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 .
Response to Arguments
Applicant’s arguments, see pages 6-8, filed 3/23/26, with respect to the rejection(s) of claim(s) 1-3, 5, 9-11, 13-19 and 21-22 under 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendment to the independent claims to require that the one or more endonuclease recognition sites are recognized and cleaved by at least one endonuclease.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 2, 9, 10, 13-16, and 21 are rejected under 35 U.S.C. 103 as being unpatentable over May (US 20150376586, of record) taken with Addgene blog plasmids 101, restriction cloning, pages 1-15, 2/18/16 (Addgene).
May et al. make and use an CRISPR-Cas complex (engineered nucleoprotein complex) comprising a single guide RNA and Cas to reduce off-target activity of the complex. See pages 11-27 and 75-76. May et al. further disclose: A composition comprising an engineered nucleoprotein complex, wherein said engineered nucleoprotein complex comprises: (a) a Cas9 polypeptide; and (b) a engineered nucleic acid-targeting nucleic acid wherein said engineered nucleic acid-targeting nucleic acid comprises an engineered region selected from the group consisting of: an engineered stem loop duplex structure, an engineered bulge region, an engineered hairpin located 3' of the stem loop duplex structure, and any combination thereof; wherein said engineered nucleoprotein complex results in a modification of a target region of a genomic DNA; wherein said engineered nucleoprotein complex has a decreased ability to modify a DNA molecule in a region that is not said target region as compared to a control nucleoprotein complex that does not comprise an engineered stem loop duplex structure, an engineered bulge region, and an engineered hairpin located 3' of the stem loop duplex structure (for example, see claim 1). A spacer segment of a nucleic acid-targeting nucleic acid can be modified to hybridize to any desired sequence within a target nucleic acid. For example, the sequence within the target nucleic acid is involved in cancer, cell growth, DNA replication, DNA repair, HLA genes, cell surface proteins, T-cell receptors, immunoglobulin superfamily genes, tumor suppressor genes, microRNA genes, long non-coding RNA genes, transcription factors, globins, viral proteins, mitochondrial genes (paragraph 191). A nucleic acid affinity tag can comprise a ribozyme sequence (paragraph 44). Suitable ribozymes can include peptidyl transferase 23S rRNA, RNaseP, Group I introns, Group II introns, GIR1 branching ribozyme, leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3 ribozymes, VS ribozymes, glmS ribozyme, CoTC ribozyme, and synthetic ribozymes (paragraph 44).
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The spacer sequence 140 can be a variable portion of the nucleic acid-targeting nucleic acid. The spacer sequence 140 can be 5' of a first duplex 145. The first duplex 145 comprises a region of hybridization between a minimum CRISPR repeat 146 and minimum tracrRNA sequence 147. 146 comprises the crRNA and the tracrRNA. The crRNA is responsible for binding to the target-specific DNA region and the tracrRNA is responsible for the activation of the Cas endonuclease. The first duplex 145 can be interrupted by a bulge 150. The bulge 150 can comprise unpaired nucleotides. The bulge 150 can facilitate the recruitment of a site-directed polypeptide to the nucleic acid- targeting nucleic acid. The bulge 150 can be followed by a first stem 155. The first stem 155 comprises a linker sequence linking the minimum CRISPR repeat 146 and the
minimum tracrRNA sequence 147. The last paired nucleotide at the 3' end of the firs duplex 145 can be connected to a second linker sequence 160. The second linker 160 can comprise a nexus. The second linker 160 can link the first duplex 145 to a mid-tracrRNA 165. The mid-tracrRNA 165 can, in some embodiments, comprise one or more hairpin regions. For example, the mid-tracrRNA 165 can comprise a second stem 170 and a third stem 175. See paragraph 174. The stem loop structure or a linker of the sgRNA can comprise an aptamer, a ribozyme, a protein-interacting hairpin, a CRISPR array, an intron or an exon (paragraphs 217-236). The sgRNA can have a tracrRNA extension sequence can comprise a moiety (e.g., stability control sequence, ribozyme, endoribonuclease binding sequence). The extension sequence can include a riboswitch sequence to allow for regulated stability and/or regulated accessibility by proteins and protein complexes.
