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 .
Election/Restrictions and Status of Claims
Applicant’s election without traverse of Group I encompassing claims 1-2, 4-6, 8-12, 25-26, 49-52, 55, and 62, drawn to a protecting oligonucleotide in the reply filed on 3/23/2026 is acknowledged.
Applicant’s further election of
Group A: a crRNA sequence and modification pattern of
mN#mN#mN#mNmNmNmNmNmNmNfNfNfNfNrN#rN#fNfNrN#mNmGrU#rU#rU#f
UfAmGmAmGmCmUmAmU#mG#mC#mU (SEQ ID NO:3) (crRNA 20)
as recited in claim 51 and
Group B: a protecting oligonucleotide modification pattern of
mUmAmAmAmAmCmNmNmNmNmNmNmNmN (RC09)
as recited in claim 52
for further prosecution in the same reply filed on 3/23/2026 is also acknowledged.
Applicants have also:
cancelled non-elected claims 58-59 in the reply filed on 3/23/2026, making the election requirement moot.
Added new claims 64- 68, which will be added to elected Group I.
No claims are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Accordingly, claims 1-2, 4-6, 8-12, 25-26, 49-52, 55, 62, and 64- 68 are under consideration.
Applicant is reminded that upon the cancellation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Priority
This application claims priority to provisional applications 63/336,340 and 63/438,842. Therefore, it is entitled to the 29th April 2022 priority date of the earliest provisional application.
Claim Objections
Claims 2,are objected to because of the following informalities:
Claim 2 should have a colon after comprises a modification of; i.e., comprises a modification of:
Similarly, claims 26, 55, 65, 67, and 68 should have a colon after the recitation of of.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 11 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 11 requires an action. However, the claim does not recite an active method step. Further the claim depends from claim 25 which is a composition claim. MPEP § 2173.05(q) states attempts to claim a process without setting forth any steps involved in the process generally raises an issue of indefiniteness under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph. For example, a claim which reads: "[a] process for using monoclonal antibodies of claim 4 to isolate and purify human fibroblast interferon" was held to be indefinite because it merely recites a use without any active, positive steps delimiting how this use is actually practiced. Ex parte Erlich, 3 USPQ2d 1011 (Bd. Pat. App. & Inter. 1986). In the instant case, claim 11 recites the double stranded oligonucleotide of claim [[1]]25, wherein the binding of a trans-activating crRNA (tracrRNA) to the guide sequence portion of the crRNA dissociates the protecting oligonucleotide from the crRNA, which is not an active method step.
Claim 11 could also be interpreted as a functional limitation of the double stranded oligo. If so, the claim is indefinite because it is not clear if the tracrRNA is part of the oligonucleotide instead of the protecting oligo.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 8, 11, and 52 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
MPEP 608.01(n) Dependent Claims [R-01.2024] further states:
In accordance with 35 U.S.C. 112(d), or pre-AIA 35 U.S.C. 112, fourth paragraph, a claim in dependent form shall contain:
(i) a reference to a claim previously set forth, and
(ii) then specify a further limitation of the subject matter claimed.
In the instant case, claims 8 and 11 depend upon claim 25, which is NOT previously set forth, rather follows claims 8 and 11 in the claim listing. Similarly, claim 52 depends upon claim 64, which is NOT previously set forth, rather follows claim 52 in the claim listing.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(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 1-2, 4-6, 8-12, 25-26, 49-50, 55, 62, and 64- 68 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1-2, 4-6, 8-12, 25-26, 49-50, 55, 62, and 64- 68 encompass a large genera of:
chemically modified protecting RNAs as they encompass protecting RNAs that are complementary to any guide sequence in any organism.
chemically modified guide RNAs as they encompass guide sequences that are complementary to any target sequence in any organism.
Claims 1-2, 4-6, 8-12, 25-26, 49-50, 55, 62, and 64- 68 also encompass a large genera of possible modifications to the protecting and guide RNA and large genera of modification patterns. Claims 49 and 55 encompass a system of altering expression of any target gene in any target cell with the large genera of chemically modified protecting RNAs duplexed with chemically modified guide RNAs that are directed to any target polynucleotide sequence in any organism and wherein both the chemically modified protecting RNAs and guide RNAs comprise a large genera of chemical modification patterns.
