interference against Chen or Kim; and the 37.C.F.R. 1.131 Declaration filed on 03/08/2024 should be deemed to be sufficient to overcome the 102(e) and 103(a) rejections of record over Chen and Kim. Additionally, with respect to amended claim 61 and new claim 62, Applicant argued that none of the previously cited art teach or suggest “the Cas9 protein is Streptococcus thermophilus LMD-9 CRISPR1 Cas9” and “the PAM comprises the sequence NNAGAAW”.
First, according to MPEP 2136.05(a), when the claims of the reference U.S. patent or U.S. patent application publication and the application are directed to the same invention or are obvious variants, an affidavit or declaration under 37 CFR 1.131(a) is not an acceptable method of overcoming the rejection. Please refer to the above modified rejections with Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256) and Kim et al (US 2023/0374524 with an effective filing date of 10/23/2012 for the provisional application 61/717,324). Claims in both of these US patent application publications are directed to the same invention or obvious variants of the presently claimed invention.
Second, with respect to the new limitations recited in claims 61-62 please refer to the supplemental teachings of Doudna et al (US 2014/0068797 with an effective filing date of 05/25/2012; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008) in the above modified 103(a) rejections.
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).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 1, 10, 47-50 and 57-60 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 2-14 and 17-21 of copending Application No. 19/028,841 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because an engineered CRISPR-Cas system comprising the elements (a)-(b) recited in independent claim 2, 20, or 21 in the copending Application No. 19/028,841 anticipates the claimed genus in the application being examined and, therefore, a patent to the genus would, necessarily, extend the rights of the species or sub- should the genus issue as a patent after the species of sub-genus.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1, 17, 51-52, 54-56 and 61-62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 2-14 and 17-21 of copending Application No. 19/028,841 in view of Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256), Doudna et al (US 2014/0068797; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008).
The claims of the present application differ from claims 2-14 and 17-21 of copending Application No. 19/028,841 in reciting specifically at least anyone of the following features: the engineered composition comprising two or more RNAs each with an engineered sequence capable of hybridizing to a different target sequence in a eukaryotic cell; the eukaryotic cell is a human cell; the one or more is linked or fused to N-terminus and/or C-terminus of the Cas9 protein; and the Cas9 protein is Streptococcus thermophilus LMD-9 CRISPR1 Cas9 and the PAM comprises the sequence NNAGAAW.
At about the effective filing date of the present application (12/12/2012), Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Additionally, Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus (e.g., LMD-9, YP_820832.1); L. innocua, Campylobacter jejuni and N. meningitidis can be used (at least paragraphs [0595], [0596]; and Fig. 8).
Moreover, Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease (at least Abstract; page 1410, right column, first full paragraph; and Figs. 5 and 7).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify an engineered CRISPR-Cas system in claims 2-14 and 17-21 of copending Application No. 19/028,841 to have the recited features of the presently claimed engineered composition in light of the teachings of Chen et al, Doudna et al and Horvath et al as set forth above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the modifications because: (i) Chen et al already taught the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences; targeted genome modification of a eukaryotic cell such as human 562 cells; and an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or in an internal location of the endonuclease); (ii) Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus such as LMD-9 and YP_820832.1 can also be used; and (iii) Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease. Moreover, it would have been obvious for an ordinary skilled artisan to also incorporate at least one NLS at both the N-terminus and C-terminus of the Cas9 protein to facilitate or enhance transport of the Cas9 protein into the nuclei of eukaryotic cells with a reasonable expectation of success. This is because since Chen et al already taught explicitly that the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells, including the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or an internal location of the endonuclease; and this teaching indicates or suggests that it is unimportant whether the at least one NLS is located at the N-terminus or C-terminus of the Cas9 protein to retain the functionality of the at least one NLS to promote entry of Cas9 into the nuclei of eukaryotic cells.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 10, 17 and 47-62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 2-15 of copending Application No. 19/025,692 in view of Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256), Doudna et al (US 2014/0068797; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008).
