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
Applicant's election with traverse of Invention I (e.g. claims 1-3, 5-18 and 24-26) drawn to preparing segmented nucleic acids joined by triazole linkers; and
“a guide RNA” as the type of segmented nucleic acid corresponding to Species (A); and “a 3’-azido modified segment” as the type of modification at the 3’-end of segment 1 corresponding to Species B in the reply filed on January 20, 2026 is acknowledged.
The Examiner respectfully notes the elected species of a 3’-azido modified segment for segment 1 corresponding to Species B as discussed above read on claims 1 and 25, see claim 1, lines 3-4 and claim 25, lines 3-4; as the Examiner further notes each of claims 15-17 and 26 recite within lines 3-4 the limitation “synthesis of segment 1 contains an alkynyl modification at its 3’-end”.
Therefore, the Examiner notes the elected species (A) and (B) as discussed above encompass only claims 1-3, 5-10, 14, 18 and 24-25.
The traversal is on the ground(s) that:
(A) Examples 2 and 5 in the specification (see paragraphs [000152] and [000161]-[000162]) clearly demonstrate the use of an example azido support (3’-Azido CPG 1000Å) for synthesis of 3’-azido modified long oligonucleotides; where
modified segments to be ligated within claim 1 can be prepared by direct synthesis using appropriate solid supports (e.g. azido support) or widely used post synthesis modifications; wherein
using azido solid supports are based on Applicant’s surprising discovery that azido supports are useful for direct synthesis of 3’-azido modified long nucleotides; and
synthesis of nucleic acids on solid supports is a predominant practice; as solid supports are critical reagents in oligonucleotide synthesis and “post synthesis”, wherein post synthesis modifications require multiple repetitive and expensive separation and purification steps, resulting in significantly reduced overall yields and substantially increased production costs. See Applicant’s remarks, filed January 20, 2026, pg. 9, paragraphs 1-2.
This argument is not found persuasive because the Examiner respectfully reiterates claim 1 is drawn to preparing segmented nucleic acids joined by triazole linkers; wherein claim 1 of Invention I does not recite nor require use of a solid support within the preparation method, as evidenced by claim 2, line 2, which is the first recitation of the limitation “a solid support”.
Additionally, the Examiner respectfully notes the solid support recited within claim 2 does not require the structural limitations of the solid support recited within claims 27-28.
Furthermore, the Examiner respectfully notes the preparation of the segmented nucleic acids of claim 1 can be performed using different designs and modes of operation as taught by Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892), where Zhong teaches two RNA modules are synthesized chemically by phosphoramidite chemistry and ligation chemistry either on solid support(s) or in solution, see paragraph [0082]; and synthetic methods include ligation via formation of a 1,2,3-triazole crosslinker, see paragraph [0083].
Applicant argues:
(B) Species (A) of this invention has at least one common non-natural structural element; they are all segmented nucleic acids comprising one or more triazole links formed by a click reaction, see Applicant’s remarks, filed January 20, 2026, pg. 10, paragraph 3.
This is not found persuasive because the Examiner respectfully notes claim 1 recites “preparation of segmented nucleic acids” which the Examiner reasonably interprets the phrase “nucleic acids” to mean any nucleic acid.
Therefore, the preparation method of claim 1 does not require the nucleic acid be a ribozyme, required in claim 11; an aptamer required in claim 12, or a guide RNA of human ADAR1 or ADAR2 required in claim 14.
Thus, when searching and considering the limitation “nucleic acid” the Examiner respectfully notes searching and considering one type of nucleic acid does not necessarily include within the search results the additional structures as discussed above, for example searching structures corresponding to a ribozyme do not necessarily encompass aptamers or guide RNA of human ADAR1 or ADAR2 as discussed above.
(C) Species (B) are various embodiments of this invention, namely making terminal modifications of nucleic acid segments for click reaction-based ligations, see Applicant’s remarks, pg. 10, last paragraph of the page.
This is not found persuasive because the Examiner respectfully notes each nucleic acid segment requires a specific structural element (e.g. an azido, an alkyne, or an amino) at either the 5’-end and/or the 3’-end of the segment, and thus when searching for an azido modification at the 3’-end of segment 1 does not necessarily encompass searching and considering segment 1 having different structural modifications for example alkyne or amino modifications which can used within chemical ligation techniques; for example in click reactions as argued by Applicant within argument (C) above.
(D) Species (C) are various embodiments of this invention where the azido supports of claim 27 are used to make 3’-azido modified nucleic acid segments; and wherein Claim 28 further limits the limitation “-LL-“ of claim 27 to the structures comprising C=O and NH of two amide bonds as shown in claim 28, see Applicant’s remarks, pg. 11, paragraph 3.
