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 4-2-26.
Claims 1, 2, 11, 17, 19, 20, 36, 52, 139, 152, 157, 176, 182-188 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Withdrawn Objections/Rejections
Any objections or rejections not repeated in this Office action are hereby withdrawn.
Maintained Rejections
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 52 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
In claim 52, lines 4-10, crRNA molecules with sequences are recited, but no accompanying SEQ ID Nos. are listed in the claim.
Appropriate correction is required.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 2, 11, 19, 20, 32, 51, 52, 139, 152, 157, 176, 182-188 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention for the reasons set forth in the Office action mailed 10-16-25 and for the reasons set forth below.
The breadth of the claims
The claims are broadly drawn to chemically modified guide RNA comprising: (a) a CRISPR RNA (crRNA} portion comprising (i) a 3' end and a 5' end, (ii) a guide sequence of 10-30 nucleotides in length capable of hybridizing to a target polynucleotide sequence, and a repeat sequence of any length; and (b) a trans-activating CRISPR RNA (tracrRNA} portion comprising a 3' end and a 5' end of any lengths, and an anti-repeat nucleotide sequence of any length that is complementary to the repeat sequence, wherein the crRNA portion comprises at least 90% modified nucleotides, of which at least positions 4-6, positions 15, 16, and 19, and/or positions 22-24 from the 5' end comprise a 2'-deoxy chemical modification, which tracrRNA portion comprises at least 80% modified nucleotides optionally also comprising modification of the phosphate group optionally comprising a phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, phosphotriester, or a phosphorothioate modification, which crRNA portion optionally comprises a phosphorothioate chemical modification at positions 1-3 or positions 1-6 from the 5' end, and optionally further comprising at least one moiety conjugated to the chemically modified guide RNA, and which chemically modified guide RNA optionally further comprises a nucleotide or non-nucleotide loop or linker linking the 3' end of the crRNA portion to the 5' end of the tracrRNA portion, which RNA-guided nuclease is a Cas9 nuclease, which modifications of the ribose group optionally comprise 2'-O-methyl, 2'-fluoro, 2'-O- (2-methoxyethyl) (MOE), 2'-NH2 (2'-amino), 4'-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2'-(S)-constrained ethyl (S-cEt), a constrained MOE, and a 2'-0,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANc), and optionally comprising one or more modifications of a nucleobase optionally comprising 2-thiouridine, 4-thiouridine, N6- methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5- substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups, and which crRNA portion optionally comprises between 1 and 20 phosphorothioate modifications, wherein the nucleotides at positions 4-6 from the 5' end of the crRNA portion comprise the 2'-deoxy chemical modification, or wherein the nucleotides at positions 15, 16, and 19 from the 5' end of the crRNA portion comprise a 2'-deoxy chemical modification, or wherein the nucleotides at positions 22-24 from the 5' end of the crRNA portion comprise a 2'-deoxy chemical modification, or optionally comprising a 2'-fluoro chemical modification at one or more nucleotides of positions 11-19 from the 5' end of the crRNA portion.
Applicant’s Arguments
Applicant argues the following:
The disclosure describes the chemically modified guide RNA of the instant claims with adequate specificity-whether through structural features, physical and chemical properties, functional characteristics supported by structure-function correlation, drawings, and actual reduction to practice-such that one of ordinary skill in the art can reasonably conclude that the full scope of the claimed subject matter is supported. When viewed in light of the level of skill in the art, the specification conveys possession of each claimed embodiments and the genus defined by claim 1.