Knowledge in the Prior Art:
guide RNAs:
Jiang et al. (Science, Vol. 348, Published 16 June 2015, pages 1477-1481) teach the target choice for CRISPR system relies on base pairing between the DNA and the 20nt guide RNA sequence, as well as the presence of a protospacer adjacent motif (PAM) proximal to the target site, and the target complementarity of a “seed” sequence within the protecting segment of crRNAs is critical for DNA recognition and cleavage (Left column, page 1477).
Lee K. et al., (eLife 6:e25312., 02 May 2017, downloaded from the internet from < https://elifesciences.org/articles/25312> on 13th May 2026, 17 pgs.), teach that outside of base modifications and backbone modifications, little is known about the chemical modifications that are tolerated by the gRNA of the CRISPR/Cas9 system, and it is uncertain if the gRNA tolerate modifications with chemical moieties that are structurally unrelated to nucleotide bases, and how big the modifications can be without affecting functional of Cas9 or donor DNA (Intro page 1). Lee K. et al., teach Cas9 gRNA tolerates large chemical modifications at their terminal positions, however is more tolerant to large modifications on the 5’ end than the 3’ end of the crRNA (pages 2 and 4).
Oligonucleotides in a duplex:
Liu (US 20150071901). Liu teaches that protecting a functional single-stranded nucleic acid (guide sequence) by hybridizing it to a complementary strand (protecting oligonucleotide), inhibits its function. For e.g., Liu found that mRNA-sensing guide RNAs are inactive when their targeting portion is double stranded in a stem loop and become active when mRNA-binding destroys the double-stranded stem thereby liberating the targeting portion (whole document, particularly Fig. 2C).
When claims 1-2, 4-6, 8-12, 25-26, 49-50, 55, 62, and 64- 68 are analyzed in light of the specification, the instant invention encompasses a large genera of chemically modified guide and protecting RNAs having a large genera of protecting sequences that hybridize to a large genera of target polynucleotide sequences as well as encompassing a large genera of types of chemical modifications and arrangement of modifications in the protecting RNA. The instant specification discloses a variety of chemical modifications to the ribose group [0106-0107], phosphate group [0108-0109] and nucleobase [0110], and possible combinations of modifications [027]. Table 1, page 25, Table 2, pages 25-34, Table 3 pages 40-42, and Table 4, pages 42-43 disclose chemical modification patterns and their sequences for crRNAs and protecting oligonucleotides.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure. In the instant case, the specification discloses sequences and modification patterns as seen in Tables 1-4. However, many of the protecting oligonucleotides and crRNA sequences encompass a large number of species of possible sequences in the 20 nucleotide guide and complementary protecting oligonucleotides that will target a large genera of target genes.
Protecting oligonucleotides: Exemplary sequences, RC01 – RC07 in Table 1, all include at least 4 nucleotides as “N” which is complementary to the guide region of the crRNA for targeting the specific gene, and therefore encompasses millions of species of target sequences targeting any target region of any gene of any organism. Table 3 pages 40-42 disclose specific sequences, SEQ ID NOs:135-151 that duplex with the corresponding to the crRNAs described below.
crRNAs: Exemplary sequences, SEQ ID NOs:5-126 in Table 2, all include the first 20 nucleotides as “N” which is the guide region of the crRNA for targeting the specific gene, and therefore encompasses millions of species of target sequences targeting any target region of any gene of any organism. Table 4 pages 42-43 disclose specific sequences (SEQ ID NO: 152-155) for targeting specific regions of specific genes including MCV1 and GFP. However, this is not a representative number of species of the large genus of chemically modified protecting RNA complementary to crRNA capable of hybridizing to any target polynucleotide sequence.
Therefore, the specification does not describe the essential structure of a representative number of species of the large genus of chemically modified protecting RNAs that encompasses targeting a large genus of genes and comprising a large genus of chemical modification patterns.
Next, then, it is determined whether a representative number of species have been sufficiently described by other relevant identifying characteristics, (i.e., other than nucleotide sequence), specific features and functional attributes that would distinguish different members of the claimed genus. In the instant case, there are no other features or functional attributes that would distinguish the different members of the genus.
