DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/29/2025 has been entered.
Response to Amendment/Status of Claims
Receipt of Arguments/Remarks filed on 09/08/2025 is acknowledged. Claim 8 stands cancelled. Claims 1 and 9 were amended. Claims 1-7 and 9-11 are pending and under examination..
Response to Arguments
Applicant’s arguments and amendments, see pages 5-10, filed 09/08/2025, with respect to the 35 U.S.C. 102(a)(1) rejections of claims 1,2,6 and 9-11 as anticipated by WO 2015113922; the 35 U.S.C. 102(a)(1) rejections of claims 1,6,7 and 9-11 as anticipated by WO 2014043544; claims 2-4 rejected under 35 U.S.C. 103 as unpatentable over ‘544 in view of Bartel; claims 1,6,7 and 9-11 under 35 U.S.C. 103 as unpatentable over WO 2011117353 in view of Lee et al.; claim 2 rejected under 35 U.S.C. 103 as unpatentable over ‘353 in view of Lee and Chen et al.; and claims 3 and 4 as unpatentable over ‘353 in view of Lee and Bartel, have been fully considered and are persuasive. Applicant has amended claims 1 and 9 to limit the miRNA target site sequence to a sequence complementary to bases in positions 2-7 or 3-8 from the 5’ end of miRNA and having complementary G:A wobble base pairs, in which the complementary G:A wobble base pairs are not taught by the cited references. Therefore, the rejections have been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendments to claims 1 and 9 to require the miRNA target site sequence to have complementary G:A wobble base pairs, and therefore, a case of obviousness is being made.
In the final office action dated 06/06/2025, claim 5 was indicated as being objected to as being dependent on a rejected base claim but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. However, upon further consideration, the examiner is rejecting claim 5 under a case of obviousness regarding the recited sequence. See the 103 rejections below.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
However, there is no English translation of the non-English language foreign priority
Application.
Claim Interpretation
The abasic spacer in claims 1-11 is disclosed in the instant specification as “a substitute for maintaining a space instead of a single nucleotide, and may connect neighboring nucleotides in the manner that can maintain the space. The abasic spacer is preferably an organic compound, more preferably, a phosphate group
(-H2P04) or a sulfuric acid group (-H2PSO4)-containing (linked) hydrocarbon chain, and most preferably, an alkyl group having at least three carbon atoms (C3 spacer) or an alkyl group having at least three carbon atoms (C3 spacer) or an alkyl group having at least 6 carbon atoms (C6 spacer), but the present invention is not limited thereto. In addition, the abasic spacer may include an abasic nucleotide, and such an abasic nucleotide-type spacer generally refers to a single nucleotide analogue having no base, and refers to all types of modification that cannot bind with all other biological bases comprehensively originating from RNA, including an abasic deoxyribonucleotide spacer (dSpacer) and an abasic ribonucleotide spacer (rSpacer). According to an exemplary embodiment of the present invention, the abasic spacer may be an rSpacer molecule having an Ribo nucleotide as a backbone” (page 14, lines 15-22 to page 15, lines 1-6).
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.
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 1,2,6 and 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2015113922 (‘922), Published 6 August 2015, cited on an IDS dated 05/26/2023, in view of Jadhav et al. (US 20180208927, Published 26 July 2018).
