DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on April 1, 2026 has been entered.
Application Status and Withdrawn Rejections
Applicant’s amendments filed March 23, 2026, amending claims 1, 28 and 37, and canceling claim 29 are acknowledged. Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32, 36-37 and 78 are pending and under examination.
Claims 1 and 37 were amended to add the limitation “wherein the DNA binding nuclease, RT and integrase enzyme are linked via a linker. The amendment to claims 1 and 37 overcomes the §102 and nonstatutory double patenting rejections over the composition and method claims in the previous office action. However, §102 rejection over claim 78 is maintained. Additionally, new §103 rejections of claims 1 and its dependent claims and claim 37 are recited below. The amendment to claim 28 overcomes the §112(a) rejection.
Any other rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 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 –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim 78 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021). This is a new maintained rejection.
Regarding claim 78, Anzalone teaches the pegRNAs HEK3_attP_A_43 and HEK3_attP_B_44 (i.e., a pair of gRNAs) (Supp Table 1). Anzalone teaches that each of the pegRNAs comprises a spacer sequence, a scaffold sequence, and a 3’ extension, which comprises a primer binding sequence and an RT template (Supp Table 1). Anzalone teaches the pegRNAs bind to PE2, which comprises the DNA-binding Cas9 nickase (Fig 1a). Anzalone teaches the HEK3_attP_A_43 and HEK3_attP_B_44 pegRNAs comprise together the attP integration recognition sequence (Fig 1c; Supp Table 1). Anzalone teaches that the attP sequences that were integrated into the targeted sequence could hybridize to each other (i.e., form a double stranded nucleic acid) (Fig. 1C). Because each of the integrated sequences were complementary to a reverse transcription template sequence in one of the guide RNAs, the reverse transcription template sequences in the guide RNAs must have been capable of forming a double stranded nucleic acid (Fig 1a-c).
Response to Arguments - §102
Applicant argues that the amendment to claims 1 and 37 overcome the § 102 rejection over Anzalone (Remarks, pages 8-9). This argument has been fully considered and is persuasive as it pertains to claims 1, its dependent claims, and claim 37. However, claim 78 was not amended and does not require a nuclease, RT enzyme, or integrase. Applicant does not provide arguments specifically for claim 78. The § 102 rejection over Anzalone is maintained.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32 and 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021, of record) as evidenced by Anzalone2 (Anzalone et al., Nature (2019), 576: 149-157, of record) and in view of Ioannidi (Ioannidi et al., "Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases." BioRxiv (2021): 2021-11, available online November 1, 2021).
Ioannidi was published within one year of the effective filing date of the claimed invention and names Gootenberg and Abudayyeh, the two inventors of the current application. However, Ioannidi names 15 additional authors. Therefore, Ioannidi constitutes prior art under § 102(a)(1).
Regarding claims 1, 30 and 32, Anzalone teaches a composition comprising PE2, Bxb1, 2x pegRNA comprising an attP sequence, wherein the composition is used for the purpose of integrating an exogenous sequence in the genome of a cell (Fig 3a). Anzalone teaches PE2 is a Prime Editing enzyme comprised of a Cas9 nickase (i.e., a DNA binding nickase) and a reverse transcriptase (RT) (page 731, ¶5). Anzalone does not teach whether in PE2, how Cas9 and RT are linked. However, Anzalone2 teaches in all the prime editors, Cas9 and RT are linked through a flexible linker (¶ spanning pages 151-152). Anzalone teaches Bxb1 is an integrase (page 732, ¶3). Anzalone teaches that each pegRNA comprises a spacer sequence that hybridizes to the target DNA, hairpins that bind to the Cas9 nickase (i.e., a scaffold sequence), a portion that hybridizes to the nicked DNA (i.e., a primer binding sequence), and a portion that is used by the RT as a template to transcribe a sequence to be integrated into the target DNA (Fig. 1a, green = pegRNA; red = reverse transcribed from template sequence; Supp Table 1). Anzalone teaches two pegRNAs that each comprise a portion of the attP sequence (i.e., a first integration recognition sequence) (Fig 1c; page 732, ¶3; Supp Table 1). Anzalone teaches that the two pegRNAs each with a portion of the attP integration recognition sequence together encode the entire attP integration recognition sequence (Fig. 1c; Supp Table 1). Anzalone teaches that the attP sequences that were integrated into the targeted sequence could hybridize to each other (i.e., form a double stranded nucleic acid) (Fig. 1C). Because each of the integrated sequences was complementary to the reverse transcription template sequences in one of the guide RNAs, the reverse transcription template sequences in the guide RNAs must have also been complementary to each other (i.e., they were capable of forming a double stranded nucleic acid) (Fig 1a-c).