This argument is rendered moot in view of the election of Invention I as discussed above.
Although, the Examiner does respectfully reiterate to Applicant the argument of no common structural element for the recitation of “-LL-“ as recited within Formula I of claim 27, see the restriction/election requirement mailed October 20, 2025, pg. 6, paragraph 1.
Therefore, arguments (A)-(D) have been fully considered but are not found persuasive for the reasons discussed above. Thus, the Examiner respectfully notes the requirement is still deemed proper and is therefore made FINAL.
Claims 11-13, 15-17, and 26-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species or invention where applicable, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on January 20, 2026.
Claim Status
The claim set and Applicant’s remarks filed January 20, 2026 have been entered.
Claims 4 and 19-23 are canceled.
Claims 11-13, 15-17, and 26-28 are withdrawn from further consideration as being drawn to either a nonelected species or invention where applicable as discussed in greater detail within the Election/Restrictions section above.
Thus, claims 1-3, 5-10, 14, 18 and 24-25 as filed are examined on the merits herein.
Claim Objections
Claim 6 is objected to because of the following informalities:
Claim 6, line 1, recites 3’-aimino nucleic acid”, which the Examiner reasonably interprets “aimino” is the misspelling of “amino” as evidenced by claim 1, line 6 which recites “amino at its 3’-end”. Thus, to promote clarity the Examiner respectfully suggests replacing “aimino” with “amino” as discussed above.
The Examiner encourages the Applicant to review the entire claim set for additional typos or potential grammatical errors within the claims and to resolve them where applicable.
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.
Claims 1-3, 5-10, 14, and 25 are 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.
The phrases:
“at a position close to its 5'-end” recited in line 6, claim 1;
“at a position close to its 3'-end” recited in lines 6-7 in claim 1;
“at a position close to its 5'-end” recited in lines 5-6 in claim 25; and
“at a position close to its 3'-end” recited in lines 6-7 in claim 25 are all relative terms by reciting the phrase “close to” in relation to a position in the nucleic acid which renders these claims indefinite. The phrase “close to” is not defined by either claim 1 or claim 25, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The Examiner respectfully notes these limitations refer to segment 2 which is a segment of nucleic acid containing 8-200 nucleotides (nt) in length, see claim 1, lines 5-7 and claim 25, lines 5-7.
The Examiner also notes segment 2 recites the limitation “containing” within line 5 of claim 1 and line 5 of claim 25, which the Examiner reasonably interprets the phrase “containing” is synonymous with the word “comprises” and is thereof inclusive or open-ended and does not exclude additional, unrecited elements. See MPEP 2111.03(I).
The Examiner also notes the specification only exemplifies “close to its 3’-end” as any one or several nucleotides, or its full sequence (see pg. 16, paragraph [00090]).
Therefore, the Examiner respectfully notes the specification does not clearly define nor provide guidance to one of ordinary skill in the art in how the limitation “close to” is to be interpreted; as the specification does not define an exact nucleotide position nor a range of nucleotide positions wherein segment 2 contains an alkyne close to its 5’-end or an amino close to its 3’-end as recited within claim 1, lines 5-7 and claim 25, lines 5-7.
Consequently, the Examiner reasonably interprets “close to” in view of the exemplification as discussed above to mean the alkyne or amino could be present anywhere on the applicable nucleic acid segment or may be connected to a structure connected to the applicable nucleic acid segment as discussed above.
Furthermore, the Examiner notes the meaning of the phrase “close to” is relative to the length and structure of the nucleic acid segment, for example segment 2 which is previously discussed above contains 8 to 200 nt; and wherein “close to” is exemplified to be any one or several nucleotides, or its full sequence as discussed above.
Therefore, the metes and bounds of the phrase “close to” as recited within claim 1 and claim 25 above and in view of the specification as disclosed above is unclear and indefinite as to how the phrase “close to” is to be interpreted when referring to the alkyne or amino in segment 2 as recited in claim 1 and claim 25 as discussed above; as the Examiner respectfully notes the phrase “close to” is not clearly defined by the specification as discussed above.
Thus, in view of the forgoing reasons above, claim 1 and claim 25 are unclear and indefinite.
Claims 2-3, 5-10 and 14 are included within this rejection as these claims either depend from or rely on independent claim 1.
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.
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.
(I) Claims 1-2, 6-7, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892).