May et al. teaches or suggests a sgRNA comprising a spacer sequence, a lower and upper stem, a bulge, a nexus and hairpins and inserting a ribozyme into a stem loop, linker or a tracrRNA extension sequence of the sgRNA, but does not specifically teach a sgRNA comprising an inactivation sequence (e.g., ribozyme) inserted into the lower or upper stem or hairpins, wherein the inactivation sequence is flanked by one or more endonuclease recognition sequences.
Paragraphs 70-71 of the as-filed specification provide a definition for the term 'inactivation sequence'. The term refers to a nucleic acid sequence that causes the sgRNA to be inactive via (i) steric hindrance of Cas binding through bulky structures that, e.g., disrupt sgRNA folding, intrinsic RNA structures or recruited protein binding; (ii) cis- or trans-ribozyme insertion; or (iii) cellular (endogenous or exogenous) RNase targeting a Cas or specific nuclease to the DNA of each inactivation sequence. The broadest reasonable interpretation of the term embraces any nucleotide sequence that requires another molecule to be present to activate the sequence. e.g., ribozyme, aptamer, endonuclease binding site.
The limitation "activatable" in the pre-amble of the instant claims does not add any structural limitations to the claimed sgRNA because it recites a purpose or an intended use or could be an inherent function of the sgRNA having the recited structural limitations.
The as-filed specification in paragraph 70 defines the term 'endonuclease recognition sites':
a nucleic acid sequence or structural motif recognized by an endonuclease, which cleaves the nucleic acid at or near the structural motif.
While May suggests that a moiety can be an endoribonuclease binding sequence, May does not specifically teach flanking a cis-acting ribozyme with endonuclease recognition sites that are recognized and cleaved by at least one endonuclease.
In view of the broadest reasonable interpretation (BRI) of the claimed invention, the claimed invention embraces a DNA comprising the CRISPR/Cas system or sgRNA because the term ‘endonuclease’ reads on nuclease that cleave DNA or RNA. Furthermore, instant claim 19 embraces a nucleic acid (DNA or RNA) comprising the sgRNA inserted into the 5’ or 3 UTR region. Also, see page 26 of the as-filed specification. Claim 19 indicates that the instant claims could embrace a DNA or RNA sequence. Also the claimed invention embraces endonuclease recognition sites that are cleaved and recognized by Cas9 (instant claim 22) and Cas9 is known to cut DNA sequences not RNA sequences.
In view of the BRI, adding restriction sites to a desired nucleotides to a make a construct was well known to one of ordinary skill in the art as taught by Addgene (pages 1-15). See also MPEP 2141 II.C. Rationales to support rejections under 35 U.S.C. 103 recites, “Prior art is not limited to the references being applied, but includes the understanding of one of ordinary skill in the art.”
It would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date to combine the teaching of May taken with Addgene to flank the inactivation sequence in the hairpin or stem of the complex with one or more restriction enzymes that are recognized and cleaved by at least one endonuclease to make a DNA construct for in vitro transcription or cloning purposes, namely to arrive at the claimed invention. In addition, it would have been obvious to add an additional endonuclease binding site as taught by May and Addgene to fine-tune using the complex to edit or modulate transcription of a genome in a cell or to use additional endonuclease for substituting different ribozymes.
Furthermore, it would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date to insert a ribozyme in the upper stem or hairpin of the sgRNA to increase stability control of the CRISPR-Cas complex as taught by May, namely to arrive at the claimed invention. A person of ordinary skill in the art would have been motivated to use the complex to modify transcription of a genome in a cell to reduce off-target effects of the complex in the cell as taught by May (pages 2 and 5). May further teaches a genomic DNA comprising the complex (page 2). A vector comprising a promoter can comprise the complex, the sgRNA or the Cas polypeptide (paragraphs 250-254). May teaches making different sgRNA target different genes in a cell lines (pages 38-40). May et al. teach making and using two or more sgRNA in the complex to control modification or transcription of another target sequence. In view of the working examples of May, it would have been obvious to study the off-target effect of two different genes in a cell. In addition, one of ordinary could use two different gRNAs, wherein one targets the sense strand and the other targets the antisense strand to induce a double stranded break and improve efficiency of editing a genome in the cell.