Applicant’s attention is directed to the guidelines for the Examination of Patent Applications Under the 35 U.S.C. 112(a) or Pre-AIA 35 U.S.C. 112, first paragraph, "Written Description" Requirement (MPEP 2163).
Dependent claims 9 and 10 are additionally rejected for the requirement of a particular Tm, the disclosure does not have written description on this limitation.
Page 35 of the instant specification discloses high affinity interaction (of guide with target) may be enhanced by increasing the GC nucleotide content in the duplex repeat nucleotide sequence and the anti-repeat nucleotide sequence of crRNA and tracrRNA to increase binding affinity between the repeat and anti-repeat nucleotide sequences, and that modifications such as 2’-Fluoro and 2’-O-methyl modifications can be introduced which increase the melting temperature of the duplex.
However, claims 9-10 depend upon claim 8, which in turn depends upon claim 25. None of the claims that claims 9-10 depend upon require “tracrRNA”. Therefore, the description of altering Tm with a tracrRNA is irrelevant.
In conclusion, Applicant’s disclosure of SEQ ID NOs: 135-151 and SEQ ID NO: 152-155 in the instant disclosure is not deemed sufficient to reasonably convey to one skilled in the art that Applicant was in possession of the claimed broad genus at the time the application was filed. Thus, it is concluded that the written description requirement is not satisfied for the claimed genus.
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.
Claim(s) 1 and 12 are is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhang (WO 2016/094867 A1).
Regarding claim 1, Zhang teaches a guide RNA (para 00738, “protected sgRNA”) and contacting the cell with a chemically modified (para 0032) “protector RNA” nucleic acid (Fig 1; para 00137 “The protector strand can be either a separate RNA transcript or strand (also referred to herein as dual pgRNA)”) to form PROTECTED GUIDE RNAS (PGRNAS) (title). Zhangs’ protector strand binds the crRNA (pg. 4 para 0014 “In one aspect, the invention provides for hybridizing a “protector RNA” to a guide sequence, wherein the “protector RNA” is an RNA strand complementary to the 5’ end of the sgRNA, to thereby generate a partially double-stranded sgRNA.). The guide RNA has further nucleotides that may be appended to the 5’ end (extensions may be added to the 5’ end, abstract) as shown in Fig. 1. Fig. 1 also shows the other end (not appended to extension) to be free. Both the sgRNA and the nucleic acid bound to it are chemically modified (para 0032) nucleic acids (para 0032: In an embodiment of the invention, the protector sequence of the pgRNA comprises chemically modified bases. In an embodiment of the invention, the guide sequence comprise chemically modified bases. In another embodiment of the invention, both the extension sequence and the protector sequence comprise chemically modified bases. In another embodiment of the invention, the extension sequence, the protector sequence, and the guide sequence comprise chemically modified bases.). The protected guide improves specificity of target binding or improves stability as compared to naturally occurring CRISPR-Cas (para 0009). The working examples show EMX1.3 sgRNA which is hybridized to complementary sequences (Example 1). Thus, Zhang’s “protector strand” reads on instant protecting oligonucleotide.
Regarding claim 12, Zhang teaches at least one moiety conjugated to the protecting oligonucleotide ([00507] For a convenient detection of the probe-target complexes formed during the hybridization assay, the nucleotide probes are conjugated to a detectable label.).
Thus, Zhang anticipates instant claims 1 and 12.
Claim(s) 1-2 and 4-6 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Dong (US 2020/0056167 A1).
Regarding claim 1, Dong teaches a system including compositions and method for use in a CRISPR genome editing system (abstract). The system includes a chemically modified nucleic acid, comprising at least one chemically modified nucleotide, complementary to the guide RNA [0016] – [0017]. The crRNA comprising a 5' end and a 3' end (Fig. 3). In an embodiment of the invention, [0013] wherein the chemically modified nucleic acid inhibits the activity of the nuclease of the CRISPR genome editing system. Thus, Dong’s “nucleic acid” reads on instant protecting oligonucleotide.
Regarding claim 2, Dong teaches in some embodiments the nucleic acid is RNA [0024] and the modifications comprise modifications of a ribose group, a phosphate group, a nucleobase, ([0120]-[0134]; Table 1), or a combination thereof (claims 17-22 and 27-29).