Regarding claims 1 and 6, ‘922 teaches antimiR oligomers and designs thereof, which comprise a contiguous nucleotide sequence fully complementary, or essentially complementary (i.e. may comprise one or two mismatches) to a microRNA sequence or subsequence thereof (Page 69, lines 8 and 13-16). ‘922 recites an oligomeric compound comprising a first region of a contiguous sequence of 7-26 phosphorothioate linked nucleosides (A), a second region of a contiguous sequence of 7-26 phosphorothioate linked nucleosides (A’), a functional group such as a conjugate moiety covalently linked to a region A or A’ via a biocleavable linker (B’), such as a biocleavable linker comprising a peptide linker, e.g. a lysine linker, wherein the first and/or second regions target microRNA targets, and wherein the first and second regions are 8-10 nucleotides in length (Claims 16 and 19). The lysine linker contains 6 carbons and is therefore an abasic C6 spacer. ‘922 teaches in some embodiments the first or second 3’ nucleobase of the oligomer corresponds to the second 5’ nucleotide of the microRNA sequence. ‘922 teaches the antimir, where nucleobase units 2-7 of the oligomer as measured from the 3’ end of the oligomer are complementary to the microRNA seed region sequence (page 70, lines 7-9). Therefore, as ‘922 teaches nucleobase units 2-7 of the oligomer as measured from the 3’ end of the oligomer are complementary to the microRNA seed region sequence, and that the second 3’ nucleobase of the oligomer corresponds to the second 5’ nucleotide of the miRNA sequence, ‘922 teaches the limitation of the miRNA target site sequence is a sequence complementary to bases in positions 2-7 from the 5’ end miRNA.
While ‘922 teaches the antimiR oligomers which comprise a contiguous nucleotide sequence that is essentially complementary (may comprise one or two mismatches) to a microRNA sequence or subsequence thereof, ‘922 does not teach the miRNA target site sequence has complementary G:A wobble base pairs.
Before the effective filing date, Jadhav et al. taught multi-targeted molecules comprising at least two nucleic acid based effector molecules covalently or non-covalently linked to each other, capable of modulating expression of a target (paragraph 0004). Jadhav et al. taught the nucleic acid based effector molecules capable of modulating gene expression of a target gene as anti-microRNAs or antimirs, supermirs, and antagomirs (paragraph 0005) . Jadhav et al. taught at least two antimirs covalently linked to each other via a non-nucleotide based linker (paragraph 0265), and also taught at least two antagomirs covalently linked to each other via a non-nucleotide based linker (paragraph 0267). Jadhav et al. taught the effector molecules can be optimized for RNA interference by decreasing the free energy of the duplex association by introducing thermally destabilizing modifications in the sense strand at a site opposite to the seed region of the antisense strand (i.e. at positions 2-8 of the 5’ end of the antisense strand) which can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand (paragraph 0376). Jadhav et al. taught the thermally destabilizing modification can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand within the dsRNA duplex, and exemplary mismatch base pairs include G:A (paragraph 0382).
Regarding claim 2, ‘922 recites the oligomeric compound, wherein the at least one or both of the first and second regions target a microRNA selected from the group consisting of miR ID NO 40-976 (claim 20). ‘922 teaches the target microRNA may be miR-1, which is SEQ ID NO: 113 and is cited in the SEQ ID NO range in claim 20 (Table 2, page 115). ‘922 teaches miR-1 is related to cardiac arrythmia (page 67).
Regarding claim 9, ‘922 teaches methods of synthesis and manufacture of the oligomer of the invention by sequential synthesis of oligomer region A’, region B, second oligomer region A’, optionally followed by the addition of the third region C, optionally via a linker Y (page 108, lines 4-9).
‘922 does not teach the miRNA target site sequence has complementary G:A wobble base pairs.
Jadhav et al. cures this deficiency as discussed above regarding claim 1.
Regarding claim 10, ‘922 recites a method of inhibiting the expression of a target gene in a cell by administering the oligomeric compound according to any one of the preceding claims to a cell which is expressing said target gene, suitably in an amount effective to reduce the expression of the target gene in said cell (Claim 31) and claims 16 and 20 recite the target is microRNA.