Anzalone provides the Cas9-RT fusion and Bxb1 to cells in different plasmids (Fig 3) and therefore does not teach Cas9-RT-Bxb1 all linked together via linkers.
Ioannidi teaches a composition for introducing a Bxb1 attB site at a targeted genome site using Cas9-RT-gRNA-mediated prime editing followed by integration of a gene of interest at the attB sequence using Bxb1 (Fig 1). Ioannidi teaches producing the Cas9, RT and Bxb1 integrase as a single fusion protein with linkers in between SpCas9, RT and Bxb1 (i.e., wherein the DNA binding nickase, the RT and the integrase enzyme are linked via a linker) (Fig 1a, ¶ spanning pages 2-3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified Anzalone’s twinPE method by including the Bxb1 with the Cas9-RT in a single fusion protein. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that Anzalone’s twinPE proteins could be linked by linkers because Ioannidi demonstrates the functionality of such a fusion protein. One would have been motivated to do so to 1) simplify the plasmid delivery protocol by reducing the number of plasmids needed from four to three, and 2) promote integration by positioning the Bxb1 integrase at the targeted site of attB introduction.
Regarding claim 3, Anzalone teaches the portion of the pegRNA that hybridizes with the nicked DNA (i.e., the primer binding sequence) is 12 nucleotides (Supp Table 1, compare HEK3 attP_B_44 and HEK3_attP_A_43 sequence to HEK3 site in Fig. 1c).
Regarding claim 5, Anzalone teaches the attP integration sequence is 43-44 nucleotides in length (Supp Table 1, compare HEK3 attP_B_44 and HEK3_attP_A_43 sequence to HEK3 site in Fig. 1c).
Regarding claim 6, the Specification defines “about” as within an acceptable error range for that particular value” ([00078]). Anzalone teaches pegRNAs for insertion of the attP site in which the reverse transcription template is 35 and 36 nucleotides in length (i.e., about 1-34 nucleotides in length) (Fig 1c, A35 + B36). Examiner interprets 35 and 36 nucleotides as being within an acceptable error range for the length of the template sequence.
Regarding claim 7, Anzalone teaches the spacer sequences of the pegRNA(A) and pegRNA(B) are 20 nucleotides in length (Supp table 1).
Regarding claim 10, Anzalone teaches the scaffold sequences are 76 nucleotides in length (Supp Table 1).
Regarding claim 11, the specification does not provide a definition for “an extended sequence”, but appears to use it to described the sequence that the RT produces when reverse transcribing the template portion of the guide RNA ([00032]). As such, “a first extended sequence” and “a second extended sequence” is interpreted as a nucleotide sequence that would be produced by extension of the priming sequence by RT. As indicated above for claim 1, the pair of pegRNAs in Anzalone comprise a template sequence that is reverse transcribed, and which encodes complementary sequences (i.e., the extended sequence) (Fig 1a,c).
Regarding claim 12, Anzalone teaches the sequences that are reverse transcribed from the template portions of the pair of pegRNAs are complementary to each other and overlap by 21-37 base pairs (i.e., at 5-60 complementary nucleotides with respect to each other) forming a duplex and results in insertion of the integration recognition sequence into the HEK3 genome site (Fig 1a-c).
Regarding claim 21, Anzalone teaches each of the pegRNAs in the pair comprise from 5’ to 3’ in order a spacer sequence, the scaffold sequence, the integration sequence and the primer binding sequence (Fig 1a, Supp Table 1).
