DETAILED ACTION
The examiner prosecuting this application has changed.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
This application is a 371 of PCT/US20/65696 12/17/2020 which claims benefit of 62/949,433 12/17/2019. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(a) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 62/949,433 fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Application No. 62/949,433 does not support the DNA ligase selected from T4 ligase, T7 ligase, and T3 ligase in claim 8 in the instant application. Claim 8 has an effective filing date of 12/17/2020.
Status of Application/Amendment/Claims
This Office action is in response to the communications filed on August 14, 2025.
Currently, claims 1-20 are pending in the instant application.
Claims 1 and 14 are amended.
Claims 15-20 are newly added. Applicant’s statement that “support for the claim amendments and new claims can be found throughout the application, as filed, for example, at paragraphs [0020], [0043], [0054], [0067], [0080], [0086], [0117], Figures 1-5, 12, and 15-17, and the original claims as filed,” and that “no new matter has been added,” is acknowledged.
The examiner finds support for all limitation in all currently pending claims. Accordingly, claims 1-20 are under examination on the merits in the instant application.
The following rejections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application.
Withdrawn Objections and Rejections
Any objections or rejections not repeated in this Office action are hereby withdrawn.
New Objections/Rejections Necessitated by Amendment
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 and 7-13 are rejected under 35 U.S.C. 103 as being unpatentable over Liu D. et al., (US11447770B1) in view of DeLoache W. et al., (WO2018013990A1), Bier E. et al., (WO2017049266A2) and Lohman G. et. al., (Nucleic Acids Research, Volume 42, Issue 3, Pages 1831–1844, published 06 November 2013).
Liu teaches prime editing: a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit, see abstract and claims. Liu further teaches “because templated DNA synthesis offers single nucleotide precision for the modification of any nucleotide, including insertions and deletions, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.” See column 4, lines 29-33.
Regarding claims 1 and 9, Liu teaches “ A method for site-specific modification of a double-stranded target DNA sequence, the method comprising: contacting the double-stranded target DNA sequence, which comprises a first strand and a second strand, with a prime editing system, wherein the prime editing system comprises:
(i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, wherein the napDNAbp is a Cas9 nickase, a Cas12a nickase, or a Cas12b1 nickase, and
(ii) a prime editing guide RNA (PEgRNA) comprising:
(a) a spacer sequence that comprises a region of complementarity to the first strand of the double-stranded target DNA sequence;
(b) an extension arm that comprises a DNA synthesis template and a primer binding site in a 5′ to 3′ orientation, wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in the second strand of the double-stranded target DNA sequence, and wherein the DNA synthesis template encodes one or more nucleotide changes compared to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence, and
(c) a gRNA core that interacts with the napDNAbp;
wherein the contacting results in:
nicking the second strand of the double-stranded target DNA sequence to form a free 3′ end at the nick site;
annealing the primer binding site with the region of the second strand of the double-stranded target DNA sequence upstream of the nick site;
synthesizing a single strand of DNA encoded by the DNA synthesis template from the free 3′ end of the second strand of the double-stranded target DNA sequence; and
replacing the region downstream of the nick site in the second strand of the double-stranded target DNA sequence with the single strand of DNA, thereby modifying the sequence of the double-stranded target DNA sequence. See claim 1.