For instance, the application provides a detailed description of actual reduction to practice of chemically modified guide RNA, and how the synthesized compounds were used to identify and further optimize heavily modified patterns of crRNA portions to retain high genome editing efficiencies. For example, the application describes: (i) the design and synthesis of 178 crRNAs and 114 tracrRNAs (Example 1 and Tables 2 and 6); (ii) the screening of guide RNA crRNA / tracrRNA pairs by nucleofecting HEK293T cells with 20 picomoles of 3xNLS-SpyCas9 and 25 picomoles of crRNA:tracrRNA guides and then measuring the cellular indel rate (Example 2 and Figures 2-8); (iii) the screening of guide RNA crRNA / tracrRNA pairs comprising conjugates targeting endogenous human genes (Example 3 and Figure 9); (iv) the screening of crRNAs with varied phosphorothioate content (Example 4 and Figure 10); and (v) the in vivo testing of guide RNA crRNA / tracrRNA pairs in mTmG transgenic mouse (Example 7 and Figures 13 and 14). Furthermore, the accompanying figures demonstrate that the claimed subject matter provides guide RNA with higher editing efficiencies compared with guide RNA crRNA / tracrRNA pairs lacking heavy chemical modifications.
For instance, Figure 5 depicts a screen of crRNA patterns C23-C44, showing crRNAs C29, C39 and C40 demonstrate efficacy similar to that of a previously developed crRNA, C20. These new crRNA are fully modified in the sense that every nucleotide that does not have a ribose modification has a phosphodiester linkage modification. In contrast, C20 still contains six unmodified ribose residues. Figure 7 and 8 shows that heavily modified tracrRNA (>80% modifications), paired with crRNA of claim 1, show equivalent or higher activity than T2 (~80% modifications). Moreover, Figure 10 confirms across a larger and diverse panel of crRNAs sequences (crRNA C52-C93) that guide RNAs tolerate least up to 20 phosphorothioate modifications. Together with the modification patterns detailed in Tables 1, 2, and 6, these results establish that Applicant was in possession of the full scope of the instant claims. The structure and function analyses reveal robust crRNA chemical modification patterns and positions that permit empirical refinement in sufficient detail to convey to a skilled person that the inventors had possession of the claimed subject matter at the time of filing.
Response to Applicant’s Arguments
Applicant is correct that the specification teaches a multitude of modified crRNA and tracrRNA molecules. The lengths of these modified oligonucleotides provided in the instant specification, however, are 67 and 36 nucleotides. The claims rejected here read broadly on any lengths of the 3’ and 5’ ends of the crRNA, no length limitations on the repeat and anti-repeat sequences, and tracrRNAs of any lengths.
Teachings in the specification
The specification teaches the following on page 3:
…existing guide RNAs suffer from several limitations, which limit their utility in therapeutic applications. For example, existing guide RNAs may be subject to rapid degradation in circulation and within cells. Moreover, chemical modifications of guide RNAs may reduce stability and editing efficiency. Accordingly, there exists a need in the art for optimized guide RNAs that retain efficient genome editing activity in vivo and ex vivo when paired with a CRISPR nuclease, such as Cas9.
The specification teaches that a pMCSG7 vector was used to express Cas9 from Streptococcus pyogenes, and contained three nuclear localization signals. This enzyme was used for screening the constructs for target cleavage.
And on pages 134-138, the specification teaches the following:
[0246] Screening of New Chemical Modification Patterns [0247] Structure-guided and systematic approaches were used to introduce 2'-OMe-RNA, 2'-F-RNA, 2’-deoxy, and PS modifications throughout guide RNAs. These modifications were chosen because they have been shown to improve stability, efficacy, and immunotoxicity associated with RNA. The strategy described herein yielded active RNP complexes with both extensively and fully modified versions of crRNAs and tracrRNAs. Figure 4 and Figure 5 depict a screen of crRNA patterns C23-C44, targeting both the MCV lasite and the MCV 1b site. The crRNAs C29, C39 and C40 demonstrate efficacy similar to that of the previously developed crRNA, C20. The crRNAs C20, C29, and C39 are fully modified in the sense that every nucleotide that does not have a ribose modification has a phosphodiester linkage modification. However, C20 still contains six unmodified ribose residues, while the new crRNA C39 only has three unmodified riboses, and C29 has only one unmodified ribose C40 is the newly developed, fully modified crRNA with no unmodified riboses in its composition. C45 is also a fully modified molecule with no unmodified ribose moieties. Like C40, this composition is expected to be very stable in vivo, though its activity is diminished somewhat in comparison to crRNA C20.
