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 11-12-25.
Claims 1-6, 23-31, 34, 40, 61, 85, 86, and 129 are pending in the instant application.
Response to Arguments and Amendments
Withdrawn Objections/Rejections
Any objections or rejections not repeated in this Office action are hereby withdrawn.
New Rejection In Light of the Joint Research Agreement Statement
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.
Claims 1-6, 23-31, 34, 40, 61, 85, 86, and 129 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dayie, K.T. (Int’l. J. Mol. Sci, Vol. 9, pages 1214-1240 (2008)), Stark et al (RNA, Vol. 12, pages 2014-2019 (2006)), Palumbo et al (ChemBioChem, Vol. 21, pages 1633-1640 (2020)), and Bullard et al (Biochem. J., Vol. 398, pages 135-144 (2006)), the combination further in view of Daugharthy et al (US 2021/0381049).
The claims are drawn to methods of synthesizing a guide RNA (gRNA) comprising providing a first RNA fragment comprising a terminal region comprising a 5’ phosphate moiety, and a second RNA fragment comprising a terminal region comprising a 3’ hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both, comprises at least a portion of a sequence capable of binding to an RNA-guided endonuclease, providing a splint DNA oligonucleotide comprising a first portion complementary to the first RNA fragment at the terminal region comprising a 5’ phosphate moiety and a second portion complementary to the second RNA fragment at the terminal region comprising a 3’ hydroxyl group, hybridizing the first RNA fragment, the second RNA fragment, and the splint DNA oligonucleotide together to form a first complex, contacting the first and second RNA fragments with a ligase to form a gRNA at a ligation site present between the first and second RNA fragments in the first complex to form a second complex, digesting the splint DNA oligonucleotide with a DNase to obtain the gRNA, which digesting the splint DNA oligonucleotide with the DNase to obtain the gRNA comprises contacting the second complex with the DNase to digest the splint DNA oligonucleotide, which digesting the splint DNA oligonucleotide with the DNase to obtain the gRNA comprises dissociating the gRNA and the splint DNA oligonucleotide; and digesting the dissociated splint DNA oligonucleotide with the DNase to obtain gRNA, which DNase optionally comprises DNase I, DNase II, or a bacterial DNase, separating the digested splint DNA oligonucleotide products from the gRNA which hybridizing the first RNA fragment, the second RNA fragment and a splint DNA oligonucleotide is carried out in the presence of one or more RNase inhibitors, which first RNA fragment, second RNA fragment, or both are optionally 10 to 90 nucleotides in length, wherein the ratio between the length of the first RNA fragment and the length of the second RNA fragment is at least 20:80, or which first RNA fragment is about 20 to 50 nucleotides in length, and the second RNA fragment is about 80 to 50 nucleotides in length, which 5’ phosphate moiety is 5’-phosphate or 5’-phosphorothioate, and which ligase optionally comprises T4 DNA ligase, T4 RNA ligase I, or T4 RNA ligase II, which splint DNA oligonucleotide is 20 to 100 nucleotides in length and is optionally attached to a solid support, or which ligation site corresponds to a site in a tetraloop portion of a stem-loop structure in the gRNA or a site in a helix portion of a stem-loop structure in the gRNA, or which first or second RNA fragment, or both, comprise one or more modifications in the backbone, or base modifications, one or more phosphorothioate linkages, or combinations thereof.
The claims are also drawn to a method of synthesizing a guide RNA (gRNA), the method hybridizing a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint DNA oligonucleotide and a second splint DNA oligonucleotide to form a complex, wherein (a) the first RNA fragment comprises a terminal region comprising a 3’ hydroxyl group, (b) the second RNA fragment comprises a first terminal region comprising a 5’ phosphate moiety and a second terminal region comprising a 3’ hydroxyl group, (c) a third RNA fragment comprises a terminal region comprising a 5’ phosphate moiety, (d) the first splint DNA oligonucleotide comprises (i) a first portion complementary to the terminal region comprising the 3’ hydroxyl group of the first RNA fragment; and (ii) a second portion complementary to the first terminal region comprising the 5’ phosphate moiety of the second RNA fragment, (e) the second splint DNA oligonucleotide comprises (i) a first portion complementary to the second terminal region comprising the 3’ hydroxyl group of the second RNA fragment; and (ii) a second portion complementary to the terminal region comprising the 5’ phosphate moiety of the third RNA fragment, which complex comprises (i) a first ligation site present between the 3’ hydroxyl group of the first RNA fragment and the 5’ phosphate group of the second RNA fragment, and (i) a second ligation site present between the 3’ hydroxyl group of the second RNA fragment and the 5’ phosphate group of the third RNA fragment, and wherein each of the first splint DNA oligonucleotide and the second splint DNA oligonucleotide is no more than 32 nucleotides in length; and ligating the first and second RNA fragments, and the second and third RNA fragments, respectively, with a ligase at the first and second ligation sites in the complex, thereby synthesizing a gRNA, which gRNA is a single gRNA (sgRNA).