Regarding claims 22-24, 27 and 36, Anzalone teaches that PE2 was originally designed and reported in Anzalone et al., Nature (2019), 576: 149–157 (2019), herein referred to as Anzalone2. Although Anzalone teaches that PE2 comprises a Cas9 nickase and an RT, Anzalone is silent regarding the precise species make-up of the PE2. Anzalone2 teaches that PE2 comprises Cas9(H840A) fused to the RT from M-MLV which comprises five amino acid substitutions relative to wild type M-MLV, including D200N, L603W, T330P, T306K and W313F (page 152, ¶6). Therefore, the PE2 in Anzalone’s composition with two pegRNAs and Bxb1 inherently comprised a Cas9-H840A nickase, an RT derived from M-MLV, and an RT that had mutations relative to the wild-type RT sequence including D200N, L603W, T330P, T306K and W313F.
Regarding claim 28, Anzalone teaches pegRNA 3’ extension sequences of HEK3_attP_A_43 and HEK3_attP_B_44 (Supp Table 1), which were used to insert the attP integration recognition sequence at the HEK3 locus (Fig 1c). Using the TwinPE mechanism detailed in Fig 1a and the sequence of the HEK3 locus shown in Fig 1c, the sequence of the attP integration recognition sequence was deduced and represented by the bolded sequence below. The capital letters are not present in the pegRNA sequences, but were deduced via complementarity to the extension sequence of the other pegRNA. The underlined nucleotides are the primer binding sequence in each pegRNA.
AGGTTTgtctggtcaaccaccgcggtctcagtggtgtacggtacaaacctccgggatactgg
gtctgactcgtgctccaaacagaccagttggtggcgccagagtcaccacatgccatGTTTGGA
The attP sequence deduced from Anzalone’s teaching (bolded above) comprises a sequence that is 100% identical to SEQ ID NO 396.
Regarding claim 37, the teachings of Anzalone regarding the composition comprising PE2 (i.e., a Cas9 DNA-binding nickase fused to an RT) and two pegRNAs that each comprise a spacer, scaffold, RT template and primer binding site are recited above for claim 1. Anzalone teaches (a) the overall mechanism of twinPE using PE2 and the pegRNAs (Fig 1a). Regarding (a), Anzalone teaches that each of the two molecules of PE2 bind one of the pegRNAs that target the PE2 to the target location in the cell genome (Fig 1a). Anzalone teaches the Cas9 nickase in PE2 nicks one strand of the target DNA (Fig 1a). Anzalone teaches the RT reverse transcribes the RT template sequence in each of the pegRNAs (Fig 1a, red lines). Anzalone teaches the reverse transcribed sequences overlap each other by at least 5 nucleotides forming a duplex, which is then inserted into the genome of the cell (Fig 1a). Anzalone teaches that the twinPE mechanism can be used to insert attP (i.e., an integration recognition sequence) into the targeted genome (Fig 1c). Anzalone teaches that in the case of attP insertion, a portion of the attP sequence is reverse transcribed from the RT template sequence in each of the pegRNAs such that the portions are complementary to each other and overlap by 21-37 bp (Fig 1c). Because each of the integrated sequences were complementary to the reverse transcription templates sequences in one of the guide RNAs, the reverse transcription template sequences in the guide RNAs must have been capable of forming a double stranded nucleic acid (Fig 1a-c). Regarding (b), Anzalone teaches also providing a (i) a donor DNA comprising a gene of interest linked to an attB sequence (i.e., a sequence that is complementary to the attP integration sequence), and (ii) Bxb1 (i.e., an integrase) to the cell (Fig 3c). Anzalone teaches Bxb1 integrates (i.e., incorporates by integration) the donor gene into the targeted genome site at the attP site (Fig 3c). Regarding the fusion of Cas9, RT and Bxb1, the teachings of Ioannidi and the obviousness of fusing the three protein components of Anzalone’s twinPE system are recited above as for claim 1.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32 and 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021, of record) as evidenced by Anzalone2 (Anzalone et al., Nature (2019), 576: 149-157, of record) and in view of Scott (US 20230023791 A1, priority to January 14, 2022).