Regarding claim 1-2, 6-7 and 14, Zhong teaches chemically ligated guide RNA (e.g. guide RNA, required in claim 7, line 1) oligonucleotides which comprise two functional modules (crgRNA and tracrgRNA) (e.g. segmented nucleic acids, required in claim 1, line 1) joined by non-nucleotide chemical linkers (nNt-linker), their complexes with CRISPR-Cas9 (e.g. CRISPR-Cas RNP complex, required in claim 7, line 2), and discloses processes and methods for preparation of these compounds (e.g. method of preparation, required in claim 1, line 1), see abstract.
Zhong teaches tracrgRNA is a ligated dual oligonucleotide, comprising tracrgRNA1 and tracrgRNA2, or multiple oligonucleotides, see paragraph [0027].
Zhong teaches the ligation between crgRNA and tracrgRNA (ligation 1) is through the formation of a triazole (e.g. by triazole linkers, required in claim 1, line 2) by Cu(I) catalyzed [2+3] cycloaddition, see paragraph [0111].
Zhong teaches “crgRNA” refers to crRNA equipped with chemical functions for conjugation/ligation, see paragraph [0068].
Zhong teaches “tracrgRNA” refers to tracrRNA equipped with chemical functions for conjugation/ligation, see paragraph [0069].
Zhong teaches “chemical ligation” refers to joining together of synthetic oligonucleotides via an nNt-linker by chemical methods such as click ligation (the azide-alkyne reaction to produce a triazole linkage) and formations of other chemical groups, see paragraph [0071].
Zhong teaches the two RNA modules (crgRNA and tracrgRNA) are synthesized chemically by phosphoramidite chemistry either on solid support(s) or in solution (e.g. synthesis, required in claim 1, line 3 and line 5); where non-limiting examples of compounds include oligonucleotide azides (e.g. azido modification, required claim 1, line 3) and oligonucleotide alkynes (e.g. an alkyne, required in claim 1, line 5), see paragraph [0082].
Zhong teaches step 1: synthesis of crgRNA and tracrgRNA where non-limiting examples of structures are given below and include a tracrgRNA which includes an alkyne at the 5’-end depicted as
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548
528
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, see pg. 4, paragraph [0022], tracrgRNA; as well as a crgRNA which includes an azide at the 3’-end depicted as,
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480
686
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, see pg. 5, first recited compound, crgRNA.
The Examiner notes the tracrgRNA is an oligonucleotide of 61 nt in length corresponding to segment (2) of 8-200 nt in length required in claim 1, line 5; and the crgRNA is an oligonucleotide of 32 nt in length corresponding to segment (1) of 8-200 nt in length in claim 1, line 3.
The Examiner notes the two compounds above correspond to the limitation where segment 1 contains an azido modification at its 3’-end required in claim 1, lines 3-4; and segment 2 contains an alkyne at its 5’-end required in claim 1, lines 5-6.
Zhong teaches step 2: chemical ligation of crgRNA and tracrgRNA depicted as,
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544
860
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, where the Examiner notes conjugation of crgRNA (e.g. segment 1, required in claim 1, line 1 and line 8) and tracrgRNA (e.g. segment 2, required in claim 1, line 4 and line 8) are conjugated by reaction between said azido and alkyne to form a two-segmented nucleic acid linked by the resulting triazole corresponding to the limitations within claim 1, lines 8-9.
Zhong teaches oligonucleotide amines used in ligation via formation of a 1,2,3-triazole linker via direct modification of the fully deprotected oligonucleotide amine such as ON-1, depicted as
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504
730
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, see paragraphs [0083]-[0084].
The Examiner notes the oligonucleotide amine exemplified as ON-1 as depicted above contains an amino at the 3’-end corresponding the limitation of “an amino at its 3’-end” required in claim 1, lines 6-7.
The Examiner notes ON-1’s amino at its 3’-end is transformed into an azido as depicted in ON-2 above corresponding to the limitation recited in claim 1, lines 10-11.
Zhong teaches chemical ligations for ligation between crgRNA and tracrgRNA (ligation1) are applicable to formation of ligated dual tracrgRNA between tracrgRNA1 and tracrgRNA2 (ligation2), see paragraph [0091].
Zhong teaches the two ligations can be formed by the same chemistry such as an azide-alkyne [3+2] cycloaddition, see paragraph [0096].
The Examiner notes the two preceding paragraphs correspond to the limitation of “conjugation of azido two-segmented nucleic acid d) to another segment between said azido and an alkyne in said another segment”, required in claim 1, lines 12-13.
Zhong exemplifies purification of the oligonucleotide is carried out by reversed-phrase HPLC on a Gilson system using an XBridgeTM BEH300 Prep C18 10µM 10x250 mm column (Waters) (e.g. separate the segmented nucleic acid from unreacted shorter segments and chemical reagents, required in claim 1, lines 16-17), see paragraph [0143].
