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 .
Status of Claims
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 13, 15 – 17, 20 – 23, 26 – 38, and 40 are currently pending. Claims 13 have been amended in the Applicant’s amendment filed October 13, 2025. No claims have been added or canceled in the Applicant’s amendment filed October 13, 2025.
Applicant's election of Species without traverse as follows:
Species (A): gRNA internucleotide linkage modifications: phosphonoacetate internucleotide linkage (instant claim 3);
Species (B): 2’-gRNA modifications: 2’O-methyl (instant claim 4); and
Species (C): 2’/3’-gRNA modifications: 2’O-methyl-3’phosphonoacetate (instant claim 5), was previously acknowledged in the reply filed June 21, 2023.
Claim 34 was previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
The restriction requirement was previously made FINAL.
The claims will be examined insofar as they read on the elected species.
Therefore, claims 13, 15 – 17, 20 – 23 and 26 – 33, 35 – 38, and 40 are under consideration to which the following grounds of rejection are applicable. Claims 13 and 20 are independent claims.
The examiner acknowledges receiving the Declaration (hereinafter referred to as the “Ryan declaration”) under 37 C.F.R. § 1.132 filed on 5 June, 2025 and executed by Daniel E Ryan.
Information Disclosure Statements
The information disclosure statements (IDS) submitted on October 13, 2025, November 11, 2025, December 15, 2025 and March 13, 2026 has been considered. An initialed copy of the IDS accompanies this Office Action.
Priority
The present application filed July 15, 2020 claims the benefit of CON 15/493,129, filed April 20, 2017, which claims the benefit of Provisional Application 62/347,553, filed June 8, 2016.
Therefore, the earliest priority date is June 8, 2016.
Maintained Objections/Rejections
Nonstatutory Double Patenting
The rejection of claims 13, 15-17, 20 – 23, 26-33, 35 – 38 and 40 on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 23 of US Patent No. US10767175 B2 in view of US Patent No. US2015004192 A1 is maintained
Patent claim 1 of US10767175 B2 recites a synthetic guide RNA comprising a crRNA, comprising a guide sequence and a stem sequence, and a tracrRNA. The guide sequence consists of 20 – N nucleotides, wherein N is an integer between – 10 and 3, and wherein the guide sequence comprises a modification at the 4-N, 5-N, 7-N, 9-N, 10-N, or 11-N position. Claim 1 further teaches that the modification is a DP, DSP, MSP, MS, MOE, MOES, MOEP, or MOESP. The instant claim 13 of the application recites a synthetic guide RNA comprising a crRNA, comprising a guide sequence and a stem sequence, and a tracrRNA. Further, claim 13 teaches that the guide sequence consists of 20-N nucleotides, where N is an integer between -10 and 6, and there is a modification at any position 4-N to 20-N, and wherein the modification is not at position 15-N of the guide sequence. Claim 13 teaches that the guide sequence comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification, wherein dependent claim 17 further teaches that modification is a SP, MS, MP, MSP, DP, or DSP.
Claim 1 of the instant invention has been amended to recite : wherein when said modified synthetic guide RNA is complexed with a Cas protein, said complex has a specificity score greater than a a specificity score of a control synthetic guide RNA complexed with a Cas protein, wherein said control synthetic guide RNA has the same nucleotide sequence as the modified synthetic guide RNA and does not have any internal modified nucleotides”
Claim 1 of US10767175 B2 does not require a specificity score when the modified synthetic guide RNA is complexed with a Cas protein.
US Patent No. US2015004192 A1 teaches compositions, systems, and methods to evaluate and characterize the sequence specificity of the Cas9 endonucleases (Paragraph [0005]). US Patent No. US2015004192 A1 teaches that the sgRNA comprises 5 – 50 nucleotides (Paragraph [0011]). US Patent No. US2015004192 A1 teaches that specificity scores to quantify the enrichment level of each base pair at each position in the post-selection library relative to the pre-selection library and the formula to calculate the specificity score (Paragraph [0165] and [0124]).
It would have been obvious to one of ordinary skill in the art, before the effective filing date, to have use the specificity score calculator (of US2015004192 A1) to measure the specificity of the synthetic guide RNA and Cas9 (of US10767175 B2) to optimize the sgRNA and Cas9 functionality and binding to the target site.
Response to arguments as they relate to Nonstatutory Double Patenting filed on October 15, 2025.