The Scott reference claims priority to four provisional US applications. The point citations provided in the rejection below are fully supported in the 62/299695 Application, filed January 14, 2022. Therefore, Scott qualifies as prior art under § 102(a)(2).
Regarding claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32 and 36-37, the teachings of Anzalone and Anzalone2 are provided above in paragraphs 14-15 and 18-28 and are incorporated here. Briefly, Anzalone teaches twin Prime Editing (twinPE), which requires every limitation of the claimed compositions and method except for the fusion of the Bxb1 integrase to the Cas9-RT which are already linked by a linker. Anzalone does not teach a single fusion protein wherein the integrase is linked to the Cas9-RT fusion protein.
Scott teaches methods of prime editing using Cas12i2, (i.e., a different class II Cas effector protein) fused to RT via a linker, and a guide RNA (Figs 1, 6). Scott teaches the CRISPR nuclease can be fused to an integrase ([0136]). Scott teaches the CRISPR nuclease that is fused to an integrase is further fused to a reverse transcriptase ([0136]). Scott teaches the integrase is Bxb1 ([0136]). Thus, Scott teaches a Cas-RT-Bxb1 fusion protein.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified Anzalone’s twinPE method by including Bxb1 with Cas9-RT in a single fusion protein. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that Anzalone’s twinPE proteins could be fused together because Scott teaches a fusion protein comprising a CRISPR Cas effector, RT and Bxb1. One would have been motivated to do so to 1) simplify the plasmid deliver protocol, by reducing the number of plasmids needed from four to three, and 2) promote integration by positioning the Bxb1 integrase at the site of attB introduction.
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 filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual 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/apply/applying-online/eterminal-disclaimer.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32, 36-37 and 78 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-22 of U.S. Patent No. 11827881 in view of Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021).
Patented claim 1 recites a method of site-specifically integrating an exogenous nucleic acid sequence into a mammalian cell genome or intracellular target nucleic acid, the method comprising: (a) incorporating at least one integration sequence at a specific target site in the cell genome or intracellular target nucleic acid by introducing ex vivo into a mammalian cell: (i) an expressible polynucleotide construct encoding an editing polypeptide, wherein the editing polypeptide comprises a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity; and (ii) at least two guide RNAs (gRNAs) (i.e., a gRNA pair) each comprising a targeting sequence, a primer binding sequence, and a complement of the at least one integration sequence, wherein each of the at least two gRNAs interacts with the expressed editing polypeptide to direct the editing polypeptide to the specific target site of the cell genome or intracellular target nucleic acid, wherein the DNA binding nuclease domain nicks a strand of the cell genome or intracellular target nucleic acid to form a nicked site, and wherein the reverse transcriptase domain reverse transcribes the complement of the at least one integration sequence within each of the gRNAs and thereby incorporates the at least one integration sequence into the nicked site, thereby incorporating the at least one integration sequence at the specific target site of the cell genome or intracellular target nucleic acid; and (b) integrating an exogenous nucleic acid sequence into the cell genome or intracellular target nucleic acid by introducing into the cell: (i) the exogenous nucleic acid sequence linked to a sequence that is an integration cognate to the at least one site-specifically incorporated-integration sequence; and (ii) an expressible polynucleotide construct encoding an integration enzyme, wherein the integration enzyme integrates the exogenous nucleic acid sequence into the cell genome or the intracellular target nucleic acid at the at least one site-specifically incorporated integration sequence, thereby site-specifically integrating the exogenous nucleic acid sequence into the cell genome or the intracellular target nucleic acid, wherein the expressible polynucleotide encoding the editing polypeptide, the at least two gRNAs, the expressible polynucleotide construct encoding the integration enzyme, and the exogenous nucleic acid sequence are introduced into the mammalian cell concurrently. Patented claim 3 recites wherein the DNA binding nickase domain is linked to the reverse transcriptase domain by a linker. Patented claim 5 recites wherein the linked DNA binding nickase-reverse transcriptase domains are further linked to the integration enzyme. Patented claim 10 recites the integration enzyme is Bxb1. Patented claim 11 recites the integration sequences are attB or attP. Patented claim 12 recites wherein the DNA binding nuclease domain comprising a nickase activity is selected from Cas9-D10A, Cas9-H840A, and Cas12a/b nickase. Patented claims 13-15 recites wherein the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) and comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. Patented claim 21 recites steps the same as claim 1, but also that the integration sequence in an attB or attP sequence. Patented claim 22 recites wherein the integration sequence is 40, 42, 44 or 46 base pairs.