Although, Zhong does not (a) depict tracrgRNA having an amino at its 3’-end, required in claim 1, lines 6-7; (b) teach said alcohol is substituted within an azido, required in claim 2, line 2; and (c) teach addition of the 5’-alkynyl modifier to the detritylated oligonucleotide on solid support, see claim 5, lines 5-6.
However, in the same field of endeavor of producing segmented nucleic acids, with respect to limitations (a) and (c), the Examiner notes Zhong explicitly teaches oligonucleotide amines used in ligation via formation of a 1,2,3-triazole linker as discussed above, wherein said amine is at the 3’-end of an oligonucleotide, exemplified as ON-1, and transformed into an azido, as exemplified as ON-2, as discussed above.
Zhong further exemplifies an alkyne function is introduced at the 5’-end nucleotide of the tracrgRNA exemplified by the non-limiting example ON-12 by solid phase synthesis (e.g. preparation of 5’-alkynyl, 3’-amino nucleic acid of claim 1, step b), see claim 6, lines 1-2), see paragraph [0112].
Zhong exemplifies the synthesis of ON-12 depicted as:
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308
324
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566
550
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508
538
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, see pp. 21-22, paragraph [0112].
The Examiner notes the synthesis scheme above depicts the addition of the 2’-alknyl modifier on solid support; and wherein the oligonucleotide is detritylated, wherein DMTr is removed and reintroduces the -OH group at the 5’-position of the last nucleotide recited within ON-12; and wherein ON-12 is cleaved from the solid support as depicted above. The Examiner respectfully notes DMTr is taught as a hydroxyl (e.g. -OH) protecting group in the synthesis of ON-12 above and is therefore within the scope of the artisan to use these teachings to synthesize the 5’-alkynyl modifier on the tracrgRNA molecule depicted and discussed on pg. 12 of this office action, first recited structure and taught by Zhong on pg. 4, paragraph [0022], thereby corresponding to the limitation recited in claim 6, lines 5-6.
The Examiner notes within the reaction scheme above, the second recited compound within the reaction scheme comprises a cyanoethyl phosphite which results in a phosphate after cleavage of the cyanoethyl group as depicted above, and thus corresponds to the limitation recited in claim 6, lines 7-9.
Zhong exemplifies oligonucleotide synthesis via solid support in Example 1 (see pg. 32), where compound 2 is prepared by treating compound 1 with 2-azidoethanol in dimethylacetamide at 120°C, at the presence of BF3.OEt2, and attached to an amino-functionalized support, see paragraph [0133], depicted as
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642
394
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, see paragraph [0132].
The Examiner notes the exemplification of Zhong above in producing compound 2 as discussed above corresponds to the limitation of claim 6, lines 3-4.
It would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have added an amino at the 3’-end of the tracgRNA as taught by Zhong above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated in order to produce the chemically ligated guide RNA oligonucleotide which comprise two functional modules (crgRNA and tracrgRNA) as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success to have added an amino at the 3’-end of the tracgRNA as taught by Zhong above, because Zhong explicitly teaches oligonucleotide amines used in ligation via formation of a 1,2,3-triazole linker via direct modification of the fully deprotected oligonucleotide amine by transformation into an azide as exemplified in ON-1 and ON-2 as taught by Zhong above.
With respect to limitation (b), Zhong teaches cleavage of oligonucleotides from the solid support and deprotection are achieved by exposure to concentrated aqueous ammonia/ethanol (3/1 v/v) for 2 h at room temperature followed by heating in a sealed tube for 45 min at 55°C, see paragraph [0141].
Zhong exemplifies azide and alkyne modifications are introduced as phosphoramidites in chemical synthesis of the RNAs, wherein Zhong exemplifies an alkyne modification is introduced as a phosphoramidite by chemical modification of a 5’-OH as depicted in ON-3, see paragraph [0085].
Therefore, with respect to the limitation of “step a) is performed on an alcohol attached to a support”; required in claim 2, lines 1-2; the Examiner reasonably interprets this to be a physical limitation of the solid support attaching the oligonucleotide of segment 1 of claim 1 to a solid support. Since Zhong teaches the two RNA modules (crgRNA and tracrgRNA) are synthesized chemically by phosphoramidite chemistry on solid support(s), the physical limitation as discussed above is met by the teachings of Zhong as discussed above.