Regarding claim 11, the preamble recites “a gene transfer system” which is an intended use, and the body of the claim does not recite any additional steps or components of that of claim 1, and therefore comprises the same limitations as claim 1.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the oligomeric antimir compound of ‘922 wherein nucleobase units 2-7 of the oligomer as measured from the 3’ end of the oligomer are complementary to the microRNA seed region sequence to have complementary G:A base pairs based on the teachings of Jadhav et al. There would be a reasonable expectation of success, because both ‘922 and Jadhav et al. pertain to nucleic acid molecules comprising multiple nucleic acid sequences that target miRNAs joined by non-nucleotide linkers, and would amount to combining prior art elements according to known methods to yield predictable results. In addition, ‘922 suggests that the antimiR oligomers comprise a contiguous nucleotide sequence that is essentially complementary (may comprise one or two mismatches) to a microRNA sequence or subsequence thereof. All the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. While Jadhav et al. does not call the G:A base pair a wobble base pair, Jadhav et al. teaches the G:A as a mismatch and the benefits thereof. One of ordinary skill in the art would have been motivated to provide a G:A base pair in the sequence complementary to bases in positions 2-7 of the miRNA, because Jadhav et al. taught that the effector molecules can be optimized for RNA interference by decreasing the free energy of the duplex association by introducing thermally destabilizing modifications in the sense strand at a site opposite to the seed region of the antisense strand (i.e. at positions 2-8 of the 5’ end of the antisense strand) which can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand (paragraph 0376), and taught the thermally destabilizing modification can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand within the dsRNA duplex, and exemplary mismatch base pairs include G:A (paragraph 0382). Therefore, an ordinary artisan could have provided a complementary G:A base pair in the antimir sequence that would function as a wobble base pair, that is complementary to bases in positions 2-7 from the 5’ end of the miRNA based on the teachings of Jadhav et al. in order to introduce thermally destabilizing modifications to optimize the inhibiting activity the oligomeric antimir compound of ‘922.
Accordingly, the limitations of claims 1,2,6 and 9-11 would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claims 3-5 are rejected under 35 U.S.C. 103 as being unpatentable over ‘922 and Jadhav et al., as applied to claims 1,2,6 and 9-11 above, and further in view of Bartel (Cell 136, Published 23 January 2009, pages 215-230) cited on an IDS dated 11/30/2020.
The teachings of ‘922 and Jadhav et al. as applicable to claims 1,2,6 and 9-11 are described above.
‘922 and Jadhav et al. do not teach the modified nucleic acid inhibiting miRNA contains a 5’-ACAUUCCA-3’ or 5’-AAAUUCCA-3’ sequence, or wherein the modified nucleic acid inhibiting miRNA consecutively comprises at least two 5’-ACAUUCCA-3’ sequences or at least two 5’-AAAUUCCA-3’ sequences, and at least one abasic spacer before and after the sequence.
Before the effective filing date, Bartel teaches the understanding of miRNA target recognition and miRNA target sites. Bartel teaches mammalian targets can be predicted by searching for conserved 7 nt matches in aligned regions of vertebrate 3’-UTRs, and prediction specificity increases when requiring an 8 nt match or multiple matches to the same miRNA, and that enough genomes have been sequenced and aligned such that these targets with single sites can now be predicted with confidence that most are authentic (page 218, left column). Bartel teaches using a three-step protocol for predicting evolutionary conserved targets for metazoan miRNA: (1) identify the two 7 nt matches to the seed region (Figures 1A and 1B), for example, miR-1 with the sequence of 5’-UGGAAUGUAAAGAAGUAUGUA would recognize the CAUUCCA match and the ACAUUCC match, (2) use available whole-genome alignments to compile orthologous 3’UTRs, (3) search within the orthologous UTRs for conserved occurrence of either 7 nt match which are the predicted regulatory sites (page 218, left column). Bartel teaches that a search for conserved 8 nt sites comprised of both 7 nt motifs (e.g. ACAUUCCA in the case of miR-1, Figure 1C) yields greater prediction specificity, and a search for conserved 6 nt seed matches (Fig 1D) yields greater sensitivity (page 217-218).