The patented claims do not recite both gRNAs having the recited elements such that they are capable of forming a double stranded nucleic acid. The patented claims do not recite lengths for the primer binding sequence, the RT template sequence, the spacer sequence or the scaffold sequences. The patented claims do not recite specific sequences for the scaffold or the integration sequence. The patented claims do not recite that the reverse transcribed sequence comprising the integration sequence on each of the gRNAs overlap with each other. The patented claims do not recite the nucleic acids encoding Cas9-RT and encoding integrase as operably linked such that they would form a linked Cas9-RT-integrase fusion protein.
The teachings of Anzalone are recited above in paragraphs 14-15 and 18-28 and are incorporated here. Briefly, Anzalone teaches the twinPE method for incorporating an attP site into a targeted genome site using a Cas9n-RT enzyme, and two gRNAs each comprising a spacer, scaffold, a primer binding site, and an RT template comprising overlapping portions of the integration sequence such that the pair of guide RNAs comprise complementary sequence and can form a double stranded nucleic acid.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified the patented method/composition by designing the RT template such that the reverse transcribed sequences from the two gRNAs overlap forming a duplex for insertion of the integration site. It would have amounted to designing prime editing RNAs by known design principles to yield predictable results. The skilled artisan would have predicted that the RT template can be designed as such and been motivated to do so because Anzalone teaches that the twinPE method is successful at integrating the desired integration recognition sequence. Regarding the specific lengths and sequence of the gRNA segments, the skilled artisan would have predicted that such lengths and sequences could be incorporated into the patented gRNAs because Anzalone teaches those lengths and attP sequences in the very similar twinPE method for attP sequence insertion.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32, 36-37 and 78 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-22 of U.S. Patent No. 11952571 in view of Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021).
Patented claim 1 recites a system capable of site-specifically integrating an exogenous nucleic acid into a mammalian cell genome at a desired target site, wherein the system comprises, in a single composition: (a) a nucleic acid encoding a DNA binding nickase domain linked to a reverse transcriptase domain; (b) a nucleic acid encoding at least two guide RNAs (gRNAs) (i.e., a pair of gRNAs) comprising, from 3′ to 5′, i. a primer binding sequence, ii. a sequence complementary to one strand of an integration recognition sequence (i.e., an RT template), and iii. a target binding sequence (i.e., a spacer sequence), wherein the gRNA is capable of guiding the linked nickase-reverse transcriptase domains to the genomic target site; (c) nucleic acid encoding an integration enzyme; and (d) an exogenous nucleic acid linked to a sequence that is an integration cognate of the integration recognition sequence. Patented claim 3 recites wherein the DNA binding nickase domain is linked to the reverse transcriptase domain by a linker. Patented claim 5 recites wherein the linked DNA binding nickase-reverse transcriptase domains are further linked to the integration enzyme. Patented claim 6 recites wherein the DNA binding nickase domain is selected from Cas9-D10A, Cas9-H840A, and Cas12a/b nickase. Patented claims 7-9 recite wherein the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) and comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. Patented claims 13-14 recites wherein the integration enzyme is Bxb1. Patented claims 15-16 recite wherein the integration recognition sequence is an attB sequence, an attP sequence.