Accordingly, with respect to the limitation “wherein said alcohol is substituted within an azido group, and subsequent global deprotection gives segment 1 containing azido modification at its 3’-end”, required in claim 2, lines 2-3; the Examiner reasonably interprets this limitation to be a physical consequence of (i) cleavage of the synthesized oligonucleotide attached via its 3’-alcohol to solid support as taught by Zhong above; (ii) after cleaving the synthesized oligonucleotide from solid support as taught by Zhong above modifying the alcohol at the oligonucleotide’s 3’-end with an azido group by introducing an azide containing phosphoramidite as taught by Zhong above and exemplified by crgRNA of Zhong as depicted above; and (iii) deprotection of the oligonucleotide as taught by Zhong above.
Therefore, the Examiner respectfully notes the teachings of Zhong as discussed above meet the recitation of “said alcohol is substituted within an azido”, as required in claim 2, line 2 within limitation (b) above.
With respect to the limitation “said segmented nucleic acid of claim 1 is an RNA conjugate”; required in claim 14; the Examiner reasonably interprets this limitation to be a physical limitation of the segmented nucleic acids prepared in claim 1. Since Zhong teaches synthesizing the segmented nucleic acids of claim 1, specifically a guide RNA comprising ligating at least crgRNA and tracrgRNA as discussed above, the physical limitation is met by the method of Zhong.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have added limitations (a)-(c) as taught by Zhong above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated in order to produce the chemically ligated guide RNA oligonucleotide which comprise two functional modules (crgRNA and tracrgRNA) as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success to have added limitations (a)-(c) as taught by Zhong above, because Zhong teaches limitations (a)-(c) within the method of Zhong as discussed in greater detail above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(II) Claims 3, 18 and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892) as applied to claims 1-2, 6-7 and 14 above, and further in view of Krasheninina et al. (Published 05 January 2021, Angewandte Chemical International Edition, Vol. 60, Issue 13, pp. 6970-6974, PTO-892).
Zhong addresses claims 1-2, 6-7 and 14 as written above. Zhong further teaches the production of ON-11, depicted as
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208
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, see paragraph [0138]; wherein after Fmoc cleavage, the resulting 3’-end aminoethyl (e.g. amine, required in claim 24, pg. 6, line 1) oligonucleotide is then treated with NHS ester of 6-azido caproic acid (e.g. NHS ester by formation of an amide, required in claim 24, lines 1-2) in DMF, see paragraph [0140].
Zhong teaches tracrgRNA is a ligated dual oligonucleotide (via ligation 2, the inner ligation between tracrgRNA1 and tracrgRNA2) where non limiting examples include:
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240
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, see paragraph [0080], where the Examiner notes the tracrgRNA2 comprises 24 nt (e.g. segment 3 of 8-200 nt in length, required in claim 25, lines 8-9).
Although, Zhong does not teach (i) step d) is executed via a diazotransfer reaction with fluorosulfuryl azide, required in claim 3, line 2, claim 18, lines 3-4; or (ii) segment 3 containing an alkyne modification at its 5’-end, required in claim 25, lines 8-9.
However, in the same field of endeavor of chemical transformations of amines within nucleic acids, with respect to limitation (i), Krasheninina teaches amine to azide conversion on native RNA via metal-free diazotransfer opens new avenues for RNA manipulation, see pg. 6970, title.
Krasheninina teaches a metal-free diazotransfer on native RNA containing an aliphatic primary amino group using the diazotizing reagent fluorosulfuryl azide (FSO2N3) (e.g. the diazotransfer with fluorosulfuryl azide, required in claim 3), see pg. 6970, abstract.
Krasheninina teaches the reaction provides the corresponding azide modified RNA in nearly quantitative yields without affecting the nucleobase amino groups. The obtained azido-RNA can then be further processed utilizing well-established biorthogonal reactions, such as azide-alkyne cycloadditions (Click). See pg. 6970, abstract.
With respect to the limitation “segmented nucleic acid conjugates”, recited in claim 18, line 1; the Examiner reasonably interprets this limitation to be a physical limitation of ligating at least two segments of nucleic acids. Since Zhong teaches two ligations via azide-alkyne [3+2] cycloadditions of a guide RNA which comprises crgRNA and tracrgRNA (ligation1) and forming a ligated dual tracrgRNA between tracrgRNA1 and tracrgRNA2 (ligation2), the physical limitation as discussed above is met by the method of Zhong as discussed above.
With respect to limitation (ii), it would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have added an alknyl modification at the 5’-end of the tracrgRNA2 as taught by Zhong above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated in order to produce the chemically ligated guide RNA oligonucleotide which comprise two functional modules, crgRNA and tracrgRNA, wherein said tracrgRNA is a ligated dual tracrgRNA between tracrgRNA1 and tracrgRNA2 as taught by Zhong above.