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, that the oligomeric antimir compound of ‘922 targeting miR-1 modified by complementary G:A base pairs of Jadhav et al., would comprise a 5’-ACAUUCCA-3’ sequence based on teaching of Bartel regarding the three-step protocol for predicting evolutionary conserved targets using miR-1. There would be a reasonable expectation of success because ‘922 teaches the antimir compound may target miR-1 and Bartel teaches the miR-1 sequence and the corresponding matches in the seed region, and would amount to simple substitution of one known element for another to obtain predictable results. One of ordinary skill in the art would have been motivated to provide the oligomeric antimir compound of ‘922 that is taught as targeting miR-1, modified by the complementary G:A base pairs of Jadhav et al. with a 5’-ACAUUCCA-3’ sequence based on Bartel’s simple three-step protocol to predict evolutionarily conserved targets for a miRNA, and which identifies the sequence ACAUUCCA as a seed matched site. It would have been obvious to one of ordinary skill in the art to provide the oligomeric antimir compound of ‘922 that is taught as targeting miR-1 modified by complementary G:A base pairs of Jadhav et al., with at least 2 consecutive ACAUUCCA sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule. One of ordinary skill in the art would have been motivated to do so because ‘922 taught at least one or both of the first and second regions target a microRNA which may be miR-1 and Bartel teaches prediction specificity increases when requiring multiple matches to the same miRNA.
It would have been obvious to one of ordinary skill in the art to provide the oligomeric antimir compound of ‘922 that is taught as targeting miR-1 modified by complementary G:A base pairs of Jadhav et al., with at least 2 consecutive 5’-AAAUUCCA-3’ sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule based on teaching of Bartel regarding the three-step protocol for predicting evolutionary conserved targets using miR-1 as an example. There would be a reasonable expectation of success because ‘922 teaches the antimir compound may target miR-1 and Bartel teaches the miR-1 sequence and the corresponding matches in the seed region. While Bartel does not teach the exact sequence of “AAAUUCCA”, Bartel does teach “ACAUUCCA”, which has one different nucleotide. An ordinary artisan could have arrived at the sequence “AAAUUCCA” from the sequence “ACAUUCCA” by replacing the 2nd ‘C’ nucleotide with ‘A’ based on the teachings of Jadhav et al. regarding introducing thermally destabilizing modifications to optimize the inhibiting activity, and which modifications can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand and exemplary mismatch base pairs include G:A (paragraph 0382). One of ordinary skill in the art would have been motivated to provide at least 2 consecutive AAAUUCCA sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule because ‘922 taught at least one or both of the first and second regions target a microRNA which may be miR-1 and Bartel teaches prediction specificity increases when requiring multiple matches to the same miRNA.
Accordingly, the limitations of claims 3-5 would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over ‘922 and Jadhav et al., as applied to claims 1,2,6 and 9-11 above, and further in view of Wang (Bioinformatics, 2014 Jan 26; 30(10):1377-1383).
The teaching of ‘922 and Jadhav et al. as applicable to claims 1,2,6 and 9-11 is described above.
‘922 and Jadhav et al. do not teach wherein the seed region of the miRNA is a sequence between positions 1 to 8 from the 5’ end of the miRNA.
Before the effective filing date, Wang taught using high-throughput experimental data from a CLASH study (crosslinking, ligation and sequence of hybrids) as an opportunity to characterize miRNA target recognition patterns which may provide guidance for improved miRNA target prediction (Abstract). Wang taught canonical seed is defined as any 6-mer sequence within positions 2–8 or 7-mer sequence within positions 1–8 of the miRNA (page 1378, left column, Section 2.2; Table 1, page 1379).
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the teachings regarding the miRNA seed region of ‘922 to comprise a sequence between positions 1 to 8 from the 5’ end of the miRNA based on the teachings of Wang with a reasonable expectation of success. There would be a reasonable expectation of success as this would amount to simple substitution of the miRNA seed region of ‘922 with the miR seed region of positions 1-8 of the miRNA of Wang. One of ordinary skill in the art would have been motivated to do so because Wang taught canonical seed is defined as any 6-mer sequence within positions 2–8 or 7-mer sequence within positions 1–8 of the miRNA (page 1378, left column, Section 2.2; Table 1, page 1379).
Accordingly, the limitations of claim 7 would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claims 1,6,7 and 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2011117353 (’353), Published 29 September 2011, cited on an IDS dated 05/26/2023, Lee et al. (Nature Communications, Published 18 December 2015, pages 1-14), cited on an IDS dated 11/30/2020, and Jadhav et al. (US 20180208927, Published 26 July 2018).
