DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 11-25-25.
Claims 8, 11, 19, 48, 50-72 are pending in the instant application.
Election/Restrictions
Claim 50 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11-25-25.
Applicant’s election without traverse of Group I, claims 8, 11, 19, 48, 51-72 in the reply filed on 11-25-25 is acknowledged.
Claim Objections
Claim 11 is objected to because of the following informalities: please remove the parentheses in line one of claim 11 (“( or DNA encoding the template RNA}”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 8, 11, 19, 48, 51-72 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The breadth of the claims:
The claims are drawn to compositions comprising systems for modifying DNA comprising a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises a reverse transcriptase (RT) domain from a retrovirus and an endonuclease domain; and a template RNA (or DNA encoding the template RNA) comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof, which template RNA (or DNA encoding the template RNA) comprises from 5' to 3' (i) a sequence that binds a target site, (ii) a sequence that binds a polypeptide comprising a reverse transcriptase (RT) or RT domain, (iii) a heterologous object sequence, and (iv) a 3' target homology domain, which heterologous object sequence comprises an alteration relative to a corresponding original sequence, wherein the alteration improves the speed, fidelity, or
speed and fidelity of target-primed reverse transcription by a reverse transcriptase (RT), or wherein the heterologous object sequence has one or both of the following
characteristics: i) does not comprise self-complementary sequences or if a self-complementary sequence is present, it has one, two, or all of the following characteristics: (1) each self-complementary sequence is no more than 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides in length, (2) the self-complementary sequence forms a hairpin comprising arms of no longer than 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides in length, or (3)
the self-complementary sequence comprises at least 1, 2, 3, 4, or 5 positions of non-complementarity with its partner sequence, ii) does not comprise a repetitive sequence or if a repetitive sequence is present, it is of no more than 12, 11, 10, 9, 8, 7, or 6 nucleotides in length, or which template RNA further comprises an RT termination moiety situated between the heterologous object sequence and either (i) or (ii).
(Previously Presented) The template RNA of claim 19, wherein the RT
termination moiety is situated between the heterologous object sequence and
which RT termination moiety optionally comprises a non-nucleic acid molecule optionally comprising a spacer optionally comprising a C3 spacer or tri/hexa-ethylene glycol spacer, or a trizole or one or more streptavidin and biotin moieties, or which
RT terminator sequence optionally comprises a sequence that adopts a secondary structure under physiological conditions optionally comprising a secondary or tertiary structure comprising one or more hairpins, which RT terminator sequence is optionally situated directly adjacent to the heterologous object sequence, or optionally directly adjacent or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 nucleotides from the sequence that binds the polypeptide, which template RNA optionally comprises one or more chemically modified nucleotides optionally comprising 1-methylguanosine, N6,N6-dimethyladenosine, or 3- methyluridine.
Teachings in the specification:
The specification teaches the following:
Fig. 1 shows the predicted structural characteristics (using RNAstructure) of an exemplary template nucleic acid (e.g., without improvement) as predicted by RNA structure. …nucleotide positions for portions of the template nucleic acid are as follows: (1) gRNA spacer, 1-20; (2) gRNA scaffold, 21-96; (3) Heterologous object sequence, 97-209; (4) 3' target region, 210-221. Figure 1 discloses SEQ ID NO: 57.
Fig. 2 shows the predicted structural characteristics of the exemplary template nucleic acid from Fig. 1 with improvements comprising a redesigned heterologous object sequence (mRNAOptimiser). Arrows in the figure point to the ends of the heterologous object sequence, and nucleotide positions for portions of the template nucleic acid are as described in Fig. 1.Figure 2 discloses SEQ ID NO: 58.
Fig. 3 shows a schematic of the structure of exemplary template RNAs.
Fig. 4 shows a diagram showing the modules of an exemplary template RNA. Individual modules of the exemplary template can be combined, re-arranged, and/or omitted, e.g., to produce a template as described herein. A = 5' homology arm; B = Ribozyme; C = 5' UTR; D = heterologous object sequence; E = 3' UTR; F = 3' homology arm.