One of ordinary skill in the art would have had a reasonable expectation of success to have added an alkynyl at the 5’-end of the tracrgRNA2 as taught by Zhong above, because Zhong explicitly teaches a tracrgRNA which includes an alkyne at the 5’-end as discussed above and taught by Zhong on pg. 4, paragraph [0022] and wherein the alkyne function is introduced at the 5’-end of tracrgRNA via solid phase support synthesis as taught by Zhong in paragraph [0112], see pp. 21-22.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have incorporated (i) the diazotizing reagent fluorosulfuryl azide of Krasheninina into the method of converting the oligonucleotide comprising a primary amine as exemplified as ON-1 as taught by Zhong into an oligonucleotide azide as exemplified as ON-2 as taught by Zhong above; and (ii) have added an alkyne modification at the 5’-end of tracrgRNA2 as taught by Zhong above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to incorporate limitations (i)-(ii) into the method of Zhong as discussed above in order to create the chemically ligated guide RNA of Zhong as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success to have incorporated limitations (i)-(ii) as discussed above into the method of Zhong, because (i) Krasheninina and Zhong are drawn to chemically modifying primary amines present within nucleic acids into azides as discussed above; wherein Krasheninina explicitly teaches the reaction provides the corresponding azide modified nucleic acid, exemplifying RNA, in nearly quantitative yields without affecting the nucleobase amino groups; and wherein Krasheninina further explicitly the obtained azido-RNA of Krasheninina can then be further processed utilizing well-established biorthogonal reactions, such as azide-alkyne cycloadditions (e.g. Click reactions); and (ii) Zhong explicitly teaches alkyne function is introduced at the 5’-end of tracrgRNA via solid phase support synthesis as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(III) Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892) as applied to claims 1-2, 6-7 and 14 above, and further in view of El-Sagheer et al. (Published 09 February 2010, Chemical Society Reviews, Vol.. 39, pp. 1388-1405, PTO-892).
Zhong addresses claims 1-2, 6-7 and 14 as written above. Although, Zhong does not teach said transformation in step d) of claim 1 is an amide formation with a ligation function-substituted NHS ester, required in claim 5, lines 1-2.
However, in the same field of endeavor of click ligations of oligonucleotides, El-Sagheer teaches convenient syntheses of the relevant azide-modified oligonucleotides in Scheme 14 depicted as
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, see pg. 1400, right column, scheme 14.
El-Sagheer teaches azide-ODN 54 was prepared (Scheme 14) by reacting 3’- amino-C7-modified ODN 53 with 4-azidobutyric acid NHS ester 24 of carboxylic acid 52 in bicarbonate buffer at pH 8.75, see pg. 1400, right column, paragraph 1.
El-Sagheer teaches Scheme 14 is a convenient synthesis for copper-catalyzed azide–alkyne cycloaddition reactions, exemplifying the template-mediated chemical ligation of two oligonucleotide strands, one with a 5’-alkyne and the other with a 3’-azide (Scheme 13), see pg. 1400, left column, paragraph 2.
The Examiner notes Scheme 14 of El-Sagheer as depicted above and the two previous paragraphs correspond to the limitation wherein said amine is transformed via an amide formation with a ligation function-substituted NHS ester as discussed above, as the azide oligonucleotide (ODN) 54 as taught by El-Sagheer in Scheme 14 above contains an amide bond with a ligation function-substituted NHS ester, exemplifying 4-azidobutyric acid NHS ester 24 of carboxylic acid 52, wherein compound 24 contains an azide-substituted NHS ester as depicted above.
With respect to the limitation of “e) is a conjugation of resulting two-segmented nucleic acid in d) to another segment between said ligation function and a compatible ligation function in said another segment, see claim 5, pg. 3, lines 1-3”; the Examiner reasonably interprets this to be a physical limitation of reacting the two-segmented nucleic acid in step d) of claim 5, lines 1-2 with another nucleic acid segment compatible with the two-segmented nucleic acid produced in step d) of claim 5, lines 1-2 as discussed above. Since Zhong teaches two ligation steps to form a three-segmented nucleic acid wherein said ligations can be formed by the same chemistry such as an azide-alkyne [3+2] cycloaddition; and wherein El-Sagheer teaches Scheme 14 which produces an oligonucleotide azide containing an amide bond exemplified within azide ODN 54 as discussed above. The Examiner notes the physical limitation as discussed above is met by the combined teachings of Zhong and El-Sagheer as discussed above.