Regarding claim 1, ‘353 teaches bivalent molecules comprising a first oligonucleotide linked to a second oligonucleotide (page 5, lines 14-15), and that the first and/or second oligonucleotide comprise an antisense sequence complementary to a cellular RNA such as microRNA (page 5, lines 19-20). ‘353 teaches the antisense sequence may be an antimir antisense sequence capable of binding to a microRNA and that the first and/or second oligonucleotide comprise a seed sequence of microRNA capable of base pairing to the complementary sequence of the seed sequence (page 5, lines 23-27) and may be used to block microRNA activity by binding to microRNA to deregulate all targets of the microRNA (page 5, lines 32-33). ‘353 teaches that preferred contiguous sequences are at least 6 nucleotides (page 8, lines 6-7), and the preferred length of the first and the second oligonucleotide is preferably more than 5 nucleotides or 6 nucleotides (page 17, line 17). ‘353 teaches the first and/or second oligonucleotide comprise a sequence selected from the group consisting of contiguous sequences that are capable of base pairing to the complementary sequence of a sequence of positions 2-7 (page 8, lines 26-35 to page 9 lines 1-20). ‘353 teaches the linking moiety may comprise abasic units and is attached to the 3’ end of the first oligonucleotide and to the 5’ end of the second oligonucleotide (page 27 lines 1-5). ‘353 teaches bivalent antimirs may be more potent than monovalent antimirs because two microRNAs will bind cooperatively to the bivalent molecule and they may have more favorable biodistribution (Example 1 page 30).
‘353 does not teach that at least one base is substituted with an abasic spacer between the miRNA target site sequences. ‘353 does not teach that the abasic spacer is an abasic ribonucleotide spacer with no base (rSpacer, rSp), an abasic deoxyribonucleotide spacer (dSpacer), a C3 spacer or a C6 spacer. ‘353 does not teach the miRNA target site sequence has complementary G:A wobble base pairs.
However, before the effective filing date, Lee et al. taught that gene silencing by RNA interference represses hundreds of off-target transcripts and that avoiding miRNA-like off-target repression is a major challenge (Abstract). Lee et al. taught using an abasic spacer that contains no base but functions as a linker, including an abasic deoxynucleotide spacer, dSpacer, as a substitute for a nucleotide in the nucleation region (position 2-6) and that the substitution prevents miRNA-like off-target repression and conserves on-target activity (page 3). Lee et al. also taught using a C3 spacer substitution elicited excellent repression activity for a perfectly matched on-target without inducing seed-mediated target repression (page 9).
Regarding the miRNA target site sequence having complementary G:A wobble base pairs, Jadhav et al. taught multi-targeted molecules comprising at least two nucleic acid based effector molecules covalently or non-covalently linked to each other, capable of modulating expression of a target (paragraph 0004). Jadhav et al. taught the nucleic acid based effector molecules capable of modulating gene expression of a target gene as anti-microRNAs or antimirs, supermirs, and antagomirs (paragraph 0005) . Jadhav et al. taught at least two antimirs covalently linked to each other via a non-nucleotide based linker (paragraph 0265), and also taught at least two antagomirs covalently linked to each other via a non-nucleotide based linker (paragraph 0267). Jadhav et al. taught the effector molecules can be optimized for RNA interference by decreasing the free energy of the duplex association by introducing thermally destabilizing modifications in the sense strand at a site opposite to the seed region of the antisense strand (i.e. at positions 2-8 of the 5’ end of the antisense strand) which can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand (paragraph 0376). Jadhav et al. taught the thermally destabilizing modification can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand within the dsRNA duplex, and exemplary mismatch base pairs include G:A (paragraph 0382).
Regarding claim 6, ‘353 teaches the first and/or second oligonucleotide of the molecule comprises a contiguous sequence that is at least 6,7 or 8 nucleotides (page 8 line 7).
Regarding claim 7, ‘353 teaches the first and/or second oligonucleotides comprises a contiguous sequence capable of base pairing to the complementary sequence of a sequence of position 1-8 (page 8, lines 26-35 and page 9, lines 8-13).
Regarding claim 9, ‘353 teaches that the linking moiety is attached during oligonucleotide synthesis and when the first oligonucleotide has been synthesized, the linking moiety is attached to the first oligonucleotide, where after the second oligonucleotide is synthesized (page 27, lines 33-35).