Regarding claims 4-6, Dong also teaches wherein the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof ([0034]: FIG. 1E Schematic illustration of RNA-crRNA duplexes; [0036]: [T]he intermediate grey letters denote 2'-fluoro modified RNA (crRNA21 and 22). The light black letters denote 2'-O-methyl modified RNA (crRNA23, 24, and 25).)
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Thus, Dong anticipates instant claims 1-2 and 4-6.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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) 25-26, 8-11, 51, and 66 are rejected under 35 U.S.C. 103 as being unpatentable over Dong (US 2020/0056167 A1) in view of Sontheimer (WO 2019183000 A1). Claim 11 is evidenced by Liu (US 20150071901).
Regarding claim 25, Dong teaches a chemically modified nucleic acid, comprising at least one chemically modified nucleotide, complementary to the crRNA (guide RNA [0016] – [0017], which are part of a CRISPR genome editing system (abstract). The crRNA comprising a 5' end and a 3' end (Fig. 3).
Regarding claim 26, Dong teaches in some embodiments the nucleic acid is RNA [0024] and the modifications comprise modifications of a ribose group, a phosphate group, a nucleobase, ([0120]-[0134]; Table 1), or a combination thereof (claims 17-22 and 27-29).
Regarding claim 66, Dong teaches wherein the protecting oligonucleotide binds to a region of the crRNA comprising at least one unmodified nucleotide (Fig. 1E).
Dong does not teach wherein the crRNA comprises at least 50% modified nucleotides (claim 25).
However, before the effective filing date of instant invention, Sontheimer had taught several heavily modified versions of gRNA that are more potent than their unmodified counterparts (summary). In particular, Sontheimer had taught wherein the crRNA comprises at least 80% modified nucleotides (claim 1). Sontheimer also taught various advantages of extensively modified RNAs such as: stability, improved potency, and/or reduced off-target effects [007].
It would have been obvious to one of ordinary skill in the art to have arrived at a crRNA that comprises at least 50% modified nucleotides in the process of optimizing the activity and stability of the crRNA with guidance from Sontheimer such as various positions of the crRNA that were amenable to modification without compromising activity. One would have been motivated to incorporate such a modified crRNA that comprises at least 50% modified nucleotides in to a duplex with a protecting oligonucleotide for the advantage of obtaining a ds oligonucleotide that was resistant to nuclease-mediated breakdown in vivo. One would have had reasonable expectation of success in doing so because both Dong and Sontheimer taught that modifications on RNA are possible and modifications render an oligonucleotide nuclease resistant. See MPEP 2144 II.
Regarding claim 8, Dong further teaches wherein the protecting oligonucleotide binds to the crRNA to form a duplex. See recitation:
[0072] In some embodiments, the complementary region
between the nucleic acid and the guide RNA comprises at
least 10 nucleotides.
Regarding claims 9-10, neither Dong nor Sontheimer discuss the Tm of the duplex or even explicitly teach that Tm is greater or less than the Tm of the unmodified nucleic acid duplex. The Tm is a property attributed to the structure of the composition. As per MPEP 2112.01, Composition, Product, and Apparatus Claims [R-10.2019] title: I. PRODUCT AND APPARATUS CLAIMS — WHEN THE STRUCTURE RECITED IN THE REFERENCE IS SUBSTANTIALLY IDENTICAL TO THAT OF THE CLAIMS, CLAIMED PROPERTIES OR FUNCTIONS ARE PRESUMED TO BE INHERENT, and considering that the structure of the composition of matter made obvious in Dong and Sontheimer is substantially identical to that of the instant application, the claimed property of the composition, namely the Tm, is inherent. Thus, the Tm of Dong and Sontheimer’s duplex is as recited.
Regarding claim 51, Sontheimer further teaches wherein the crRNA portion has the
mN#mN#mN#mNmNmNmNmNmNmNfNfNfNfNrN#rN#fNfNrN#m NmGrU#rU#rU#fUfAmGmAmGmCmUmAmU#mG#mC#mU (crRNA 20) [0032], which is the same as the recited pattern:
mN#mN#mN#mNmNmNmNmNmNmNfNfNfNfNrN#rN#fNfNrN#m
NmGrU#rU#rU#fUfAmGmAmGmCmUmAmU#mG#mC#mU (SEQ ID NO:3) (crRNA 20).