The 'wherein' clause recited in instant claims 2 and 10 set forth a function of the sgRNA without limiting the structure of the sgRNA, so absent evidence to the contrary, any prior art that makes obvious the structure of the sgRNA in claims 1 and 9 meets the structural limitations of the claim 2 and 10 and thus would necessarily have the same functions.
It would have been obvious to target a gene as taught by May or a mRNA encoded by the gene to modify expression of a protein. See also MPEP 2141(II)(C): One of ordinary skill in the art would have been motivated to combine the teaching to use a ribozyme selected from peptidyl transferase 23S rRNA, RNaseP, Group I introns, Group II introns, GIR1 branching ribozyme, leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3 ribozymes, VS ribozymes, glmS ribozyme, CoTC ribozyme, and synthetic ribozymes as taught by May (paragraph 44). See also MPEP 2143(I)B and E.
Addgene teaches to place a sequence in the proper orientation downstream of a promoter in a construct, you can add an EcoRI site just 5’ of the start of the gene and a HindIII site just 3’ of the end of the sequence. This way you can then cut the construct as well as the insert with EcoRI and HindIII and, when you mix the cut products together, the two EcoRI digested ends will anneal and the two HindIII digested ends will anneal leaving the 5’ end of your sequence just downstream of the promoter and placing the sequence in the proper orientation. It would have been a simple substitution to make a sgRNA have two endonuclease recognition sites, each of which is recognized and cleaved by different endonuclease to insert or removed a cis-acting ribozyme.
Therefore, the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Claims 3 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over May et al. ('586) and Addgene as applied to claims 1, 2, 9, 10, 13-16, and 21 above, and further in view of Lu et al. (US 20050257277).
May et al. teach using a ribozymes selected from: peptidyl transferase 23S rRNA, RNaseP, Group I introns, Group II introns, GIR1 branching ribozyme, leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3 ribozymes, vs ribozymes, glmS ribozyme, CoTC ribozyme, and synthetic ribozymes (paragraph 44) in the sgRNA of the CRISPR-Cas complex.
May et al. and Addgene do not specifically teach wherein the inactivation sequence is a cis-acting ribozyme encoded by a nucleic acid molecule of SEQ ID NO: 4.
However, Lu et al. teach a cis-acting ribozyme comprising SEQ ID NO: 4 recited in claims 4 and 16. Pages 5-6, 8-9, and 13-17 and SEQ ID NO: 1. Lu teaches that cleavage sites of ribozymes are well known in the art (page 6). A person of ordinary skill in the art would easily be able to choose the ribozyme and insert it into a nucleotide sequence which produces an RNA transcript including the ribozyme (page 6).
It would have been prima facie obvious to a person of ordinary skill in the art before the time the effective filing date to combine the teaching of May and Addgene taken with Lu to make the system have a nucleotide comprising a cis-acting ribozyme comprising SEQ ID NO: 4 as the inactivation sequence, namely to arrive at the claimed invention. It would have been a simple substitution with a reasonable expectation for success for a person of ordinary skill in the art to replace with a nucleic acid sequence at the tetraloop with a sequence comprising a cis-acting ribozyme as the inactivation sequence and upon presence of a RNA guided endonuclease, the ribozyme undergoes structural changes that activates the ribozyme triggering cleavage of the gRNA. See MPEP 2143(I)B. One of ordinary skill in the art would have been motivated to combine the teaching to study the function (e.g., efficiency or control of gene editing) of the CRISPR/Cas system in a cell line.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over May et al. ('586) and Addgene as applied to claims 1, 2, 9, 10, 13-16, and 21 above, and further in view of Cong et al. (Science 2013, 339(6121) 819-823, cited on an IDS).
May et al. teach making several sgRNA (pages 38-40).
However, May et al. and Addgene do not specifically teach an array of sgRNA, wherein one sgRNA targets another sgRNA in the array.
Cong teaches an array of sgRNAs to enable simultaneous editing of several sites within the mammalian genome. "Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology (abstract)".