Regarding claims 1 and 10, Liu teaches that “once the nick is introduced thereby producing a free 3′ hydroxyl group immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand anneals to a complementary sequence at the 3′ end of the extension arm referred to as the “primer binding site,” creating a short double-stranded region with an available 3′ hydroxyl end, which forms a substrate for the polymerase of the prime editor complex. The polymerase (e.g., reverse transcriptase) then polymerase as strand of DNA from the 3′ hydroxyl end to the end of the extension arm. The sequence of the single stranded DNA is coded for by the DNA synthesis template, which is the portion of the extension arm (i.e., excluding the primer binding site) that is “read” by the polymerase to synthesize new DNA. This polymerization effectively extends the sequence of the original 3′ hydroxyl end of the initial nick site. The DNA synthesis template encodes a single strand of DNA that comprises not only the desired edit, but also regions that are homologous to the endogenous single strand of DNA immediately downstream of the nick site on the PAM strand. Next, the encoded 3′ ended single strand of DNA (i.e., the 3′ single strand DNA flap) displaces the corresponding homologous endogenous 5′-ended single strand of DNA immediately downstream of the nick site on the PAM strand, forming a DNA intermediate having a 5′-ended single strand DNA flap, which is removed by the cell (e.g., by a flap endonuclease). The 3′-ended single strand DNA flap, which anneals to the complement of the endogenous 5′-ended single strand DNA flap, is ligated to the endogenous strand after the 5′ DNA flap is removed. The desired edit in the 3′ ended single strand DNA flap, now annealed and ligate, forms a mismatch with the complement strand, which undergoes DNA repair and/or a round of replication, thereby permanently installing the desired edit on both strands.” See column 16, lns. 35-67 and column 17, lns. 1-21, and Figs. 1A-E and M. More specifically, Liu teaches “the term “3′ replacement DNA flap” or simply, “replacement DNA flap,” refers to the strand of DNA that is synthesized by the prime editor and which is encoded by the extension arm of the prime editor PEgRNA. More in particular, the 3′ replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3′ replacement DNA flap comprises the same sequence as the 5′ endogenous DNA flap except that it also contains the edited sequence (e.g., single nucleotide change). The 3′ replacement DNA flap anneals to the target DNA, displacing or replacing the 5′ endogenous DNA flap (which can be excised, for example, by a 5′ flap endonuclease, such as FEN1 or EXO1) and then is ligated to join the 3′ end of the 3′ replacement DNA flap to the exposed 5′ hydoxyl end of endogenous DNA (exposed after excision of the 5′ endogenous DNA flap, thereby reforming a phosphodiester bond and installing the 3′ replacement DNA flap to form a heteroduplex DNA containing one edited strand and one unedited strand.
Regarding claim 11, Liu teaches that the double-stranded nucleic acid is in a cell. See claims 13-15.
Regarding claim 13, Liu teaches providing a second CRISPR guide RNA that comprises from 5' to 3': a guide sequence that hybridizes with a second nucleic acid sequence in the target strand of the double-stranded nucleic acid that is different from the nucleic acid sequence to which the first CRISPR guide RNA hybridizes. See claims 16-17.
Liu doesn’t teach providing a ligand oligonucleotide comprising a nucleic acid sequence to be incorporated into the double-stranded nucleic acid; a splint oligonucleotide comprising a first nucleic acid sequence that hybridizes with the ligand oligonucleotide and a second nucleic acid sequence that hybridizes with a nucleic acid sequence in the displaced strand of the double-stranded nucleic acid, wherein the splint oligonucleotide spans a ligation junction between one end of the ligand oligonucleotide and the displaced strand of the double-stranded nucleic acid; and a ligase selected from T4 ligase, T7 ligase, T3 ligase, and PBCV-1 DNA Ligase either separate or fused to the CRISPR protein.
DeLoache teaches “fusing a Cpf1 or other CRISPR polypeptide with a polypeptide with ligase activity,” and that "fusion of protein subunits of a complex has been performed on other systems and can be accomplished ... by one skilled in the art with knowledge of the nucleic acid sequences to be fused to the Cas9 or Cpf1," [0123]-[0130]. DeLoache further teaches "enzymatic ligases, include, but are not limited to, bacteriophage T4 ligase, T7 ligase ... Afu ligase," [120]. DeLoache also teaches “methods of gene editing using any targetable DNA nuclease (e.g., Cpf1, Cas9…),” [0102]. DeLoache further teaches that “the systems and methods … can be used with… Cas9 mutants that act as single stranded nickases,” [0112].
Bier discloses an oligonucleotide with complementarity to both the genomic region to be targeted for HDR and to sequences carried on a donor cargo vector such that it should serve as a bridging element (see paragraph [0048]). Bier also discloses an Oligo-Clamp consisting of nucleotides complementary to both the targeted genome sequence and to sequences present on the donor cargo vector such that one end of the Oligo-Clamp sequence forms a hybrid with the genome target DNA and the other portion of the Oligo-Clamp forms a stable hybrid with the donor cargo vector effecting a bridging of the donor cargo to the site of intended HDR-mediated recombination of the vector sequences into the genome (see paragraph [0060]). Bier further discloses that in the presence of an enzymatically active endonuclease (e.g., endonuclease/guide polynucleotide complex such as Cas9/gRNA), the donor cargo vector can be in a configuration to serve as a substrate for homology directed repair (HDR) wherein the nucleic acid cargo sequence can then be efficiently inserted into the nucleic acid target sequence (see paragraph [0418]). Bier’s oligo-clamp oligonucleotide reads on a splint oligonucleotide comprising a first nucleic acid sequence that hybridizes with the ligand oligonucleotide and a second nucleic acid sequence that hybridizes with a nucleic acid sequence in the displaced strand of the double-stranded nucleic acid, as required by claim 1.