Thus, Dong in view of Sontheimer make obvious instant claims 25-26, 8-11, 51, and 66.
Claim(s) 11, 49-50 and 55 are rejected under 35 U.S.C. 103 as being unpatentable over Dong (US 2020/0056167 A1) in view of Sontheimer (WO 2019183000 A1) as applied to claims 25-26, 8-10, 51, and 66 above and further in view of Liu (US 20150071901). Claim 49 is evidenced by Jiang and Doudna (Jiang and Doudna, Annu. Rev. Biophys. 2017. 46:505–29).
The double stranded oligonucleotide of claim 25 is taught above. Dong further teach the double stranded oligonucleotide is part of a genome editing system (Dong abstract). Dong further teach that the oligonucleotide of their invention, inhibits a nuclease activity of the CRISPR genome editing system [0113]. Dong further teach chemically modifying nucleobases of the mRNA encoding a Cpfl protein and/or the guide RNA of the CRISPR genome editing systems [0126]. Dong further teach Cpfl protein is a programmable endonuclease ([0139] Cpf1 (CRISPR from Prevotella and Francisella 1) is one of the CRISPR associated effector endonucleases, which induces double stranded DNA breaks under the guidance of a single CRISPR RNA (crRNA)).
Dong does not teach a trans-activating crRNA (tracrRNA) as part of the genome editing system.
However, before the effective filing date of instant invention, Liu had taught a similar strategy of inactivating a gRNA by binding it to a complementary oligonucleotide (mRNA), which control the binding and hence cleavage activity of RNA-programmable endonucleases (abstract). Liu taught that protecting a functional single-stranded nucleic acid (guide sequence) by hybridizing it to a complementary strand (protecting oligonucleotide), inhibits its function. For e.g., Liu found that mRNA-sensing guide RNAs are inactive when their targeting portion is double stranded in a stem loop and become active when mRNA-binding destroys the double-stranded stem thereby liberating the targeting portion (whole document, particularly Fig. 2C).
Thus, Liu evidenced that the modified nucleic acids that modulate the activity of genome editing of Dong, are applicable to other CRISPR systems.
Regarding claim 11, Liu evidences that protecting a functional single-stranded nucleic acid (guide sequence) by hybridizing it to a complementary strand (protecting oligonucleotide), inhibits its function. For e.g., Liu found that mRNA-sensing guide RNAs are inactive when their targeting portion is double stranded in a stem loop and become active when mRNA-binding destroys the double-stranded stem thereby liberating the targeting portion. Therefore, the recitation wherein the binding of a trans-activating crRNA (tracrRNA) to the guide sequence portion of the crRNA dissociates the protecting oligonucleotide from the crRNA is evidenced by Liu.
Regarding claims 49-50, Liu taught that in type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA) ([0037]; Figs. 1-4). Jiang and Doudna evidence that a fused guide RNA with tracrRNA comprise the following structure: 3' end of the crRNA further comprises a nucleotide loop linking the 3' end of the crRNA portion to 5' end of a trans-activating crRNA (tracrRNA) comprising an anti-repeat nucleotide sequence that is complementary to the repeat sequence portion of the crRNA. See Fig. 4B. Thus, the limitation of a trans-activating crRNA (tracrRNA) comprising a 5' end, a 3' end, and an anti-repeat nucleotide sequence that is complementary to the repeat sequence portion of the crRNA is met in Liu’s teachings.
Regarding claim 55, Liu further teaches any suitable RNA-guided nuclease may be used e.g., S. pyogenes Cas9 (SpCas9) ([0037]).
It would be prima facie obvious to utilize a ds oligonucleotide comprising a protecting oligonucleotide and a crRNA as taught by Dong in a Type II CRISPR-Cas (Cas 9) system as Liu provide the teachings and motivation to do so. Further, Liu had taught that a tracrRNA is required by the Cas9 endonuclease and Jiang and Doudna evidenced the structure of a tracrRNA as having 5’ and 3’ repeat and anti-repeat sections. One skilled in the art would be motivated to adopt a Type II CRISPR-Cas (Cas 9) system as the genome editing system because of the availability of many Cas9 proteins from various species. Generally, it is prima facie obvious to select a known material for incorporation into a composition, based on its recognized suitability for its intended use. One of ordinary skill in the art would have reasonable expectation of success because both Dong and Liu taught an endonuclease-based process works when a single oligonucleotide is hybridized via its complementary sequence to a gRNA. See MPEP 2144.07 and 2143 G.