It would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date to combine the teaching of May and Addgene taken with Cong to make a nucleic acid construct comprise an array of sgRNAs, wherein one sgRNA targets another sgRNA in the array, namely to arrive at the claimed invention. Since May and Addgene make obvious the activatable sgRNAs, it would have been obvious to one of ordinary skill in the art to make an array of activatable sgRNAs that could be designed according to Cong where the cognate sgRNA targets at least one activatable sgRNA in the array of activatable sgRNAs, to enable multiple sequences to be targeted conditionally.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Claims 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over May et al. ('586) and Addgene as applied to claims 1, 2, 9, 10, 13-16, and 21 above, and further in view of Intellia Therapeutics, Inc. (WO 2017160752, published 9/21/17 and priority to 3/14/16, cited on an IDS).
May et al. and Addgene do not specifically teach a nucleic acid comprising a coding sequence, a 5' UTR region and a 3' UTR region; and the activatable sgRNA of claim 9 is inserted into either the 5' or 3' UTR region.
The 'wherein' clause sets in instant claim 18 set forth embraces a function of the sgRNA without limiting the structure of the sgRNA, thus absent evidence to the contrary, any prior art that makes obvious the structure of the sgRNA and the method steps used in claims 13 meets the structural limitations of the claim 18 and thus would necessarily result in the sgRNA being inserted into the 3' or 5' UTR of a gene. See MPEP 2112.02.
May et al. teach inserting a sgRNA into a genomic DNA sequence (page 2), but does not specifically teach inserting the sgRNA into a 5' UTR or a 3' UTR, however, when one of ordinary skill in the art carries the method steps made obvious by May, Addgene and ‘752 they would arrive at the claimed product in claim 19. In addition, '752 teaches inserting a guide RNA in the 5' UTR or 3' UTR of a Cas9 protein transcript (pages 34-35). '752 teaches in some embodiments, the intracellular half-life of the Cas9 protein transcript may be reduced by containing the guide RNA within its 3' UTR and thereby shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be within an intron of the Cas9 protein transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the Cas9 protein and the guide RNA in close proximity on the same vector may facilitate more efficient formation of the CRISPR complex.
'752 further teaches a DNA vector encoding a CRISPR-Cas9 system that cleaves a target sequence on a target nucleic acid molecule, the system comprises a Cas9 protein and a guide RNA, wherein a first vector has the Cas9 protein operably linked to a first promoter and as second vector encoding the gRNA operably linked to a second promoter, the target sequence reduces expression of the Cas9 protein or the gRNA (for example. see pages 3-14, 19-44 and 69-77).
It would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date to combine the teaching of May and Addgene taken with '752 to the insert the sgRNA into either the 5' or 3' UTR region of a nucleic acid, namely to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to combine the teaching to observe expression of gRNA and Cas9 protein the by the same promoter. In addition, one of ordinary skill in the art would have been motivated as simple substitution (see MPEP 2143(I)B) to use the sgRNA made obvious by May that targets a nucleic acid in a cell in the DNA vector taught by '752, wherein the target sequence encodes a second sgRNA which targets a promoter in the genomic sequence of the cells to fine-tune regulation of editing or modulating transcription of a genome of a certain cell type in a population of cells.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Allowable Subject Matter
Claims 4, 12 and 22 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The prior art does not teach or suggest the SEQ ID NOs: for the activatable sgRNA set forth in instant claims 4 and 14. See the non-final rejection mailed on 7/7/25 for closest prior art to these claims.
Even though claim 22 contains a ‘wherein’ clause, the clause appears to set forth an additional structure and function of the sgRNA and further limits the structure of the sgRNA. The prior art of record would not inherently have the function of the product in claim 22 because the prior art does not teach or suggest adding one or more recognition for Cas9 to recognize and cleave. Page 48 of the specification shows that Cas9 recognition sites flanking or internal to the ribozyme sequence could convert activatable sgRNA into a function sgRNA through mutagenesis of the ribozyme (FIG. 6).
Conclusion
See attached PTO-326 for disposition of claims.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Brian Whiteman whose telephone number is (571)272-0764. The examiner can normally be reached on Monday thru Friday; 6:00 AM to 3:00PM.
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/BRIAN WHITEMAN/ Primary Examiner, Art Unit 1636
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