It would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have incorporated the teachings of El-Sagheer into the method of Zhong as discussed above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to have incorporated the teachings of El-Sagheer into the method of Zhong; in order to create the chemically ligated guide RNA via azide-alkyne [3+2] cycloaddition (e.g. click ligation) containing the crgRNA and the dual tracrgRNA comprising tracrgRNA1 and tracrgRNA2 as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success to have incorporated the teachings of El-Sagheer into the method of Zhong as discussed above as both El-Sagheer and Zhong are drawn to methods of making oligonucleotide azides; and teach said oligonucleotide azides for use in copper-catalyzed azide–alkyne cycloaddition reactions as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(IV) Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892) as applied to claims 1-2, 6-7 and 14 above, and further in view of Park et al. (Published 14 February 2017, Scientific Reports, Vol. 7, Article Number 42458, pp. 1-10, PTO-892).
Zhong addresses claims 1-2, 6-7 and 14 as written above. Zhong further teaches “guide RNA” is used interchangeably with “chimeric RNA”, “chimeric guide RNA”, “single guide RNA” and “synthetic guide RNA”, see paragraph [0063].
Although, Zhong does not teach said guide RNA comprises a DNA segment of 18-200 nt in length at its 3’-terminus, required in claims 8-9.
However, in the same field of endeavor of guide RNA, Park teaches targeted gene knock-in by CRISPR/Cas ribonucleotides in porcine zygotes, see pg. 1, title.
Park teaches two complementary sgRNA oligoDNAs (19-22 nucleotides in length depending on the guide sequence) were synthesized, annealed to form double-stranded DNA and cloned, wherein the cloned fragments were DNA sequenced to confirm their fidelity and in vitro transcribed to generate chimeric single guided (sg) RNAs, see pg. 8, materials and methods, paragraph 1.
Park teaches single stranded DNA (ssDNA) oligonucleotides (hereafter referred to as “oligo”) have proven effective in facilitating homology directed repair (HDR) driven knock-in of short stretches of sequences into porcine zygotes, see pg. 2, paragraph 3.
With respect to the limitation “the 3’-terminal segment further comprises an RNA segment of 3’-end of tracrRNA covalently tethered to the 5’-end or 3’-end of said DNA segment, required in claim 9”; is reasonably interpreted by the Examiner to be a physical limitation well within the scope of the artisan based on the combined teachings of Zhong and Park as discussed above. Since Park teaches a single guide RNA (sgRNA) tethered to an oligoDNA 19-22 nucleotides in length; and Zhong teaches the chemically ligated guide RNA as discussed above is interchangeable with single guide RNA. The physical limitation as discussed above are met by the combined teachings of Zhong and Park as discussed above.
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have modified the guide RNA as previously taught by Zhong above for integration of a DNA segment containing 19-22 nucleotides in length based on the teachings of Park above, recited and required within instant claim 8; and to have covalently tethered said DNA segment into the terminal 3’-end of a tracrRNA of Zhong, recited and required within instant claim 9, as limitations within the scope of the artisan as combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been motivated to chemically ligate as taught by Zhong; the ssDNA as taught by Park above; into the guide RNA of Zhong above in order to synthesize a guide RNA, comprising multiple-tracrgRNA, exemplified as tracrgRNA1 and tracrgRNA2 above, wherein said guide RNA taught by the combination of Zhong and Park above has a covalently attached ssDNA repair template that would repair the 3’-overhangs resulting from homology-directed repair of the double-stranded breaks (DBS) caused by the use of the CRISPR-Cas system comprising the ligated guide RNA as taught by Zhong; wherein the Examiner respectfully notes Zhong teaches the guide RNA is complexed with CRISPR-Cas9 as discussed above.
Therefore, the chemically synthesized guide RNA comprising multiple-tracrgRNAs and containing the ligated ssDNA as taught by the combination of Zhong and Park above would facilitate efficient insertion of shorter nucleotide segments, and thereby provide the capability for precise gene editing using the CRISPR-Cas9-guideRNA complexes as taught by the combination of Zhong and Park as discussed above.
One of ordinary skill in the art would have had a reasonable expectation of success as Park teaches single stranded DNA has proven effective in facilitating homology directed repair (HDR) driven knock-in of short stretches of sequences into porcine zygotes as discussed above; and Zhong teaches chemical ligation of nucleic acids via click ligation as discussed above at either the 5’-terminus or 3’-terminus of the nucleic acid with either alkyne or azide modification of said nucleic acids.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have included the modifications as recited and required within instant claims 8-9 above in view of the combined teachings of Zhong and Park as discussed above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to create the chemically synthesized guide RNA comprising multiple-tracrgRNA and containing ssDNA with the capability of homology directed repair using the CRISPR-Cas9-guideRNA complexes as taught by the combination of Zhong and Park as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success of incorporating the modifications required within instant claims 8-9 into the method of Zhong above, because both Zhong and Park are drawn to generating guide RNAs as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(V) Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Zhong (Published 28 July 2016, US-20160215275-A1, PTO-892) as applied to claims 1-2, 6-7 and 14 above, and further in view of Lee et al. (Published 02 May 2017, eLife, DOI: 10.7554/eLife.25312, pp. 1-17, PTO-892).