Thus, Dong in view of Liu make obvious instant claims 11, 49-50 and 55.
Claim(s) 65 is rejected under 35 U.S.C. 103 as being unpatentable over Dong (US 2020/0056167 A1) in view of Sontheimer (US 20210363518 A1).
The protecting oligonucleotide of claim 2 was anticipated by Dong.
Regarding claim 65, specifically, Dong taught the modification on the nucleic acid (protecting oligonucleotide) is 2' -O-methyl modified RNA (2' - O -methyl modified RNA is shown with double-underlined text (Oligo 23 in Table 1, pg. 17)).
Dong did not teach wherein (the phosphate group) modification is independently selected from the group consisting of a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, and a phosphotriester and specifically any of the above phosphate modifications in combination with ribose modification, as recited in claim 65.
However, before the effective filing date of instant invention, Sontheimer (US 20210363518 A1) had taught several heavily modified versions of gRNA that are more potent than their unmodified counterparts (summary). Sontheimer’s teachings are discussed supra and apply.
Sontheimer taught combinations [0009] of ribose mods [00110]-[0011], and of phosphate mods [0012]. Sontheimer taught several advantages for modifying guide RNAs:
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For e.g., various advantages of extensively modified RNAs such as: in vivo or ex vivo, including stability, improved potency, and/or reduced off target effects. Furthermore, in certain embodiments, the modified RNAs of the disclosure have reduced immunogenicity, e.g., a reduced ability to induce innate immune responses prevent [0007].
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have adapted the various chemistries disclosed by Sontheimer with respect to guide RNAs on to the protecting oligonucleotides according to the teachings of Dong, i.e., the protecting oligonucleotide has specific ribose and phosphate modifications, as recited in claim 65. One would have been motivated to do so for the various advantages discussed by Sontheimer with respect to modified RNA would apply to any oligonucleotide irrespective of its function as a guide RNA or not, for e.g., various advantages of extensively modified RNAs such as: in vivo or ex vivo, including stability, improved potency, and/or reduced off target effects. Furthermore, in certain embodiments, the modified RNAs of the disclosure have reduced immunogenicity, e.g., a reduced ability to induce innate immune responses prevent. It would have been obvious to one of ordinary skill in the art to have arrived at such a protecting oligonucleotide in the process of optimizing the activity and stability of the duplex formed by binding of the protecting oligonucleotide with crRNA. One would have been motivated to do so in order to obtain a protecting oligonucleotide that was resistant to nuclease-mediated breakdown in vivo, and because Sontheimer demonstrated various positions of the crRNA that were amenable to modification without comprising activity. One would have had reasonable expectation of success in doing so because both Dong and Sontheimer taught that modifications on RNA are possible and modifications render a more nuclease resistant oligonucleotide. See MPEP 2144 II.
Thus, Dong in view of Sontheimer make obvious instant claim 65.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dong (US 2020/0056167 A1) in view of Hedgpeth (US 2004/0058322 A1).
Regarding claim 12, the protecting oligonucleotide of claim 1 was anticipated by Dong. Dong did not teach either at least one moiety conjugated to the protecting oligonucleotide, rather teaches a moiety conjugated to the guide RNA, or any details about a linker.
However, before the effective filing date of instant invention, Hedgpeth had taught several advantages for conjugating a short oligonucleotide to a moiety wherein the oligonucleotide was required to form stable hybrids with complementary target sequences (abstract). One embodiment of a conjugated moiety is a covalently bound oligonucleotide and minor groove binder (minor groove binder, MGB) combination which includes an oligonucleotide having a plurality of nucleotide units, a 3'-end and a 5'-end, and a minor groove binder moiety covalently attached to at least one of said nucleotides ([0018]). The moiety can be attached to the 3' or 5' end of said oligonucleotide [0054]. Some advantages of a conjugated moiety on the short oligonucleotide are: lessening of the differences in Tm between short oligonucleotides of different base compositions, when such short oligonucleotides are conjugated to a MGB, [0323], improvements in speed, sensitivity and versatility to a variety of assays involving hybridization of oligonucleotides [0022], and increase in stability as recited below:
.