Applicant’s arguments filed October 15, 2025 have been fully considered but they are not persuasive. Applicant essentially asserts: (a) Applicant requests that the rejection be held in abeyance until allowable subject matter is agreed upon (Applicant Remarks, pg. 11, first and second paragraph paragraph).
Regarding (a), Applicant’s arguments filed October 15, 2025 did not specifically indicated how the claims of the copending applications recited supra are patentably distinct from the instant claims as required by 37 CFR 1.111(b). Thus, the claims remain rejected for the reasons already of record.
Moreover, Applicants offer to provide a terminal disclaimer upon indication by the Examiner of allowable claims. However, Applicant’s request is not a proper response to the rejections of record as it neither traverses the grounds of rejection by providing specific arguments, nor indicates that a terminal disclaimer has been filed to overcome the rejection. As such, the rejections of record stand.
Claim Rejection - 35 USC § 112(b)
The rejection of claims 13, 15 – 17, and 36 – 38, and 40 is maintained 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. This rejection has been amended based on response filed 5 June, 2025.
Claim 13 is indefinite for the recitation of “wherein said control synthetic guide RNA has the same nucleotide sequence as the modified synthetic guide RNA and does not have any internal modified nucleotides at the end of claim 13. It is unclear what the “internal modified nucleotides” are referring to, whether the nucleotides are not modified internally (with the MP modifications) or whether the modifications do not apply to the 5’ and 3’ end. Further, this term has not been defined in the as-Filed Specification. Thus, the metes and bounds of the claim cannot be determined.
Claims 15 – 17, and 36 – 39 are indefinite insofar as they ultimately depend from claim 13.
Claim Rejection - 35 USC § 112(a) Scope of Enablement
The rejection of claims 13, 15 – 17, 20 – 23, 26 – 33, and 35 – 38, and 40 is maintained under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the Specification, while being enabling for
specific guide sequences with MP modifications at positions 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, and 19 of a 20 nucleotide gRNA specific for CLTA4, IL2RG, VEGFA, and HBB, and having a specificity score greater than a control guide RNA complexed with a Cas protein, wherein the control synthetic guide RNA has the same nucleotide sequence as the modified synthetic guide RNA and does not have any internal modified nucleotides, wherein the specificity score of said complex is calculated by multiplying an ON:OFF ratio by the ON-target cleaving, binding, or nicking percentage of the modified synthetic guide RNA assayed, and wherein said ON:OFF ratio is a ratio of ON target cleaving, binding, or nicking percentage to an OFF target cleaving, binding, or nicking percentage of said complex,
it does not reasonably provide enablement for all guide sequences, specific to any and all genes, between 17 to 30 nucleotides with MP and/or MSP modifications at the positions detailed in claim 13 to have a specificity score greater than the specificity score of a control synthetic guide RNA. This rejection has been modified as necessitated by the response filed October 13, 2025.
The Specification does not enable any person skill in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims. The claims, 13, 15 – 17, and 35 – 38, and 40, when given the broadest possible interpretation, encompass a modified synthetic guide RNA comprising a CRISPR RNA (crRNA) and a trans-acting RNA (tracrRNA), wherein the crRNA comprises a guide sequence is between 17 – 30 nucleotides, and has a MP or MSP modification at specific positions, as recited in instant claim 13, and wherein the guide RNA is complexed with a Cas protein, which has a specificity score greater than a specificity score of an unmodified synthetic guide RNA complexed with a Cas protein. Claims 20 – 23, and 26 – 33 are directed to a method of enhancing the specificity of the CRISPR function, comprising providing the synthetic guide RNA of claim 13, forming a guide RNA: Cas protein complex, and contacting the target polynucleotide with the protein complex. The Specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the patent coupled with information known in the art without undue experimentation (United States v. Telectronics, Inc., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is required is not based on a single factor but is rather a conclusion reached by weighing many factors (See Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter, 1986) and In re Wands, 8USPQ2d 1400 (Fed. Cir. 1988); these factors include the following:
Nature of invention. The claims initially encompass a modified synthetic guide RNA
comprising a CRISPR RNA (crRNA) and a trans-acting RNA (tracrRNA), wherein the crRNA comprises a guide sequence is between 17 – 30 nucleotides, and has a MP or MSP modification at specific positions, as recited in instant claim 13, and wherein the guide RNA is complexed with a Cas protein, which has a specificity score greater than a specificity score of an unmodified synthetic guide RNA complexed with a Cas protein. The claims also encompass a method of enhancing the specificity of the CRISPR function, comprising providing the synthetic guide RNA of claim 13, forming a guide RNA: Cas protein complex, and contacting the target polynucleotide with the protein complex.