Bier further teaches that once an allele is cleaved by the endonuclease (e.g., Cas9/gRNA nuclease), the proximity and long regions of homology can favor homology directed repair (see paragraph [0487]). In contrast, free plasmids carrying homology sequences may not be concentrated at the cleavage site in the nucleus where they are needed to serve as templates (see paragraph [0487]). A tethering method that may increase the probability of HDR being used in cases where the homologous template is aligned with the target sequence (see paragraph [0487]). The donor cargo vector can be tethered to the nucleic acid target sequence via at least one or two bridging bivalent nucleic acid binding proteins (see paragraph [0487]). Complexes of the bivalent nucleic acid binding protein with the donor cargo vector can help target the homology arms to the genomic site where they can mediate insertion of the nucleic acid cargo sequence, overcoming the rate-limiting step of template pairing (see paragraph [0487]).
Lohman teaches efficient ligation of donor and acceptor DNA strands brought together by an RNA splint. Specifically, Lohman teaches that “single-stranded DNA molecules annealed to an RNA splint” can be efficiently ligated by PBCV-1 DNA ligase, demonstrating that RNA-splinted DNA can serve as an effective ligation substrate, see abstract.
More specifically, Lohman teaches that “a 5’-phosphorylated…DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor … are annealed to a complementary RNA or DNA splint,” and that this substrate is reacted with ligase to generate ligated products, see FIG. 1 and FIG. 1 legend. Thus, Lohman teaches a splint oligonucleotide spanning a ligation junction between a donor DNA and an acceptor DNA, wherein the splint hybridizes to both molecules and positions them for ligation, see FIG. 1 and FIG. 1 legend.
Lohman further teaches that PBCV-1 DNA ligase efficiently ligates nicked DNA substrate and exhibits high affinity and efficient turnover on RNA-splinted DNA substrates, demonstrating suitability for ligase-mediated joining of DNA strands aligned by an RNA splint, see introduction-third paragraph and abstract.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to substitute the reverse transcriptase-mediated template extension mechanism of Liu with a splint-mediated donor ligation strategy by providing a donor (ligand) oligonucleotide and a splint oligonucleotide configured to bridge the donor oligonucleotide to the nicked target strand, and ligating the donor to the target strand using a DNA ligase.
A PHOSITA would have been motivated to do so because Liu expressly teaches targeted installation of precise nucleotide changes at a nicked DNA intermediate, Bier teaches that bringing a donor (ligand) sequence into close physical proximity to a CRISPR-generated genomic nick improves editing by overcoming the rate limiting template pairing step, and Lohman teaches that once donor and acceptor DNA strands are aligned by a splint, direct ligation provided an efficient alternative mechanism for sequence installation without polymerase dependent synthesis of the donor/ligand sequence. Furthermore, providing a pre-synthesized donor/ligand sequence would allow a PHOSITA to have greater control over the exact inserted nucleic acid sequence compared to relying on in situ reverse transcription. Thus, a PHOSITA would have recognized splint-mediated ligation as a predictable alternative to reverse transcriptase-mediated strand synthesis for installing desired sequence changes at the nicked target site. Further, because Liu teaches that fusion of the editing enzyme to the Cas nickase improves localization of catalytic activity at the target site, a PHOSITA would have been motivated to substitute the reverse transcriptase domain with a DNA ligase domain in the same fusion architecture to similarly localize ligase activity to the nicked DNA substrate and improve ligation efficiency. Thus, a PHOSITA would have turned to DeLoache who expressly teaches CRISPR-ligase fusion constructs, confirming that fusion of a CRISPR-associated nuclease, such as nickase, to a ligase was a known design choice for localizing ligase activity to a target site.