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Hedgpeth teaches oligonucleotides of various lengths and Tm that have been experimentally validated to carry a conjugated moiety (Table 6, pg. 40). Hedgpeth experimentally determine the differences between unmodified and MGB-conjugated oligonucleotides and find better performance, to wit priming, for conjugated oligonucleotides ([0030], [0260]-[[0263]; FIGs. 3-4).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have conjugated a moiety to the protecting oligonucleotide of Dong according to guidance from Hedgpeth. It would have merely amounted to a simple substitution of prior art elements; i.e., move the moiety from the crRNA to the protecting oligonucleotide, according to known methods to yield predictable results. The skilled artisan would have had a reasonable expectation that conjugating a moiety to the protecting oligonucleotide could stabilize the binding of the protecting oligonucleotide with the crRNA and work as predicted because 1) Hedgpeth had experimentally demonstrated increased stability for a duplex when the short oligonucleotide was conjugated to a moiety; 2) Hedgpeth teaches oligonucleotides of various lengths conjugated to moieties that have been experimentally validated and their Tm determined. See MPEP 2143 I (B).
Thus, Dong in view of Hedgpeth makes obvious instant claim 12.
Claim(s) 62, 64, 67-68, and 52 are rejected under 35 U.S.C. 103 as being unpatentable over Dong (US 2020/0056167 A1) in view of Hedgpeth (US 2004/0058322 A1) as applied to claim 12 above, and further in view of Sontheimer (US 20210363518 A1).
The protecting oligonucleotide of claim 12 conjugated to a moiety was made obvious by Dong in view of Hedgpeth. Hedgpeth further taught that the MGB may carry other functionalities or can be substituted by a reporter group [0048]. Hedgpeth also taught that conventional techniques are used in the process of conjugating moieties to oligonucleotides [0034].
Neither Dong nor Hedgpeth had taught a linker.
However, before the effective filing date of instant invention, Sontheimer (US 20210363518 A1) had taught several heavily modified versions of gRNA that are more potent than their unmodified counterparts (summary). Sontheimer’s teachings are discussed supra and apply.
Regarding claim 62 and 64, Sontheimer taught moieties attached to terminal nucleotides of the guide RNA via a linker. Sontheimer taught moieties may be conjugated on the 5' end and/or the 3' end of a crRNA and/or a tracrRNA, as, for example, in FIGS. 14A-14D ([0197] Exemplary linkers include... are not limited to an ethylene glycol chain, an alkyl chain, a polypeptide, a polysaccharide, a block copolymer, and the like).
Sontheimer does not disclose that these are cleavable linkers. However, ADC Review, (Journal of Antibody-drug Conjugates, April 3, 2015 downloaded from the internet on 12th April 2026 from < https://www.adcreview.com/stable-linker-technologies/peg-linkers/>) evidence that at least ethylene glycol based linkers (e.g., PEG) are cleavable.
Regarding claim 67, Sontheimer taught wherein the at least one moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides analogs, nucleoside analogs, endocannabinoids, vitamins, receptor ligands, peptides, aptamers, and alkyl chains [0194].
Regarding claim 68, Sontheimer taught wherein the at least one moiety is selected from the group consisting of cholesterol, cholesterol-triethylene glycol (TEGChol), docosahexaenoic acid (DHA), docosanoic acid (DCA), lithocholic acid (LA), GalNAc, amphiphilic block copolymer (ABC), hydrophilic block copolymer (HBC), poloxamer, Cy5, and Cy3 [0195].
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have adapted the various chemistries disclosed by Sontheimer with respect to guide RNAs on to the protecting oligonucleotides according to the teachings of Hedgpeth, i.e., conjugating moieties to terminal nucleotides of the protecting oligonucleotides, and arrive at a protecting oligonucleotide conjugated to a moiety via a cleavable linker design as recited in claims 62, 64, and 67-68. One would have been motivated to do so for the various advantages discussed by Hedgpeth (in the rejection above) with respect to conjugating the short oligonucleotide with a terminal moiety, for e.g., conjugation of the smaller oligonucleotide imparts greater stability to the duplex formed. One would have had reasonable expectation of success in doing so because Hedgpeth taught that conventional techniques are used in the process of conjugating moieties to oligonucleotides. See MPEP 2144 II.