[0248] New tracrRNA chemical modification patterns were also developed. Figure 6 depicts a screen of previously described tracrRNA patterns T2, T9, T12, T17, and T18, compared to new patterns T38, T39, and T41. The different tracrRNAs were paired with C21, C39, C40, or C45. The new crRNAs C39, C40, and C45 displayed higher editing efficiencies when paired with all tracrRNAs compared to the older C21 pattern. 20 [0249] Several new tracrRNAs are more heavily modified than the previous tracrRNA T2. TracrRNA T41, T12 and T17 show higher activity than T2. TracrRNAsT9, T18, T37, T38 and T92 display similar efficiencies as T2, while T49 and T95 display slightly diminished activity than T2 (Figure 7 and Figure 8).
[0250] The loss in efficacy seen in human cells with the fully modified crRNA C45 and heavily modified tracrRNAs T49 and T95 compared to the previously developed crRNA C20 or tracrRNA T2 may be offset by higher in vivo stability. All of the newly developed RNAs are functional in multiple combinations when tested in human cells
Example 3 — Chemically modified crRNA:tracrRNA pairs with and without conjugates targeting endogenous human genes [0251] To verify that the crRNA and tracrRNA designs of the disclosure are compatible with different guide sequences, including those targeting endogenous human genes, the designs C29, C30, C40, C42, and C45 were tested by targeting the PCSK9 gene (Figure 9). The crRNAs were paired with tracrRNA T2 or T6, and T2 was further used in a non-conjugate or GalNAc-conjugate form. C29, C39, C40, and C42 were also tested in a non-conjugate or GalNAc-conjugate form. The RNA designs were tested by electroporation of Cas9 RNP in the mouse Hepa 1-6 cell line. The graphs show indel percentages based on Inference of CRISPR Edits (ICE) analysis of PCR and Sanger sequencing data of the locus. The data represent the means from three independent biological replicates and error bars represent s.e.m. [0252] These results demonstrate that the modified crRNA and tracrRNA designs are also applicable to endogenous target sites and function with conjugates on both the crRNA and tracrRNA.
Example 4 — Chemically modified crRNAs with varied phosphorothioate content [0253] Additional chemically modified crRNAs were designed, synthesized, and tested for genome editing efficiency. crRNAs C52-C93 were tested in the TLR assay with the MCV 1a target site. Each crRNA was paired with the T41 tracrRNA. 2 pmol of an RNP containing Cas9 with the various crRNAs and the tracrRNA were transfected into the TLR-MCV1 line described above and the % mCherry expression was detected as a proxy for genome editing efficiency. The crRNAs C52-C93 contained the same chemical modification pattern as C40, except with respect to phosphorothioate placement. The crRNA sequences are shown in Table 6. The screen revealed that crRNAs containing at least up to 20 phosphorothioate modifications are tolerated (Figure 10).
Example 5 — Chemically modified crRNAs containing 2’-amino RNA and/or 4’- thio RNA modifications [0254] Additional chemically modified crRNAs containing either 2’-amino RNA or 4’-thio RNA (2.e., sugar ring oxygen in ribose sugar is replaced with sulfur modifications were designed, synthesized, and tested for gene editing efficiency. crRNAs C114-C134 were tested in the TLR assay with the MCV 1a target site or MCV 1b target site, or in the mTmG reporter system, each of which is described above.