A PHOSITA would have had a reasonable expectation of success because Liu already provides the required CRISPR nicked-target intermediate and localized editing environment, Bier teaches donor bridging/splinting at the same genomic location, and Lohman experimentally demonstrated that donor and acceptor DNA strands positioned by an RNA splint can be effectively ligated by DNA ligases such as PBCV-1 DNA Ligase. Because Liu and DeLoache already demonstrated successful use of Cas nickase fusion enzymes, a PHOSITA would reasonably expect nickase-ligase fusion to localize and function predictably. Therefore, the combination merely substitutes one known sequence installation biochemistry for another known sequence installation biochemistry within the same targeted CRISPR editing framework, wherein the donor/ligand sequence is localized to the nicked target site for incorporation.
Claims 2-6 are rejected under 35 U.S.C. 103 as being unpatentable over Liu D. et al., (US11447770B1) in view of DeLoache W et al., (WO2018013990A1), Bier E. et al., (WO2017049266A2) and Lohman G. et. al., (Nucleic Acids Research, Volume 42, Issue 3, Pages 1831–1844, published 06 November 2013) as applied to claim 1 above, and further in view of Kennedy A. et al., (US20170058298A1) and Takei Y. et al., (JBC, Volume 277, Issue 26, Pages 23800-23806, published 28 June 2002).
The teachings of Liu, DeLoache, Bier, and Lohman are incorporated herein by reference to the preceding 103 rejection.
Neither Liu, DeLoache, Bier, or Lohman teach a CRISPR guide RNA wherein the splint oligonucleotide is coupled to the CRISPR guide RNA; wherein the ligand oligonucleotide is coupled to the CRISPR guide RNA; wherein at least two of the ligand oligonucleotide, the splint oligonucleotide, and the CRISPR guide RNA are coupled by a linker, wherein the linker is an inverted-base linker, a cleavable linker, or combination thereof; and wherein the ligand oligonucleotide and the splint oligonucleotide are coupled as part of the same molecule.
Kennedy discloses compounds and methods for genomic editing to modify a DNA sequence in vivo or in vitro, comprising modifying a target polynucleotide wherein the target polynucleotide may be single-stranded or double-stranded, and, in certain embodiments, is double-stranded DNA (see paragraphs [0017]. [0026], and [0129]).
Kennedy further discloses a CRISPR protein wherein the CRISPR protein can be a mutant of a wild type CRISPR protein such as the Cas9 nickases (see paragraphs [0112]). Kennedy also discloses a guide RNA wherein the guide RNA has the functionality of binding a target polynucleotide such as a double-stranded DNA or RNA (see paragraphs [0026], and [0037]).
Kennedy further discloses a donor polynucleotide which may be inserted into a target polynucleotide, for example, into the genome of a cell (see paragraph [0018]). Andrew’s donor polynucleotide may be a natural or a modified polynucleotide, an RNA-DNA chimera, or a DNA fragment, either single- or double-stranded, or a PGR amplified ssDNA or dsDNA fragment (see paragraph [0022]). Andrew’s donor polynucleotide reads on “a ligand oligonucleotide” as required by claim 1 in the instant application, because the specification defines a ligand oligonucleotide as a polynucleotide sequence comprising a donor sequence to be incorporated into target site wherein the ligand oligonucleotide should be DNA (see specification, paragraph [0049]).
Kennedy further discloses that in certain embodiments a CRISPR guide RNA comprises an adaptor segment tethered to a donor polynucleotide (see paragraph [0017]). Kennedy further discloses that the adaptor segment of the guide RNA comprises ssDNA or ssRNA adapted for hybridizing to a polynucleotide (e.g. a splint) (see paragraph [0044]).
Kennedy further discloses that a donor polynucleotide and adaptor RNA can be ligated with a DNA-RNA ligase to form a chimeric RNA-DNA where the adaptor is bound to the donor by a cleavable internucleotide linkage (see paragraphs [0028], [0052], [0080], and claims 3-4).
Takei teaches “oligodeoxynucleotides modified at both 5′- and 3′-ends with inverted thymidine (5′-,3′-inverted T) were introduced as new reagents for antisense strategies,” and “the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides,” see abstract.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to further modify the system used in Liu, DeLoache, Bier, and Lohman by coupling the splint oligonucleotide and/or ligand oligonucleotide to the CRISPR guide RNA to form a single molecule guide RNA construct comprising the guide sequence, splint oligonucleotide, and ligand oligonucleotide joined through one or more internucleotide linkages, including cleavable linkers and modified linker chemistries such as inverted-base linkers.