The patented claims do not recite the polypeptides or the gRNAs. The patented claims do not recite a method using the patented system. The patented claims do not recite lengths for the primer binding sequence, the RT template sequence, the spacer sequence or the scaffold sequences. The patented claims do not recite specific sequences for the scaffold or the integration sequence. The patented claims do not recite that the reverse transcribed sequence comprising the integration sequence on each of the gRNAs overlap with each other.
The teachings of Anzalone are recited above in paragraphs 14-15 and 18-28 and are incorporated here. Briefly, Anzalone teaches the twinPE method for incorporating an attP site into a targeted genome site using a Cas9n-RT enzyme, and two gRNAs each comprising a spacer, scaffold, a primer binding site, and an RT template comprising overlapping portions of the integration sequence such that the pair of guide RNAs comprise complementary sequence and can form a double stranded nucleic acid.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the patented system in a method for insertion of the attP sites into the genome and insertion for the exogenous sequence at the inserted attP site, in which case the proteins and gRNAs would expressed and present in a composition. First, the patented claims recite that the obvious method is the intended use of the system. Second, Anzalone teaches such a method and composition using the highly similar twinPE method. The obviousness of designing the RT template such that the reverse transcribed sequences from the two gRNAs overlap forming a duplex for insertion of the integration site and using the specific lengths and sequence of the gRNA segments is recite above in paragraph 39.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32, 36-37 and 78 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 and 10-15 of copending Application No. 17786168 in view of Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021).
Copending claim 1 recites an engineered or non-naturally occurring composition comprising: a. a Type II Cas polypeptide; b. a reverse transcriptase (RT) polypeptide selected from M-MLV, that is connected to or otherwise capable of forming a complex with the Cas polypeptide; c. a first guide molecule capable of forming a first CRISPR-Cas complex with the Cas polypeptide (i.e., comprising a scaffold sequence) and comprising: i. a guide sequence capable of directing site-specific binding of the first CRISPR-Cas complex to a first target sequence of a target polynucleotide (i.e., a spacer sequence); ii. a 3’ binding site region capable of binding to a cleaved upstream strand of the target polynucleotide (i.e., a primer binding sequence); and iii. a RT template sequence encoding a first extended sequence comprising a variant region and a 3’ homologous sequence capable of hybridization to the downstream cleaved strand of the target polynucleotide. Copending claim 15 recites the composition of claim 1, further comprising a recombinase, optionally, wherein the recombinase is connected to (i.e., linked to) the Cas polypeptide. Copending claim 53 recites wherein the recombinase is Bxb1. Copending claim 54 recites wherein the RT template encodes a recombination site for the recombinase. Copending claims 50-51 recite the Cas enzyme is Cas9, a Cas9 nickase. Copending claim 56 recites the RT is MMLV RT.
The copending claims do not a second guide RNA with the same features of the first guide RNA or a method using the copending composition. The copending claims do not recite lengths for the primer binding sequence, the RT template sequence, the integration recognition sequence, the spacer sequence, or the scaffold sequences. The copending claims do not recite specific sequences for the scaffold or the integration sequence.
The teachings of Anzalone are recited above in paragraphs 14-15 and 18-28 and are incorporated here. Briefly, Anzalone teaches the twinPE method for incorporating an attP site into a targeted genome site using a Cas9n-RT enzyme, and two gRNAs each comprising a spacer, scaffold, a primer binding site, and an RT template comprising overlapping portions of the integration sequence such that the pair of guide RNAs comprise complementary sequence and can form a double stranded nucleic acid. Anzalone teaches the Bxb1 integrase is used to integrate an exogenous sequence at the introduced attP sites.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the copending composition with an additional guide RNA with the same features in a method for insertion of the attP sites into the genome and insertion for the exogenous sequence at the inserted attP site. Anzalone teaches such a method and composition using the highly similar twinPE method. The obviousness of designing the RT template such that the reverse transcribed sequences from the two gRNAs overlap forming a duplex for insertion of the integration site and using the specific lengths and sequence of the gRNA segments is recite above in paragraph 39. It would have also been obvious to use a Cas9-H840A nickase and M-MLV RT with the instantly claimed substitutions because Anzalone teaches the PE2 prime editor is successful at integrating the attP site in the targeted genome site in cells.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 3, 5-7, 10-12, 21-24, 27-28, 30, 32, 36-37 and 78 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 and 10-15 of copending Application No. 17786168 in view of Anzalone (Anzalone et al., Nature Biotechnology (2022), 40: 731-740, published online December 9, 2021). This is a new rejection.