The claims are also drawn to a method of synthesizing a single guide RNA (sgRNA) for use with an RNA-guided endonuclease, comprising providing a first complex comprising a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint oligonucleotide, and a second splint oligonucleotide, wherein (a) the first RNA fragment comprises (i) a terminal region comprising a 3’ hydroxyl group, (b) the second RNA fragment comprises (i) a first terminal region comprising a 5’ phosphate moiety, and (ii) a second terminal region comprising a 3’ hydroxyl group, (c) the third RNA fragment comprises (i) a terminal region comprising a 5’ phosphate moiety, (d) the first splint oligonucleotide comprises (i) a first portion complementary to the terminal region comprising the 3’ hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region comprising the 5’ phosphate moiety of the second RNA fragment; and (e) the second splint oligonucleotide comprises (i) a first portion complementary to the second terminal region comprising the 3’ hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the terminal region comprising the 5’ phosphate moiety of the third RNA fragment, wherein the first complex is formed by hybridization of (a)(i) and (d)(i), (b)(i) and (d)(ii), (b)(ii) and (e)(i), and (c)(i) and (e)(ii), which first complex has a first ligation site present between the 3’ hydroxyl group of the first RNA fragment and the 5’ phosphate group of the second RNA fragment, and a second ligation site present between the 3’ hydroxyl group of the second RNA fragment and the 5’ phosphate group of the third RNA fragment, ligating the first RNA fragment and the second RNA fragment at the first ligation site and ligating the second RNA fragment and the third RNA fragment at the second ligation site to form a second complex comprising a ssRNA comprising from 5’ to 3’: a spacer sequence and an invariable sequence that binds an RNA-guided endonuclease; the invariable sequence comprising a stem loop formed between a crRNA repeat sequence and a tracrRNA anti-repeat sequence, and a 3’ tracrRNA sequence comprising at least one stem-loop; and digesting the first and second splint DNA oligonucleotides with a DNase to obtain the sgRNA, which gRNA is about 30 to about 160 nucleotides in length.
Dayie, K.T. (Int’l. J. Mol. Sci, Vol. 9, pages 1214-1240 (2008)) teach methods for RNA ligation using DNA splinted based ligation. Dayie teaches modifications to improve ligation efficiency by protecting the donor 3’-OH with 2’ACE, chemically incorporating phosphate on the donor strand to minimize 5’-end heterogeneity, and designing an optimized linker where 4-8 single stranded nucleotides are on the acceptor RNA and 1-2 nucleotides on the donor RNA. The splints had melting temperatures between 40-45oC, thereby synthesizing a 128 nucleotide RNA from three synthetic oligonucleotide pieces. Dayie also teaches the use of deoxyribozymes and two ligases requiring a 2’, 3’-diol for an acceptor fragment and a 5’-triphosphate functional group on the donor RNA fragment (see esp. pages 1228, 1230, 1231, Figures 9 and 10).