Fig. 5 shows a table listing the modules of an exemplary RNA template. Individual modules can be combined, re-arranged, and/or omitted, e.g., to produce a template. A = 5' homology arm; B = Ribozyme; C = 5' UTR; D = heterologous object sequence; E = 3' UTR; F = 3' homology arm.
Fig 6 is a diagram showing an exemplary dual template system. The system includes two template RNA molecules, each comprising, in order, a priming region, a template region, a reverse transcriptase binding sequence, and a spacer sequence. Each of the template RNAs is bound to a reverse transcriptase (RT) protein (e.g., two identical RT proteins or two different RT proteins). The two template RNAs are bound to the same target DNA, with one template RNA bound to one strand upstream of one PAM, and the other template RNA bound to the antiparallel strand upstream of a second PAM. The two template RNA-RT protein complexes are arranged to move along the target DNA toward each other.
Figs. 7A-7B are a series of diagrams showing exemplary strategies for preventing reverse transcriptase (RT) read-through into the tracrRNA sequence. The left panel shows a template RNA comprising a generic RT block. The right panel shows a template RNA comprising a biotin-streptavidin RT block, in which the priming and template regions are conjugated to a first biotin moiety, and the spacer region of the template RNA conjugated to a second biotin moiety, with both biotin moieties then bound to a single streptavidin moiety.
Figs. 8A-8B are a series of diagrams showing exemplary template RNAs each comprising a secondary structural element capable of annealing to one end of the template RNA, thereby reducing or preventing annealing between the spacer region and the priming region of the template RNA. The left panel shows a template RNA in which the secondary structural element is located at the end of the spacer region and is self-annealed to at least a portion of the spacer region. The right panel shows a template RNA in which the secondary structural element is located at the end of the priming region and is self-annealed to at least a portion of the priming region.
Fig. 9 is a diagram showing exemplary self-annealing secondary structural elements at the ends of template RNAs. The top panel shows a secondary structural element having a sequence complementary to the priming region of the template RNA, such that the resultant hairpin formed by its hybridization to the priming region contains no mismatches. The bottom panel shows a secondary structural element having a sequence having at least one mismatch relative to the priming region of the template RNA, such that the resultant hairpin formed by its hybridization to the priming region is destabilized compared to the fully complementary hairpin.
Fig. 10 is a diagram showing an exemplary self-annealing secondary structural element at the end of a template RNA, in which the secondary structural element further comprises a ligand- activated self-cleaving ribozyme (e.g., an aptazyme). When the aptazyme binds to a particular ligand, a cleavage event is triggered that cleaves the self-annealing region of the secondary structural element off from the remainder of the template RNA.
Fig. 11A depicts exemplary positions for chemical modifications in a template RNA. The template RNA comprises (from 5' to 3') (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide comprising an RT domain, (iii) a heterologous object sequence (labeled "template"), and (iv) a 3' target homology domain (labeled "priming"). A Template RNA may comprise chemical modifications at one or more of the positions shown.
Fig. 11B depicts exemplary patterns of chemical modification in a template RNA, e.g., in (i) the sequence that binds a target site in the DNA and (ii) the sequence that binds the polypeptide comprising an RT domain. Top left diagram (SEQ ID NO: 59),
Fig. 11C depicts exemplary patterns of chemical modification in a template RNA, e.g., in (iii) the heterologous object sequence and (iv) the 3' target homology domain (labeled "priming"). In some embodiments, (iii) comprises a region (labeled "nick to edit") that extends from the end of (iv) to the position corresponding to the location of the nick in the target DNA. In some embodiments, (iii) comprises a region (labeled "edit") corresponding to the sequence desired to be inserted into the target DNA. In some embodiments, (iii) comprises a region (labeled "RT homology") that has homology (e.g., identity) to the corresponding region of the target DNA. Unmodified nucleotides are shown in white, and modified nucleotides are shown in gray.
Fig. 11D depicts exemplary patterns of chemical modification in a template RNA, e.g., in (iii) the heterologous object sequence and (iv) the 3' target homology domain.
Fig. 12 depicts a template RNA and corresponding protein, with oligonucleotides that pair with specific regions of the template RNA and disrupt pairing within the template RNA.