‘353 does not teach the miRNA target site sequence has complementary G:A wobble base pairs.
Jadhav et al. cures this deficiency as discussed above regarding claim 1.
Regarding claim 10, ‘353 teaches using the molecules of the invention for modulating microRNA regulation by blocking a microRNA or blocking a microRNA binding site in a target RNA in vitro (page 29, lines 21-24), and transfecting the oligonucleotides into HUH7 cells expressing microRNA-122 (Example 4, page 37 and Fig 1 and 2). Regarding claim 11, ‘353 teaches a vector construct comprising two microRNA-122 binding sites (Example 4 page 34).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the bivalent molecule of ‘353 with the dSpacer or C3 spacer of Lee et al. between the two miRNA target site sequences, for the purpose of preventing off target repression and conserving on-target activity, and to have complementary G:A base pairs based on the teachings of Jadhav et al. with a reasonable expectation of success. There would be a reasonable expectation of success, because both ‘353 and Jadhav et al. pertain to nucleic acid molecules comprising multiple nucleic acid sequences that target miRNAs joined by non-nucleotide linkers, and would amount to combining prior art elements according to known methods to yield predictable results. All the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. One of ordinary skill in the art would have been motivated to substitute a base with an abasic spacer between the two miRNA target site sequences, and for the abasic spacer to be dSpacer or C3 spacer, as Lee et al. teach using an abasic spacer that contains no base but functions as a linker, including an abasic deoxynucleotide spacer, dSpacer, as a substitute for a nucleotide in the nucleation region (position 2-6) and that the substitution prevents miRNA-like off-target repression and conserves on-target activity (page 3) as well as using a C3 spacer substitution elicited excellent repression activity for a perfectly matched on-target without inducing seed-mediated target repression (page 9).
Regarding the complementary G:A wobble base pairs, while Jadhav et al. does not call the G:A base pair a wobble base pair, Jadhav et al. teaches the G:A as a mismatch and the benefits thereof. One of ordinary skill in the art would have been motivated to modify the bivalent molecule of ‘353 to have complementary G:A wobble base pairs, because Jadhav et al. taught that the effector molecules can be optimized for RNA interference by decreasing the free energy of the duplex association by introducing thermally destabilizing modifications in the sense strand at a site opposite to the seed region of the antisense strand (i.e. at positions 2-8 of the 5’ end of the antisense strand) which can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand (paragraph 0376), and taught the thermally destabilizing modification can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand within the dsRNA duplex, and exemplary mismatch base pairs include G:A (paragraph 0382). Therefore, an ordinary artisan could have provided a complementary G:A base pair in the sequence that would function as a wobble base pair, that is complementary to bases in positions 2-7 from the 5’ end of the miRNA based on the teachings of Jadhav et al. in order to introduce thermally destabilizing modifications to optimize the inhibiting activity the bivalent molecule of ‘353.
It would be obvious for the miRNA target site sequence to comprise 6-8 nucleotides complementary to a seed region of miRNA to arrive at the instant invention with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to provide a miRNA target site sequence comprising 6-8 nts complementary to a seed target region of miRNA, as ‘353 teaches that the first and/or second oligonucleotide comprise a seed sequence of microRNA capable of base pairing to the complementary sequence of the seed sequence (page 5, lines 23-27) and that preferred contiguous sequences are at least 6 nucleotides (page 8, lines 6-7), and would make obvious the limitations of claim 6.
It would be obvious that the seed region is a sequence between positions 1-8 from the 5’ end of the miRNA to arrive at the instant invention with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to provide a modified nucleic acid, wherein the seed region is a sequence between positions 1-8 from the 5’ end of the miRNA, as ‘353 teaches the first and/or second oligonucleotides comprises a contiguous sequence capable of base pairing to the complementary sequence of a sequence of position 1-8 (page 8, lines 26-35 and page 9, lines 8-13), and would make obvious the limitations of claim 7.