Thus, Dong and Hedgpeth in view of Sontheimer make obvious instant claims 62, 64, 67- and 68.
The protecting oligonucleotide conjugated to a moiety via a linker of claim 64 was made obvious by Dong and Hedgpeth in view of Sontheimer.
Regarding claim 52, Hedgpeth teaches various sequences that are conjugated to moieties. The table shows that conjugation of an oligonucleotide (ODN) dramatically increases the stability of the hybrid formed between the oligonucleotide and its target. Increased stability
(i.e., increased degree of hybridization) is indicated by a higher Tm. Hedgpeth shows that the sequences are complementary to M13mp19 (phage genome).
See below:
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Hedgpeth then study 2'-O-Me-RNA modified duplexes. As seen from the table below conjugated 2'-O-Me-RNA modified backbone forms more stable duplexes with other conjugated 2'-O-Me-RNA modified backbone than with DNA.
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The bolded nucleotides in the alignment below show the similarity of a DNA version of the recited RC09 with Hedgpeth’s sequence.
RESULT 44
US-10-127-936-2
(NOTE: this sequence has 3 duplicates in the database searched)
Sequence 2, US/10127936
Publication No. US20040058322A1
GENERAL INFORMATION
APPLICANT: Meyer, Rich
TITLE OF INVENTION: IMPROVED HYBRIDIZATION AND
TITLE OF INVENTION: MISMATCH DISCRIMINATION USING OLIGONUCLEOTIDES
TITLE OF INVENTION: CONJUGATED TO MINOR GROOVE BINDERS
CURRENT APPLICATION NUMBER: US/10/127,936
CURRENT FILING DATE: 22-Apr-2002
PRIOR APPLICATION NUMBER: US/09/640,953
PRIOR FILING DATE: 16-Aug-2000
PRIOR APPLICATION NUMBER: US/09/054,832
PRIOR FILING DATE: 03-APR-1998
PRIOR APPLICATION NUMBER: 08/415,370
PRIOR FILING DATE: 03-APR-1995
NUMBER OF SEQ ID NOS: 40
SEQ ID NO 2
LENGTH: 12
TYPE: DNA
Query Match 62.7%; Score 9.4; Length 12;
Best Local Similarity 90.9%;
Matches 10; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 TAAAACCGAGG 11
|||||| ||||
Db 2 TAAAACAGAGG 12
Therefore, it would be prima facie obvious to modify Hedgpeth’s SEQ ID NO: 2 by modifying with 2'-O-Me-RNA as Hedgpeth provide the teachings and motivation to do so. Further, Hedgpeth had taught that short oligonucleotides are stabilized in duplexes when conjugated and further such modified RNA-RNA hybrids are more stable than DNA-RNA hybrids. One skilled in the art would have a reasonable expectation of success because the combination of modified sequences used in the method would be performing the same function in the Dong as it was in the method of Hedgpeth. One of ordinary skill in the art would have performed length optimization of the known sequences and determine the right Tm as Tm determination is obvious and routine, and also demonstrated by Hedgpeth. While arriving at a particular SEQ ID NO may not be obvious, reaching any other sequence would have been obvious. If carrying out such optimization leads to anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization. See MPEP 2143(I) A, G, and MPEP 2144(II). One of ordinary skill in the art would have a reasonable expectation of success since both Hedgpeth and Dong teach stabilizing duplexes for further functional activity.
Length and 2'-O-Me-RNA modification of Hedgpeth’s SEQ ID NO: 2 shown in Table 6 would result in sequences sufficiently close to RC09.
Thus, Dong and Hedgpeth in view of Sontheimer make obvious instant claim 52.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Relevant Prior Art Not relied Upon
The following art is made note of and not currently relied on, but is relevant to applicants invention. Notebaart (WO 2020/065062 A1) teaches oligonucleotides of rational design and which are antisense to target sequence of the guide RNA, and function to protect the gRNA. See Fig. 1.
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The closest prior art is applied above.
Conclusion
No claims are allowed.
Correspondence
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SHABANA S. MEYERING, Ph.D.
Examiner
Art Unit 1635
/SHABANA S MEYERING/ Examiner, Art Unit 1635
/CATHERINE KONOPKA/ Primary Examiner, Art Unit 1635
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