Fig. 13 depicts methods of making a template RNA. Different segments of the template RNA can be synthesized (e.g., by solid phase synthesis) and then assembled, e.g., by click chemistry or splint ligation.
Fig. 14 depicts a circular template RNA.
Fig. 15 depicts a splint oligonucleotide for making a circular template RNA.
The specification does not adequately describe the broad genus of compositions claimed, comprising the various systems for modifying DNA, comprising a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises a reverse transcriptase (RT) domain from a retrovirus and an endonuclease domain; and a template RNA (or DNA encoding the template RNA) comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof.
The predicted structural characteristics (using RNAstructure) of exemplary template nucleic acids, as predicted by RNA structure, .as taught in the specification, and the reduction to practice of a few sequences taught in the examples in the specification, are not representative of the broad genus claimed. The specification fails to provide a representative number of species, and does not indicate what distinguishing attributes are concisely shared by the members of this broad genus.
For the reasons stated above, the instant rejection for lacking adequate written description is proper.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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(s) 8 and 11 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Rubens et al (WO 2020/047124 A1).
The applied reference has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Rubens et al (WO 2020/047124 A1) teach systems for modifying DNA comprising a polypeptide or nucleic acid encoding a retroviral reverse transcriptase, a template comprising from 5’ to 3’ a sequence that binds a target site in the target DNA, a sequence that binds the polypeptide, a heterologous object sequence, a 3’ target homology domain and a region capable of hybridizing to any sequences regions of the template (see esp. pages 2-11, 40-42, 45, 45, 47-53, Tables 1-3)
Claim(s) 8, 11, 48 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Anzalone et al (Nature, Vol. 576, pages 140-178 (2019)).
Anzalone et al (Nature, Vol. 576, pages 140-178 (2019)) teach template RNA referred to as a pegRNA in Fig. 1c:
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Anzalone et al teach nanoparticles comprising pegRNA comprising from 5’ to 3’ (i) a spacer sequence that hybridizes to a target site, (ii) a hairpin sequence that binds a polypeptide comprising a Cas9 nickase fused to a reverse transcriptase, (iii) a RT template including an edit (heterologous object sequence of the claim), and (iv) a primer binding site (sequence homologous to the non-target strand (3’ target homology domain of the claim).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim(s) 8, 11, 19, 48, 51-72 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu, L. (US 2014/0113375) and Anzalone et al (Nature, Vol. 576, pages 149-179 (2019)), the combination in view of Lambowitz et al (US 2014/0004569).
The claims are drawn to lipid nanoparticle (LNP) compositions comprising systems for modifying DNA comprising:(a) a polypeptide or a nucleic acid encoding the polypeptide, which polypeptide comprises a reverse transcriptase (RT) domain from a retrovirus, and an endonuclease domain; and(b) a template RNA, or DNA encoding the template RNA, comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof.
The claims are also drawn to lipid nanoparticle (LNP) compositions comprising template RNA or DNA encoding the template comprising from 5' to 3' (i) a sequence that binds a target site, (ii) a sequence that binds a polypeptide comprising a reverse transcriptase (RT) or RT domain, (iii) a heterologous object sequence, and (iv) a 3' target homology domain, which heterologous object sequence comprises an alteration relative to a corresponding original sequence, wherein the alteration improves the speed, fidelity, or speed and fidelity of target-primed reverse transcription by a reverse transcriptase (RT),or wherein the heterologous object sequence has one or both of the following characteristics: i) does not comprise self-complementary sequences or if a self-complementary sequence is present, it has one, two, or all of the following characteristics:(1) each self-complementary sequence is no more than 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides in length, (2) the self-complementary sequence forms a hairpin comprising arms of no longer than 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides in length, or (3) the self-complementary sequence comprises at least 1, 2, 3, 4, or 5 positions of non-complementarity with its partner sequence, ii) does not comprise a repetitive sequence of no more than 12, 11, 10, 9, 8, 7, or 6 nucleotides in length, which template comprises from 5' to 3' (i) a sequence that binds a target site, (ii) a sequence that binds a polypeptide comprising a reverse transcriptase (RT) domain, (iii) a heterologous object sequence, and (iv) a 3' target homology domain, and which template optionally further comprises an RT termination moiety situated between the heterologous object sequence and either (i) or (ii). or which system comprises a polypeptide or a nucleic acid encoding a polypeptide comprising a reverse transcriptase (RT) domain from a retrovirus, an endonuclease domain; and further comprising a template RNA, or DNA encoding the template RNA, comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof, and which self-complementary sequence optionally forms a hairpin, which template RNA optionally comprises one or more chemically modified nucleotides optionally comprising 1-methylguanosine, N6,N6-dimethyladenosine, or 3- methyluridine.