A PHOSITA would have been motivated to do so because Kennedy expressly teaches that tethering donor polynucleotides to CRISPR guide RNAs through adaptor-mediated architectures localizes the donor sequence to the target editing site and improves editing efficiency by increasing the local donor concentration at the cleavage site. A PHOSITA would have recognized that converting Kennedy’s donor-localization strategy into a single molecule construct by directly coupling the splint and/or ligand to the guide RNA would improve co-delivery, molecular stability, and stochiometric control. Further, Kennedy teaches use f cleavable internucleotide linkages, and Takei teaches inverted thymidine as a known oligonucleotide modification useful for improving oligonucleotide stability and performance. Thus, a PHOSITA would have been motivated to employ known linker chemistries, including cleavable and inverted-base linkages, as routine design choices when constructing the claimed single molecule guide RNA architecture.
A PHOSITA would have had a reasonable expectation of success because Liu already teaches a functional CRISPR nickase editing system employing a 3’-extended guide RNA to localize editing components at a nicked target site, bier teaches the utility of bridging donor sequences to the target site using an oligonucleotide that has complementarity to a sequence at the target site and a sequence on the donor/ligand oligonucleotide, Lohman experimentally demonstrates that donor and acceptor nucleic acid strands aligned by an RNA splint can be efficiently ligated, and Kennedy expressly teaches guide RNA architectures in which donor sequences are tethered through adaptor-mediated ligation to form chimeric RNA-DNA constructs, Further, Kennedy teaches cleavable internucleotide linkage and Takei demonstrates that inverted-base oligonucleotide modifications were known and predictable oligonucleotide design choices. Accordingly, a PHOSITA would have reasonably expected that directly coupling the splint and/or ligand/donor oligonucleotide to the guide RNA through such known linkage strategies would predictably retain donor-localization and ligation functionality while improving construct stability and delivery.
Claims 14-20 are rejected under 35 U.S.C. 103 as being unpatentable over Liu D. et al., (US11447770B1) in view of Bier E. et al., (WO2017049266A2), Kennedy A. et al., (US20170058298A1) and Takei Y. et al., (JBC, Volume 277, Issue 26, Pages 23800-23806, published 28 June 2002).
Liu’s teaching are herein incorporated in full by reference to the preceding 103 rejections.
Briefly, for convenience, Liu teaches PRIME editing which employs a 3’-extended guide RNA (PEgRNA) comprising a spacer sequence that hybridizes with a target nucleic acid sequence of a double-stranded target DNA sequence and an extension arm comprising a primer binding site and a DNA synthesis template in a 5’ to 3’ orientation (see claim 1, col. 16, lines 35-67; col17, lines 1-21). The primer binding site comprises a region complimentary to a sequences upstream of a nick site in the target DNA, and the DNAS synthesis template encodes the desired nucleotide changes to be incorporated into the target DNA (claim 1).
Liu’s PEgRNA therefore teaches a CRISPR guide RNA comprising:
A guide sequence that hybridizes with a target nucleic acid sequence of a double-stranded nucleic acid sequence; and
A 3’-extention comprising a first sequence complimentary to a target DNA (the primer binding site) and a second sequence encoding donor information (the DNA synthesis template), which together teach the claimed splint/ligand architecture.
However, Liu does not explicitly teach a separate ligand oligonucleotide and splint oligonucleotide, nor expressly teach that these components are coupled by linkers as part of a single molecule.
The teachings of Bier, Kennedy, and Takei are herein incorporated in full by refence to the preceding 103 rejections.
Briefly, for convenience, Bier discloses an oligonucleotide clamp comprising a first sequence complimentary to a genomic target sequence at the cut site of a Cas protein and a second sequence complimentary to a donor cargo sequence, such that one portion hybridizes to the target genomic DNA and another portion hybridizes to the donor cargo vector, thereby bridging the donor sequence to the intended genomic editing site, [0048], [0060], and [0418]. Bier’s oligo-clamp therefore teaches a splint oligonucleotide comprising a first nucleic acid sequence complimentary to a ligand oligonucleotide and a second nucleic acid sequence complimentary to a target nucleic acid sequence.