As shown in Figure 11A, crRNAs C116-C118 and C122-C134 was paired with the T2 tracrRNA. 5 pmol of an RNP containing Cas9 with the various crRNAs and the tracrRNA were transfected into the TLR-MCV1 line described above and the % mCherry expression was detected. As shown in Figure 11B, crRNAs C116-C118 and C122-C134 were used in a modified TLR-MCV1 assay in which an unmodified tracrRNA and SpCas9 were stably expressed as well. 100 pmol of each crRNA was transfected into the cell line and the % mCherry expression was detected.
Finally, as shown in Figure 11C, crRNAs C114-C127 were tested in the mT'mG reporter assay described above with 5 pmol of an RNP containing Cas9 with the various crRNAs and the T2 tracrRNA. The crRNA sequences are shown in Table 6. Each crRNA tested either had one or more 2’-amino ribose modifications or one or more 4’-thio RNA modifications. The screen revealed that crRNAs containing one or more 2’-amino ribose modifications or one or more 4’-thio RNA modifications maintain effective gene editing activity, while possessing additional chemical modifications that can improve stability.
Example 6 — Chemically modified tracrRNAs containing 4’-thio RNA modifications [0255] Additional chemically modified tracrRNAs containing 4’-thio RNA modifications were designed, synthesized, and tested for gene editing efficiency. tracrRNAs T107-T116 were tested in the TLR assay or in the mTmG reporter system, each of which is described above. Each of T107-T116 had the same chemical modification pattern as T2, except a 4’-thio RNA modification was introduced at one or more of the unmodified residues. 5 pmol of an RNP containing Cas9 with the various tracrRNAs and the C20 crRNA were transfected into the TLR-MCV1 line or mTmG line and the fluorescence was detected. The tracrRNA sequences are shown in Table 2. As shown in Figure 12, all of the tracrRNAs tested retained effective gene editing activity. The inclusion of 4°-thio RNA modifications at previously unmodified positions provides tracRNAs that are closer to being 100% chemically modified. T107 for example, has a modification at all but 5 nucleotides.
Example 7 — In vivo gene editing [0256] The various chemically modified guide RNAs have displayed substantial gene editing activity in vitro while possessing enhanced stability (e.g.,serum stability). The in vivo activity of select chemically modified guide RNAs was next determined in the mTmG transgenic mouse. RNPs made up of select crRNAs and tracrRNAs, along with Cas9, were intrastriatally (IS) injected into the mouse at a dose of 150-200 pmol. Six days following injection, mouse brain tissue was stained to detect 15 GFP expression. The guide RNA crRNA/ tracrRNA pairs were used: C20 / T2, C29 / T2, C20 / T41, and C29 / T41.
As shown in Figure 13, GFP was expressed in brain tissue from mice receiving a C20 / T2 containing RNP. As shown in Figure 14, GFP was expressed in brain tissue from mice receiving a C20 / T41 containing RNP. The data shows that the chemically modified guide RNAs are capable of gene editing activity in vivo.
[Emphases added}
As stated previously, the teachings in the specification are not representative of the expansive genus of molecules claimed. The specification fails to provide the requisite guidance for making and using the large genus of modulatory agents instantly claimed, and further whereby off target activity is reduced and/or on target activity is increased relative to an unmodified guide RNA.
Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus of modulators, and because the claimed genus is highly variant, the description provided is insufficient, one of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the myriad of modulatory agents instantly claimed.
Thus, Applicant was not in possession of the broadly claimed genus.
Allowable Subject Matter
Claims 17 and 36 are objected to as being dependent upon 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.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - Sequences appearing in the specification are not identified by sequence identifiers (i.e., “SEQ ID NO:X” or the like) in accordance with 37 CFR 1.831(c).
There are no sequence identifiers associated with the sequences listed in Table 1, pages 83-92, Table 2, pages 92-102, Table 3, page 104, Table, page 104, text on pages 106-107, Table 5, pages 108-109, Table 6, pages 112-123, and claim 52.
Please provide accompanying SEQ ID Nos. for these sequences.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Conclusion
THIS ACTION IS MADE FINAL. 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.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free).
Jane Zara
5-14-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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