Liu, L. (US 2014/0113375) teach lipid nanoparticle (LNP) compositions comprising systems for modifying DNA comprising:(a) a polypeptide or a nucleic acid encoding the polypeptide, which polypeptide comprises a reverse transcriptase (RT) domain from a retrovirus, and an endonuclease domain; and(b) a template RNA, or DNA encoding the template RNA, comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof, or which forms a hairpin, which template comprises from 5' to 3' (i) a sequence that binds a target site, (ii) a sequence that binds a polypeptide comprising a reverse transcriptase (RT) domain, (iii) a heterologous object sequence, and (iv) a 3' target homology domain, and which template optionally further comprises an RT termination moiety situated between the heterologous object sequence and either (i) or (ii). or which system comprises a polypeptide or a nucleic acid encoding a polypeptide comprising a reverse transcriptase (RT) domain from a retrovirus, an endonuclease domain, and further comprising a template RNA, or DNA encoding the template RNA, comprising from 5' to 3' (i) a sequence that binds a target site in the DNA, (ii) a sequence that binds the polypeptide, (iii) a heterologous object sequence, (iv) a 3' target homology domain, and (v) a region capable of hybridizing to any of (i), (ii), (iii), or (iv), or portions or combinations thereof, and which self-complementary sequence optionally forms a hairpin, and which RT termination moiety optionally comprises a spacer ((see esp. para 0044, 0055, 0059, 0077, Figs. 1-12, pages 2-3, 6).
Anzalone et al (Nature, Vol. 576, pages 149-179 (2019)) teach the following template RNA referred to as a pegRNA in Fig. 1c:
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With regard to claim 11, Anzalone et al teach the pegRNA comprises from 5’ to 3’ (i) a spacer sequence that hybridizes to a target site, (ii) a hairpin sequence that bind a polypeptide comprising a Cas9 nickase fused to a reverse transcriptase, (iii) a RT template including an edit (heterologous object sequence of the claim), and (iv) a primer binding site (sequence homologous to the non-target strand (3’ target homology domain of the claim).
The primary references do not teach terminal blocking agents.
Lambowitz et al (US 2014/0004569) teach blocking agents to impede further recopying by reverse transcriptase:
[0051] Certain embodiments of the methods described herein can include a blocking agent at the 3' end of the complimentary oligonucleotide template strand to terminate the oligonucleotide and impede further recopying by the reverse transcriptase. The blocking agent impedes the reverse transcriptase from using this oligonucleotide as a target. Examples of suitable blocking agents include 3'-amino-modifier C3 and 3'-amino-modifier C7, both of which contain branched linkers in which the amino group is protected with the fluorenylmethoxycarbonyl (Fmoc) group. Other potential 3' modifiers could be thiol groups; DPTA (3,3'-(hydroxynitrosohydrazino)bis-1-propanamine), which can be also used to conjugate the oligonucleotide to gold surfaces; spacer phosphoamidite modifiers; or glycerol. Spacer modifiers could be made photocleavable. Use of blocking agents to prevent recopying is understood by those skilled in the art, and therefore other blocking agents may be employed with the methods described herein. See also claim 27.
It would have been obvious to design and optimize the instantly claimed systems and templates because the prior art taught all of the components as claimed, and optimizing the positions and spaces of the well known and routinely components involves routine experimentation, as illustrated in the teachings of Anzalone and Liu.
For these and the aforementioned reasons, the instant invention would have been obvious to one of ordinary skill in the art before the effective filing date of the instant invention.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
4-30-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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