Scope of the invention. The claims initially encompass a modified synthetic guide RNA and
method of enhancing the specificity of the CRISPR function.
Number of working examples and guidance. In the instant case, Applicant teaches several
examples disclosing the modifications made to sgRNAs in the as-Filed Specification. Example 3 illustrates that a series of 32 sgRNA were made for targeting the CTLA locus, wherein the sgRNA contain one or more specificity enhancing modifications. Example 3 teaches MP modifications in the guide sequence at nucleotide 1, nucleotides 1-2, nucleotides 1-3, nucleotides 1-4, and nucleotides 1-5, respectively, of the 20 nucleotide sequence (Paragraph [0277]). Related Figure 8B teaches the specificity score for in vitro cleavage for CLTA1, wherein there are various different specific modifications that are made in the CLTA1 guide sequence, both at the 5’ end and that 3’ end (Paragraph [0278]). MP modification gave the largest desired effect of decreasing off-target cleavage while retaining high levels of on-target cleavage (Paragraph [0278]).
Example 4 illustrates single or triple MP modifications were “walked” across the 20-nt guide sequence to see which modified positions could yield improvements in specificity as judged by on-target versus off-target cleavage activities for targeting CLTA4 (Paragraph [0279]). Example 4 teaches that experimental sgRNAs contained a 2′-O-methyl-3′-PACE (“MP”) modification at one or more positions in the guide sequence, in addition to having an MP modification at nucleotide 1 and also at the penultimate nucleotide in the tracrRNA region at the 3′ end of the sgRNA (Paragraph [0279]). The specificity score for in vitro cleavage of CTLA4 target polynucleotide are show in Figure 9B, wherein there are multiple modifications made to the guide sequence, the first one is at the 3’ end, and a MP modification at another location in the guide sequence. The as-Filed Specification teaches that a general strategy for improving specificity is to incorporate 1, 2, 3, 4 or 5 MP modifications at consecutive phosphodiester inter-nucleotide linkages at the 5′ end of a guide sequence in a gRNA (Paragraph [0279]).
Example 5 illustrates single or triple MP modifications were “walked” across the 20-nt guide sequence to see which modified positions could yield improvements in specificity as judged by on-target versus off-target cleavage activities for targeting IL2RG (Paragraph [0279]). Example 4 teaches that experimental sgRNAs contained a 2′-O-methyl-3′-PACE (“MP”) modification at one or more positions in the guide sequence, in addition to having an MP modification at nucleotide 1 and also at the penultimate nucleotide in the tracrRNA region at the 3′ end of the sgRNA (Paragraph [0279]). The specificity score for in vitro cleavage of IL2RG target polynucleotide are show in Figure 10, wherein there are multiple modifications made to the guide sequence, the first one is at the 3’ end, and a MP modification at another location in the guide sequence.
Example 6 illustrates a modification “walk” done with an MP modification installed at incremental positions across a guide sequence targeted to HBB gene in the 20-nucleotide guide sequence. The specificity score for in vitro cleavage of HBB target polynucleotide are show in Figure 11, wherein there are multiple modifications made to the guide sequence, the first one is at the 3’ end, and a MP modification at another location in the guide sequence.
The disclosure is not deemed to be descriptive of the all guide RNAs ranging from 17 to 30 nucleotides having a specificity score that is greater than a specificity score of an unmodified synthetic guide RNA complexed with a Cas protein. The as-Filed Specification teaches four working examples, wherein 4 guide RNAs, targeting CLTA1, CLTA5, IL2RG, and HBB, are modified with MP modifications. However, all these modifications only occur in 20 nucleotide guide RNAs, and thus specificity score is only determined for modifications in the 20 nucleotide guide RNAs. The as-Filed Specification does not teach MSP modifications, modifying the guide RNAs of different lengths (ranging from 17 to 30 nucleotides), or determining the specificity score of the modifications made in the guide RNAs of different lengths.