The claims of the present application differ from claims 2-15 of copending Application No. 19/025,692 in reciting specifically a Cas9 protein linked or fused to one or more nuclear localization signals (NLSs) in addition to an RNA having the elements recited in independent claim 1 of the present application; the engineered composition comprising two or more RNAs each with an engineered sequence capable of hybridizing to a different target sequence in a eukaryotic cell; the eukaryotic cell is a human cell; the one or more is linked or fused to N-terminus and/or C-terminus of the Cas9 protein; and the Cas9 protein is Streptococcus thermophilus LMD-9 CRISPR1 Cas9 and the PAM comprises the sequence NNAGAAW.
At about the effective filing date of the present application (12/12/2012), Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Additionally, Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus (e.g., LMD-9, YP_820832.1); L. innocua, Campylobacter jejuni and N. meningitidis can be used (at least paragraphs [0595], [0596]; and Fig. 8).
Moreover, Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease (at least Abstract; page 1410, right column, first full paragraph; and Figs. 5 and 7).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify an engineered CRISPR-Cas system chimeric RNA in claims 2-15 of copending Application No. 19/025,692 to further include a Cas9 protein linked or fused to one or more NLSs as well as other recited features of the presently claimed engineered composition for targeted genome modification of a eukaryotic cell that includes a human cell; in light of the teachings of Chen et al, Doudna et al and Horvath et al as set forth above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the modifications because: (i) Chen et al already taught the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences; targeted genome modification of a eukaryotic cell such as human 562 cells; and an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or in an internal location of the endonuclease); (ii) Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus such as LMD-9 and YP_820832.1 can also be used; and (iii) Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease. Moreover, it would have been obvious for an ordinary skilled artisan to also incorporate at least one NLS at both the N-terminus and C-terminus of the Cas9 protein to facilitate or enhance transport of the Cas9 protein into the nuclei of eukaryotic cells with a reasonable expectation of success. This is because since Chen et al already taught explicitly that the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells, including the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or an internal location of the endonuclease; and this teaching indicates or suggests that it is unimportant whether the at least one NLS is located at the N-terminus or C-terminus of the Cas9 protein to retain the functionality of the at least one NLS to promote entry of Cas9 into the nuclei of eukaryotic cells.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 10, 17, 47-49 and 51 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 208-223 and 226-229 of copending Application No. 18/106,039; or claims 41-44 and 50-60 of copending Application No. 18/107,108 (reference application) for the same reasons already set forth in the Final Office Action dated 11/09/2023 (pages 18-19).
Although the claims at issue are not identical, they are not patentably distinct from each other because an engineered CRISPR-Cas composition for modifying a genomic locus of interest in a eukaryotic cell in claims 208-223 and 226-229 of copending Application No. 18/106,039, or an engineered CRISPR-Cas system for in vivo genome editing in a multicellular organism in claims 41-44 and 50-60 of copending Application No. 18/107,108 anticipates/encompasses the claimed genus in the application being examined and, therefore, a patent to the genus would, necessarily, extend the rights of the species or sub- should the genus issue as a patent after the species of sub-genus.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1, 10, 17 and 47-62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 208-223 and 226-229 of copending Application No. 18/106,039; or claims 41-44 and 50-60 of copending Application No. 18/107,108 (reference application) in view of Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012 for the provisional application 61/734,256), Doudna et al (US 2014/0068797; IDS) and Horvath et al (J. Bacteriology 190:1401-1412, 2008).
The claims of the present application differ from claims 208-223 and 226-229 of copending Application No. 18/106,039; or claims 41-44 and 50-60 of copending Application No. 18/107,108 in reciting specifically at least that the one or more NLSs (e.g., SV40 large T-antigen NLS comprising the sequence of SEQ ID NO: 30) is linked or fused to N-terminus and/or C-terminus of the Cas9 protein; the tracr-mate sequence comprising the sequence of SEQ ID NO: 600; and the Cas9 is Streptococcus thermophilus LMD-9 CRISPR1 Cas9 along with its recognized PAM sequence of NNAGAAW.