It would be obvious to one of ordinary skill in the art to modify the synthesis method of the oligonucleotide of ‘353 with the teaching of Lee et al. and substitute a base with an abasic spacer in-between the consecutively arranged base sequences to improve on-target activity. One of ordinary skill in the art would be motivated to substitute a base with the abasic spacer between the consecutively arranged base sequence as Lee et al. teach using an abasic spacer that contains no base but functions as a linker, including an abasic deoxynucleotide spacer, dSpacer, as a substitute for a nucleotide in the nucleation region (position 2-6) and that the substitution prevents miRNA-like off-target repression and conserves on-target activity (page 3), and would make obvious the limitations of claim 9.
It would be obvious to provide a method of inhibiting miRNA comprising introducing the modified oligonucleotide of ‘353 inhibiting miRNA into cells in vitro for the purpose of testing the activity of the modified nucleic acid in a cell in vitro. One of ordinary skill in the art would have been motivated to introduce the bivalent molecule into cells in vitro, as ‘353 teaches using the molecules of the invention for modulating microRNA regulation by blocking a microRNA or blocking a microRNA binding site in a target RNA in vitro (page 29, lines 21-24), and transfecting the oligonucleotides into HUH7 cells expressing microRNA-122 (Example 4, page 37 and Fig 1 and 2), and would make obvious the limitations of claim 10.
It would be obvious to provide a gene transfer system comprising the modified oligonucleotide inhibiting miRNA, in order to provide a means for introducing the modified nucleic acid into a cell. One of ordinary skill in the art would have been motivated incorporate the modified nucleic acid into a gene transfer system, as ‘353 teaches a vector construct comprising two microRNA-122 binding sites (Example 4 page 34), and would make obvious the limitations of claim 11.
Therefore the invention as a whole would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over ‘353, Lee et al. and Jadhav et al. as applied to claims 1,6,7 and 9-11 above, and further in view of Chen et al. (The Journal of Cell Biology, Vol. 190, Published 6 September 2010, pages 867-879), cited on an IDS Dated 05/26/2023.
The teaching of ‘353, Lee et al. and Jadhav et al. regarding claims 1,6,7 and 9-11 is described above.
‘353, Lee et al. and Jadhav et al. do not teach wherein the miRNA is miR-1.
However, before the effective filing date, Chen et al. taught that aberrant miRNA expression has been observed in muscle diseases such as cardiac and skeletal muscle hypertrophy, heart failure, and muscular dystrophy, and that expression of muscle-specific miR-1 is induced during skeletal muscle differentiation, plays a role in myoblast proliferation and differentiation, and is an important regulator of cardiomyocyte differentiation and heart development (page 867-868). Chen et al. teach knocking down miR-1 using an miRNA antagomir which resulted in decreased expression level of miR-1 in skeletal muscle (page 871, right column), and it was determined that knockdown of miR-1 enhanced muscle cell proliferation (page 872, left column), and therefore may be therapeutic tools and targets for human skeletal muscle disease (page 877, left column).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the bivalent molecule of ‘353, Lee et al. and Jadhav et al. with the teaching of Chen et al. regarding miR-1 as the target, in order to inhibit miR-1 as the miRNA. One of ordinary skill in the art would have been motivated to provide a modified bivalent molecule inhibiting miR-1, as Chen et al. taught that aberrant expression of miRNAs have been observed in muscle diseases, and knocking down miR-1 enhances muscle cell proliferation and may therefore be a therapeutic target for human skeletal muscle disease (pages 867,871,877).
Therefore the invention as a whole would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claims 3-5 are rejected under 35 U.S.C. 103 as being unpatentable over ‘353, Lee et al., Jadhav et al. and Chen et al. as applied to claim 2 above, and further in view of Bartel (Cell 136, Published 23 January 2009, pages 215-230) cited on an IDS dated 11/30/2020.
The teachings of ‘353, Lee et al., Jadhav et al. and Chen et al. as applied to claim 2 is described above.