Kennedy teaches CRISPR guide RNAs comprising an adaptor segment tethered to a donor polynucleotide, [0017]. Kennedy further teaches a splint oligonucleotide adapted for hybridizing to the adapter of the guide RNA molecule and a donor oligonucleotide, [0044]. Kennedy further teaches that a donor oligonucleotide and the adapter RNA of the gRNA may be ligated to form a chimeric RNA-DNA molecule in which the adaptor is bound to the donor by an internucleotide cleavable linkage, [0028], [0052], [0080], and claims 3-4. Thus, Kennedy teaches covalently coupling donor and a guide RNA as a single molecule construct.
Takei teaches oligodeoxynucleotides modified with inverted thymidine as known modified internucleotide architectures useful for improving oligonucleotide stability and function, thereby teaching known inverted-base linkage chemistry, see abstract.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to modify Liu’s PEgRNA construct by separating the donor-encoding and target binding functions of the PEgRNA extension arm into distinct ligand and splint oligonucleotide components, wherein the splint oligonucleotide comprises a first sequence complimentary to the ligand sequence and a second seque3nce complimentary to a target nucleic acid sequence, and coupling those components to the gRNA as a single molecule through known internucleotide linkages, including cleavable and inverted-base linkages.
A PHOSITA would have been motivated to do so because Liu teaches that extending a guide RNA with donor-encoding and target-binding sequences enables precise targeted genome editing. Further, Bier expressly teaches that donor sequences can be bridged to a genomic target near a Cas cut site using a splinting oligonucleotide have dual complementarity to both the donor and the target sequence, thereby localizing the donor at the intended editing site. Kennedy further motivates by teaching that tethering donor sequences directly to the gRNA improves co-localization, delivery efficiency, and editing efficiency by ensuring the donor sequence is physically associated with the guide RNA complex. A PHOSITA would have recognized that organizing these known functional elements into a single molecule would improve stochiometric delivery, molecular stability, and system simplicity. Further, Takei teaches that inverted-base oligonucleotide modifications were known design choices for improving oligonucleotide performance, making such linker substitutions a matter of routine optimization.
A PHOSITA would have had a reasonable expectation of success because Liu demonstrates that 3’-extended gRNAs remain functional in CRISPR editing systems; Bier demonstrates that donor and target sequences can be predictably bridged using a dual-complimentary oligonucleotide; Kennedy demonstrates that donor-linked gRNA architectures remain functional when donor sequences are tethered to guide RNAs; and Takei demonstrates that modified oligonucleotide linkages such as inverted-base linkages were well known and compatible with oligonucleotides. Accordingly, combining these known design elements would have predictably yielded the claimed CRISPR guide RNA architectures.
Response to Arguments
Applicant’s arguments, see pg. 7 “Amendments to the Specification”, filed 08/14/2025, with respect to the specification have been fully considered and are persuasive. The objection of the specification has been withdrawn.
Applicant’s arguments, see pg. 8 “Claim Rejections Under 35 U.S.C. 102”, filed 08/14/2025, with respect to claim 14 have been fully considered and are persuasive. The rejection of claim 14 has been withdrawn.
Applicant’s arguments, see pgs. 9-13 “Claim Rejections Under 35 U.S.C. 103”, filed 08/14/2025, with respect to the rejections of claims 1-13 under 35 USC. 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new grounds of rejection is made in view of Liu D. et al., (US11447770B1), DeLoache W. et al., (WO2018013990A1), Bier E. et al., (WO2017049266A2), Lohman G. et. al., (Nucleic Acids Research, Volume 42, Issue 3, Pages 1831–1844, published 06 November 2013), Kennedy A. et al., (US20170058298A1), and Takei Y. et al., (JBC, Volume 277, Issue 26, Pages 23800-23806, published 28 June 2002).
Relevant Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Anzalone et. al., (Nature volume 576, pages149–157, published 21 October 2019).
Anzalone et. al., is the seminal non-patent literature publication first describing PRIME editing. This publication and the patent publication, US11447770B1, relied upon in the 103 rejection above, disclose identical subject matter. For example, see below FIG. 1 of Anzalone:
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Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to COREY LANE BRETZ whose telephone number is (571)272-7299. The examiner can normally be reached M-F 7:30am - 6:30pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571) 272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/COREY LANE BRETZ/Patent Examiner, 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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