At about the effective filing date of the present application (12/12/2012), Chen et al already taught at least an isolated RNA-guided endonuclease (e.g., Cas9 protein derived from Streptococcus pyogenes or Streptococcus thermophilus), wherein the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells (e.g., the NLS of PKKKRKV to be located at the N-terminus or C-terminus of the endonuclease), at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence for targeted genome modification of a eukaryotic cell (e.g., human 562 cells); and wherein the endonuclease can be part of a protein-RNA complex comprising the guide RNA which can be a single molecule comprising a 5’ region that is complementary to a target site, a second region that forms a secondary structure comprising a stem (or hairpin) and a loop, and a third region at the 3’ end that remains essentially single-stranded (Abstract; Summary of the Invention; paragraphs [0006], [0016], [0018]-[0021], [0025], [0081]-[0088], and [0121]; and claims 1-21). An ordinary skilled in the art would readily recognize that at least one nuclear localization signal is meant to be one, two or more nuclear localization signals. Chen et al also taught specifically that the guide RNA comprises the sequence 5’-GUUUUAGAGCUA-3’ in crRNA/tracr-mate sequence (Table 4); the target sequence has no sequence limitation except that the sequence is immediately followed (downstream) by a PAM sequence that includes NGG and NGGNG (paragraph [0094]); and the use of an RNA-guided endonuclease with two different guide RNAs to cleave different target nucleic acid sequences (paragraphs [0016],[0073], [0075]-[0076]; support in the provisional application 61/734,256 can be found in paragraph [0004]).
Additionally, Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus (e.g., LMD-9, YP_820832.1); L. innocua, Campylobacter jejuni and N. meningitidis can be used (at least paragraphs [0595], [0596]; and Fig. 8).
Moreover, Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease (at least Abstract; page 1410, right column, first full paragraph; and Figs. 5 and 7).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify the engineered composition in claims 208-223 and 226-229 of copending Application No. 18/106,039; or claims 41-44 and 50-60 of copending Application No. 18/107,108 to have the recited features of the presently claimed engineered composition in light of the teachings of Chen et al, Doudna et al and Horvath et al as set forth above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the modifications because: (i) Chen et al taught explicit some of the features that are not recited explicitly in claims of the aforementioned copending Applications for targeted genome modification of a eukaryotic cell; (ii) Doudna et al already disclosed compositions for RNA-directed target DNA modification, and taught at least that apart from S. pyogenes SF370 Cas9, Cas9 orthologs from Streptoccoccus thermophilus such as LMD-9 and YP_820832.1 can also be used; and (iii) Horvath et al already analyzed diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus LMD-9; and disclosed that for CRISPR1, the AGAAW CRISPR motif located two nucleotides down-stream of the proto-spacer might serve as a recognition site for a CRISPR1-specific Cas nuclease. Moreover, it would have been obvious for an ordinary skilled artisan to also incorporate at least one NLS at both the N-terminus and C-terminus of the Cas9 protein to facilitate or enhance transport of the Cas9 protein into the nuclei of eukaryotic cells with a reasonable expectation of success. This is because since Chen et al already taught explicitly that the endonuclease comprises at least one nuclear localization signal (NLS) which permits the endonuclease into the nuclei of eukaryotic cells, including the NLS of PKKKRKV to be located at the N-terminus, the C-terminus, or an internal location of the endonuclease; and this teaching indicates or suggests that it is unimportant whether the at least one NLS is located at the N-terminus or C-terminus of the Cas9 protein to retain the functionality of the at least one NLS to promote entry of Cas9 into the nuclei of eukaryotic cells.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1, 10, 17 and 47-62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-17 and 35-45 of copending Application No. 17/081,387; claims 1-7, 13, 18-22 and 24-25 of copending Application No. 17/123,918; claims 1, 7-10, 12-16 and 41-43 of copending Application No. 17/612,504; claims 1-14 of co-pending Application No. 17/910,497; claims 31-42 and 44-50 of copending Application No. 17/831,745; claims 1-2, 6, 8-9, 11, 14-16, 27, 29-31, 34-35, 37-38, 43-43 and 56 of copending Application No. 17/776,269; claims 1-3, 8, 10, 12, 14, 17, 19-20, 22-23, 25, 28, 31, 33, 35 and 43 of copending Application No. 17/793,115; or claims 14-16, 18, 21-25 and 27-28 of copending Application No. 18/345,935 in view of Chen et al (US 2021/0388396 with an effective filing date of 12/06/2012