The as-Filed Specification teaches Figure 9B, which teaches the specificity score for in vitro cleavage of CLTA4. As noted in claim 13, wherein if the guide sequence is 20 nucleotides long, then the MP or MSP modification is selected from the group consisting of 4, 5, 7, 8, 10, 11, 12, 13, 14, 16, 17, 18, and 19 relative to the 5’ end of the guide sequence, and the guide RNA and Cas protein complex has a specificity score of greater than the specificity score of a control guide RNA complexed with a Cas protein. However, as seen in Figure 9B, the modifications of MP of the CTL4 target guide sequence at positions 8, 10, 13, 18, and 19 yield a specificity score that is less than the specificity score of the control (across all three specificity scores provided in the Figure 9B). A similar analysis can be done for Figures 10 and 11, which are directed to in vitro modifications of IL2RG and HBB, respectively. Thus, the disclosure does not adequately teach these 20 nucleotide guide sequences that have a specificity score of greater than the specificity score of a control sequence.
State of the art. Although the field of RNA is highly developed, the composition comprising
crRNA duplexed with a tracrRNA, wherein the crRNA comprises a guide RNA with many modifications and having a specific specificity score is not highly developed. The use of the Cas9 system is also not highly developed, and the art must therefore be considered to be poorly developed.
Unpredictability of the art. Before the effective filing date of the claimed invention, it was
known in the art that sgRNAs with +85 nucleotide tracrRNA tails (of the crRNA:tracrRNA duplex) increased the activity of Cas9 and induced higher level of indels in vivo (interpreted as the variability of the tracrRNA for optimal functionality); and that the Cas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches, as evidenced by Hsu et al. (Hsu, P. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827–832 (2013)) (pg. 827, right column, first paragraph and Abstract).
It was also known in the art that Cas9 mediated cleavage can be abolished by single mismatches at the gRNA-target site interface, particularly in the last 10 – 12 nucleotides located in the 3’ end of the 20-nucleotide gRNA targeting region, as evidenced by Fu et al. (Fu, Y., et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31, 822–826 (2013).
Additionally, there are many variabilities in the art regarding the design of the sgRNA, including the using of the NGG, a requirements of a purine/pyrimidine composition near the 3’ end of the spacer sequence, and the importance of the DNA downstream of the spacer sequence, as evidenced by Xu et al. (Xu H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 2015 Aug;25(8):1147-57. doi: 10.1101/gr.191452.115. Epub 2015 Jun 10. PMID: 26063738; PMCID: PMC4509999.) (pg. 1147, right column, first full paragraph).
Amount of Experimentation Required. Given the unpredictability of the art, the poorly
developed state of the art with regard to research on the design of the sgRNAs, the Cas9 role and function, and the sequence of the tracrRNA, the skilled artisan would have to conduct undue, and unpredicatable experimentation to practice the claimed invention and use sgRNAs that have a specificity score greater than 1.5. Further, due to the lack of guidance in the as-Filed Specification regarding the structure/function relationship of the specificity score and the sgRNA, it would require undue experimentation to use the composition and practice the breadth of the instant method as claimed.
Response to Arguments as they apply to rejection of claims 13, 15 – 17, 20 – 23, 26 -33, 35 – 38, and 40 under 35 USC § 112 (a)
Applicant’s arguments filed October 13, 2025 have been fully considered but they are not persuasive. Applicant essentially asserts (a) there was enhanced specificity chemically modified positions 4, 5, 7, 9, 10, 11, 12, 13, 14, 16, 18, and 19 (pg. 12, first paragraph); (b) enhanced specificity was found the guide sequence lengths other than 20mers (pg. 12, second paragraph); (c) modifications others than MP enhanced specificity (pg. 14, last paragraph); (d) enhanced specificity is shown for various target sequences (CTLA4, VEGA, IL2RG, and HBB) (Remarks, pg. 15, last paragraph), and (e) the data demonstrates that the claimed invention encompasses a genus of modified sequences rather than a species (pg. 16, third paragraph).
Regarding (a), Applicant argues that the data in Figures 9A, 9B, and 14 of the as-Filed Specification and data in the Ryan declaration show enhanced specificity compared to an unmodified control guide RNA. However, applicant’s arguments are not found persuasive.