Stark et al (RNA, vol. 12, pages 2014-2019 (2006) teach the synthesis of RNAs using an RNA ligase-mediated method Stark utilizes a splint design allowing for the ligation junction to mimic the natural substrate of RNA ligase, generating a 128 nucleotide RNA molecule. Stark describes DNA ligase mediated ligation which utilizes DNA ligase recognizing nicked, double stranded substrates and catalyzes the formation of phosphodiester linkage between the 5’ phosphate of one oligo (donor oligo) and the free 3’ hydroxyl of a second oligo (acceptor, which ligation requires multiple ligation steps. Stark also describes the alternative method for RNA ligation using T4 RNA ligase, which requires a free 5’ phosphate on the donor substrate, a 3’ hydroxy on the acceptor substrate, and exogenous ATP. RNA ligase is only active when the nucleotides at the splice junction are single stranded. Stark teaches the imposition of sequence specificity using a DNA splint designed to hold the donor and acceptor molecules near one another while leaving single stranded regions or linkers near the ligation junction. Stark then teaches a significant improvement on RNA ligase mediated ligation by incorporating a 2’-ACE protecting group on the 3’ terminal, optimizing linker lengths and incorporating 5’ terminal phosphates. Stark produced a 128 nucleotide RNA from three chemically synthesized oligonucleotide precursors. Stark teaches two part ligations using an acceptor oligonucleotide A that was deprotected and a donor oligonucleotide B that was phosphorylated and 2’-ACE protected, and a DNA splint oligonucleotide was mixed with the two RNA oligonucleotides in the presence of T4 ligase. Stark also teaches three part ligations as with the two step ligation but with the first step scaled up scaled up to generate product to gel purify. For a single step three part ligation, the B oligo and the A oligo were deprotected to serve as both a donor and an acceptor. All five oligonucleotides were allowed to anneal to each other (see esp. pages 2014, 2015, Figure 1 on 2015, Figure 3 on page 2017, .text pm [ages 2017-2018).
Palumbo et al (ChemBioChem, Vol. 21, pages 1633-1640 (2020)) teach
incorporating modifications for increasing stability and potency of sgRNA, including 2’O-methyl and phosphorothioate linkages. Palumbo teaches efficient and adaptable modifications of 3’-terminus of sgRNA. Palumbo synthesized sgRNA in two parts and including splint ligation, by synthesizing a 30 nucleotide fragment of the sgRNA modified at the 3’-end with a primary amine, and producing modified 3’ fragments after functionalizing the 3’-end with HGS esters. Palumbo produced a full length sgRNA using the 30 nucleotide, 3’-functionalized fragment bearing a 5’-phosphate and a 51 nucleotide RNA which hybridized to a complementary DNA and ligated using T4 DNA ligase. The modification reaction and ligation allowed for six chemically modified sgRNAs having a variety of functional groups (see esp. pages 1633-1635, Figure 1 on page 1634).
Bullard et al (Biochem. J., Vol. 398, pages 135-144 (2006)) teach ligation reactions for joining two fragments with a 5’ phosphate moiety and a 3’ hydroxyl group using a splint oligonucleotide and a ligase. Reaction rates were compared with T4 DNA ligase 1 and 2 for RNA-RNA, DNA-DNA and RNA-DNA ligations using RNA or DNA splint oligonucleotides. Splint oligonucleotides were 20 nucleotides in length and RNA fragments were 10-90 nucleotides in length (Table 1, Figures 1-5, text last paragraph page 138 – bridging paragraph on page 141).
The primary references do not teach the treatment of RNA with DNAse to remove DNA and purify the RNA molecules.
Daugharthy et al (US 2021/0381049) teach the treatment of RNA with DNAse to remove DNA and purify the RNA molecules (see esp. ¶¶ 0025, 0026).
It would have been obvious to synthesize RNA using the fragments and splints instantly claimed because the prior art had taught all of the synthetic steps and components for making medium sized RNA, as illustrated in the teachings of Dayie, Stark, Polumbo and Bullard. One of skill in the art would have been motivated to incorporate the modifications instantly claimed to enhance target binding and stability of RNA fragments. One of skill in the art would have also been motivated to remove the DNA splints after ligation of the RNA fragments in order to obtain purified, synthesized RNA. Obvious ways to remove the splints and taught in the prior art include using gel isolation, attaching the RNA to a solid surface, or degrading the DNA splints using DNAse, which DNAse treatment of RNA was routinely disclosed in the prior art, as illustrated in the teachings of Daugharthy.
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 claimed invention.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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
1-25-26
/JANE J ZARA/Primary Examiner, Art Unit 1637