‘353, Lee et al., Jadhav et al. and Chen et al. do not teach wherein the modified nucleic acid inhibiting miRNA contains a 5’-ACAUUCCA-3’ or 5’-AAAUUCCA-3’ sequence, or wherein the modified nucleic acid inhibiting miRNA consecutively comprises at least two 5’-ACAUUCCA-3’ sequences or at least two 5’-AAAUUCCA-3’ sequences, and at least one abasic spacer before and after the sequence.
However, before the effective filing date, Bartel teaches the understanding of miRNA target recognition and miRNA target sites. Bartel teaches mammalian targets can be predicted by searching for conserved 7 nt matches in aligned regions of vertebrate 3’-UTRs, and prediction specificity increases when requiring an 8 nt match or multiple matches to the same miRNA, and that enough genomes have been sequenced and aligned such that these targets with single sites can now be predicted with confidence that most are authentic (page 218, left column). Bartel teaches using a three-step protocol for predicting evolutionary conserved targets for metazoan miRNA: (1) identify the two 7 nt matches to the seed region (Figures 1A and 1B), for example, miR-1 with the sequence of 5’-UGGAAUGUAAAGAAGUAUGUA would recognize the CAUUCCA match and the ACAUUCC match, (2) use available whole-genome alignments to compile orthologous 3’UTRs, (3) search within the orthologous UTRs for conserved occurrence of either 7 nt match which are the predicted regulatory sites (page 218, left column). Bartel teaches that a search for conserved 8 nt sites comprised of both 7 nt motifs (e.g. ACAUUCCA in the case of miR-1, Figure 1C) yields greater prediction specificity, and a search for conserved 6 nt seed matches (Fig 1D) yields greater sensitivity (page 217-218).
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, that the bivalent molecule of ‘353 modified by the abasic spacer of Lee et al. and sequence having complementary G:A base pairs of Jadhav et al. that targets miR-1 according to the teaching of Chen et al., would comprise a 5’-ACAUUCCA-3’ sequence based on teaching of Bartel regarding the three-step protocol for predicting evolutionary conserved targets using miR-1 as an example. One of ordinary skill in the art would have been motivated to provide the bivalent molecule of ‘353 modified by the teachings of Lee et al., Jadhav et al. and Chen et al., with a 5’-ACAUUCCA-3’ sequence based on Bartel’s simple three-step protocol to predict evolutionarily conserved targets for a miRNA, and which identifies the sequence ACAUUCCA as a seed matched site of miR-1. It would have been obvious to one of ordinary skill in the art to provide the bivalent molecule of ‘353 modified by the teachings of Lee et al., Jadhav et al., and Chen et al. with at least 2 consecutive ACAUUCCA sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule. One of ordinary skill in the art would have been motivated to do so because Bartel teaches prediction specificity increases when requiring multiple matches to the same miRNA.
It would have been obvious to one of ordinary skill in the art to provide the bivalent molecule of ‘353 modified by the abasic spacer of Lee et al. and sequence having complementary G:A base pairs of Jadhav et al. that targets miR-1 according to Chen et al., with at least 2 consecutive 5’-AAAUUCCA-3’ sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule based on teaching of Bartel regarding the three-step protocol for predicting evolutionary conserved targets using miR-1 as an example. While Bartel does not teach the exact sequence of “AAAUUCCA”, Bartel does teach “ACAUUCCA”, which is only one nucleotide different. An ordinary artisan could have arrived at the sequence “AAAUUCCA” from the sequence “ACAUUCCA” by replacing the 2nd ‘C’ nucleotide with an ‘A’ nucleotide based on the teachings of Jadhav et al. regarding introducing thermally destabilizing modifications to optimize the inhibiting activity, and which modifications can be mismatches (i.e. noncomplementary base pairs) between the thermally destabilizing nucleotide in the opposite strand and exemplary mismatch base pairs include G:A (paragraph 0382). One of ordinary skill in the art would have been motivated to provide at least 2 consecutive AAAUUCCA sequences and at least one abasic spacer before and after the sequence to increase potency of the molecule because Bartel teaches prediction specificity increases when requiring multiple matches to the same miRNA.
Accordingly, the limitations of claims 3-5 would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Conclusion
Claims 1-7 and 9-11 are rejected.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636