Copending claim 92 recites A system capable of site-specifically integrating an exogenous nucleic acid into a mammalian cell genome at a desired target site, wherein the system comprises: (a) a nucleic acid encoding a DNA binding nickase domain linked to a reverse transcriptase domain; (b) a nucleic acid encoding a guide RNA (gRNA) comprising, from 3' to 5', a primer binding sequence, a sequence complementary to one strand of an AttB or AttP integration recognition sequence, and a target binding sequence, wherein the gRNA is capable of guiding the linked nickase reverse transcriptase domains to the genomic target site; a nucleic acid encoding a serine integration enzyme; and (c) an exogenous nucleic acid linked to a sequence that is an integration cognate of the integration recognition sequence. Copending claim 111 recites further comprising a nicking gRNA (i.e., a pair of gRNAs). Copending claims 94 recites wherein the DNA binding nickase domain is linked to the reverse transcriptase domain by a linker. Copending claim 95 recites wherein the linker is a peptide fused in-frame between the nickase and reverse transcriptase domains. Copending claim 96 recites wherein the linked DNA binding nickase-reverse transcriptase domains are further linked to the integration enzyme. Copending claim 115 recites wherein the nucleic acid sequence encoding the serine integration enzyme is linked to the nucleic acid sequence encoding a fusion protein comprised of the DNA binding nickase domain linked to the reverse transcriptase domain, thereby allowing for only a single plasmid to be used for protein expression. Copending claim 116 recites wherein the nucleic acid encoding the serine integration enzyme is linked to the fusion protein via a linker. Copending claim 97 recites wherein the DNA binding nickase domain comprises Cas9-DI0A, Cas9-H840A, nickase. Copending claim 99 recites wherein the reverse transcriptase domain is a M-MLV reverse transcriptase domain. Copending claim 104 recites wherein the serine integration enzyme comprises Bxbl.
The copending claims do not recite the nicking guide RNA having the same features of the first guide RNA or a method using the copending composition. The copending claims do not recite lengths for the primer binding sequence, the RT template sequence, the integration recognition sequence, the spacer sequence, or the scaffold sequences. The copending claims do not recite specific sequences for the scaffold or the integration sequence.
The teachings of Anzalone are recited above in paragraphs 14-15 and 18-28 and are incorporated here. Briefly, Anzalone teaches the twinPE method for incorporating an attP site into a targeted genome site using a Cas9n-RT enzyme, and two gRNAs each comprising a spacer, scaffold, a primer binding site, and an RT template comprising overlapping portions of the integration sequence such that the pair of guide RNAs comprise complementary sequence and can form a double stranded nucleic acid. Anzalone teaches the Bxb1 integrase is used to integrate an exogenous sequence at the introduced attP sites.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the copending composition with an additional guide RNA with the same features in a method for insertion of the attP sites into the genome and insertion for the exogenous sequence at the inserted attP site. Anzalone teaches such a method and composition using the highly similar twinPE method. The obviousness of designing the RT template such that the reverse transcribed sequences from the two gRNAs overlap forming a duplex for insertion of the integration site and using the specific lengths and sequence of the gRNA segments is recite above in paragraph 39. It would have also been obvious to use a Cas9-H840A nickase and M-MLV RT with the instantly claimed substitutions because Anzalone teaches the PE2 prime editor is successful at integrating the attP site in the targeted genome site in cells.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments – NSDP
Applicant indicates that upon a finding of allowability for the claims herein, a terminal disclaimer will be filed if appropriate (Remarks, page 9). Because the claims are not in condition for allowance, and no terminal disclaimers have been filed, the nonstatutory double patenting rejections remain on the record, as necessitated by amendment.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635