As noted above, the as-Filed Specification teaches Figure 9B, which teaches the specificity score for in vitro cleavage of CLTA4. As noted in claim 13, wherein if the guide sequence is 20 nucleotides long, then the MP or MSP modification is selected from the group consisting of 4, 5, 7, 8, 10, 11, 12, 13, 14, 16, 17, 18, and 19 relative to the 5’ end of the guide sequence, and the guide RNA and Cas protein complex has a specificity score of greater than the specificity score of a control guide RNA complexed with a Cas protein. However, as seen in Figure 9B, the modifications of MP of the CTL4 target guide sequence at positions 8, 10, 13, 18, and 19 yield a specificity score that is less than the specificity score of the control (across all three specificity scores provided in the Figure 9B).
Further, regarding Figure 14, regarding the IL2RG, the figure only teaches modifications at positions5, and 11, and does not teach the modifications at all the other positions that are required by the claim.
The Ryan declaration teaches Tables D1 and D2, wherein the modifications elicit enhanced specificity (pg. 8, paragraph 15). Pertaining to these two tables, the Ryan declaration teaches that the control retained an MP modification at position 1 (at the 5’ end) (pg. 7, second line). While comparing the specificity score of the sgRNA with MP modification at position 1, there are many MP positions that have a lower specificity score than that of the sgRNA with MP modification at position 1. Specifically, the specificity score of 1,19 for CLTA4 ON:OFF3 and HBB; 1,18 for CLTA4 ON:OFF3 and HBB; 1,14 for CLTA4 ON:OFF1; 1,13 for CLTA4 ON:OFF2 and ON:OFF3. Thus, not all the modifications shown here yield a specificity score higher than that compared to the control (i.e. the MP position 1).
Regarding (b) – (e), the Examiner notes that the as-Filed Specification and the Ryan declaration does teach there was enhanced specificity for guide sequence lengths other than 20mers, wherein compared to the specificity score of an unmodified control guide RNA, enhanced specificity was found the guide sequence lengths other than 20mers, and modifications others than MP enhanced specificity. However, these arguments are not convincing.
Regarding Table D3 (pg. 10), the Ryan Declaration teaches the specificity testing of MP modifications in HBB sgRNAs, wherein the HBB_1xMP (the control sgRNA) has a specificity score of 5, while the HBB sgRNAs that are modified with MP modifications at position 14, and 16 have a specificity score less than 5. The scope of the claims have not been met at positions 14 and 16 for this specific gene. The examiner acknowledges that in table D4 and D5, all the modifications do yield a specificity score higher than the control. Similarly, VEGFA_11MOE_1xMP has a specificity score of 7, while the relevant control (VEGFA_1xMP) has a specificity score of 10. In Table D12, which teaches HBB sgRNAs with various modifications, the specificity score of the sgRNA with MSP modification at position 11 is less than the specificity score of the control.
The Applicant is reminded that MPEP 715.03 teaches:
In cases where predictability is in question, on the other hand, a showing of prior completion of one or a few species within the disclosed genus is generally not sufficient to overcome the reference or activity. In re Shokal, 242 F.2d 771, 113 USPQ 283 (CCPA 1957). The test is whether the species completed by the inventor prior to the reference date or the date of the activity provided an adequate basis for inferring that the invention has generic applicability. In re Mantell, 454 F.2d 1398, 172 USPQ 530 (CCPA 1973); In re Rainer, 390 F.2d 771, 156 USPQ 334 (CCPA 1968); In re DeFano, 392 F.2d 280, 157 USPQ 192 (CCPA 1968); In re Clarke, 356 F.2d 987, 148 USPQ 665 (CCPA 1965). "[I]n the case of a small genus such as the halogens, consisting of four species, a reduction to practice of three, or perhaps even two, might serve to complete the generic invention, while in the case of a genus comprising hundreds of species, a considerably larger number of reductions to practice would probably be necessary." Shokal, 242 F.2d at 773, 113 USPQ at 285.
Given the variability in the specificity scores provided the results in the as-Filed Specification and the Ryan declaration, one of ordinary skill in the art would not expect that any and all genes would comprise a specificity score greater than the specificity score of an unmodified synthetic guide RNA complexed with a Cas protein.
Conclusion
Claims 13, 15 – 17, 20 – 23, 26 – 33, and 35 – 38, and 40 remain rejected.
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 extension fee 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to VYOMA SHUBHAM TIWARI whose telephone number is (571)272-2954. The examiner can normally be reached M-F 8:30 - 5:30 EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Maria Leavitt can be reached on (571) 272-1085. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/VYOMA SHUBHAM TIWARI/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634
Prosecution Insights