Zhong addresses claims 1-2, 6-7 and 14 as written above. Although, Zhong teaches not teach said guide RNA at its 5’-terminal segment comprises an ssDNA segment of 18-200 nt in length at its 5’-terminus, required in claim 10.
However, in the same filed of endeavor of guide RNA, Lee teaches synthetically modified guide RNA (gRNA) and donor DNA are a versatile platform for CRISPR-Cas9 engineering, see pg. 1, title.
Lee performed experiments to determine if gRNA and donor DNA could be combined into a single molecule (termed gDonor), see pg. 8, second to last paragraph from the bottom of the page. Lee exemplifies a gRNA-donor DNA conjugate (gDonor) synthesized by conjugating an azide terminated donor DNA with an alkyne modified CRISPR-targeting RNA(crRNA), and hybridizing the resulting conjugate with tracrRNA. Lee teaches gDonor was purified via gel extraction, and was synthesized with a 40% yield. See pg. 8, last paragraph of the page.
Lee teaches the donor ssDNA was 127 nucleotides (nt) in length, see pg. 12, last paragraph of the page.
Lee teaches 5’ amine-Donor ssDNA or 3’ amine-Donor ssDNA, see pg. 5, Figure 2d.
Lee teaches the activity of the gDonor was investigated by determining its ability to induce homology directed repair (HDR) in BFP-HEK cells, after electroporation with the Cas9 RNP, see pg. 8, last paragraph of the page.
Lee demonstrates in Figure 4c (see pg. 9) that the gDonor was able to convert the BFP gene to the GFP gene via HDR with an efficiency similar to unmodified gRNA and Donor DNA, and thus both the gRNA and donor DNA of the gDonor are active, see pg. 10, paragraph 1.
Lee concludes a synthesized gRNA-donor DNA conjugate (gDonor) enabled the efficient delivery of Cas9 RNP and donor DNA into cells, see pg. 11, paragraph 1.
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have modified the guide RNA as previously taught by Zhong above for integration of a ssDNA segment as taught by Lee above containing a 127nt ssDNA as a limitation within the scope of the artisan as combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been motivated to chemically ligate as taught by Zhong; the ssDNA as taught by Lee above; into the guide RNA of Zhong above in order to synthesize a guide RNA having a covalently attached ssDNA repair template that would repair the 3’-overhangs resulting from homology-directed repair of the double-stranded breaks (DBS) caused by the use of the CRISPR-Cas system comprising the ligated guide RNA; wherein the Examiner respectfully notes Zhong teaches the guide RNA is complexed with CRISPR-Cas9 as discussed above.
Therefore, the chemically synthesized guide RNA containing the ligated ssDNA as taught by the combination of Zhong and Lee above would facilitate efficient insertion of shorter nucleotide segments, and thereby provide the capability for precise gene editing using the CRISPR-Cas9-guideRNA complexes as taught by the combination of Zhong and Lee as discussed above
One of ordinary skill in the art would have had a reasonable expectation of success as Lee exemplifies the single stranded Donor DNA was able to convert the BFP gene to the GFP gene via HDR with an efficiency similar to unmodified gRNA and Donor DNA as discussed above via CRISPR-Cas9 engineering; and Zhong teaches chemical ligation of nucleic acids via click ligation as discussed above at either the 5’-terminus or 3’-terminus of the nucleic acid with either alkyne or azide modification of said nucleic acids.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have included the modifications as recited and required within instant claim 10 above in view of the combined teachings of Zhong and Lee as discussed above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to create the chemically synthesized guide RNA of Zhong as discussed above. One of ordinary skill in the art would have had a reasonable expectation of success of incorporating the modifications required within instant claim 10 into the method of Zhong above, because both Zhong and Park are drawn to generating guide RNA conjugates utilizing azide-alkyne [3+2] cycloadditions (e.g. click ligations) as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
Conclusion
No claims are allowed in this action.
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/JARET J CREWS/Examiner, Art Unit 1691
/RENEE CLAYTOR/Supervisory Patent Examiner, Art Unit 1691
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