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 .
Claim(s) 1, 11-12, 14, 25, 42, 44, 95, 102, 111, 116, 118, 120, 122, 124-125, 132-133, and 161-172.
This action is in response to the amendment, filed 08/15/2025, in which claim(s): (a) 161-172 were added; (b) 13, 108, 117, and 121 were cancelled; and (c) 111, 116, 118, 120, 122, 124, 125, 132, 133, and 166-169 were withdrawn.
Any rejection of record in the previous office actions not addressed herein is withdrawn.
New grounds of rejection are presented herein that were not necessitated by applicant’s amendment of the claims since the office action mailed 08/15/2025. Therefore, this action is not final.
Election/Restrictions
Applicant’s election without traverse of Group I (claim(s) 1, 11-14, 25, 42, 44, 95, and 102), drawn a composition comprising a single nucleic acid in the reply filed on 02/05/2025 is acknowledged. In the amendments filed on 08/15/2025, (a) claim 13 was cancelled, and (b) claim(s) 161-172 were newly added.
Applicant elected without traverse the following species: items (a) and (i) of claim 1, item (l) and the sequence pair of SEQ ID NOs: 146 and 148 of claim 11, SEQ ID NOs: 10 and 1016 of claim 42, and SEQ ID NOs: 146 and 148 of claim 44. The election reads on Group I claim(s) 1, 11-12, 14, 25, 42, 44, 95, 102, 161-165, and 170-12. Election was made without traverse in the reply filed on 02/05/2025.
Claim(s) 166-169 were withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Group(s) VII and VIII are rejoined as being drawn to (a) method of treating DMD and (b) a method of excising a portion of the dystrophin gene, reading on claim(s) 111, 116, 118, 120, 122, 124-125, and 132-133. In view of the withdrawal of the restriction requirement as to the rejoined inventions, of groups VII and VIII, applicant(s) are advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, a claim that is between the previously restricted Group VII or VIII, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application. Once the restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. See In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
The restriction requirement between Groups I and VII/VIII is still maintained.
Claim(s) 111, 116, 118, 120, 122, 124-125, and 132-133 were withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim.
Claim(s) 1, 11-12, 14, 25, 42, 44, 95, 102, 161-165 and 170-172 are under consideration.
Priority
Acknowledgement is made of Applicant’s claim for priority based on the provisional application(s) filed as 63/179,850 on 04/26/2021; 63/166,174 on 03/25/2021; 63/152,114 on 02/22/2021; and 63/076,250 on 09/09/2020.
All claims are given the priority date of 09/09/2020.
Information Disclosure Statement
Receipt of the information disclosure statement(s) on 08/15/2025 is acknowledged. The signed and initialed PTO-1449 form(s) has/have been mailed with this action.
Drawings
The drawings (replacement sheets) were received on 08/15/2025. The replacement sheet is not accepted for the following reasons.
The drawings are objected to because of the following:
The drawings submitted on 08/15/2025 do not contain a label in the top margin of “Replacement Sheet” or “New Sheet”;
Replacement sheet of Figure 7A filed on 08/15/2025, recites “Amino acids 1-45 of SEQ ID NOs: 918 or 919”; “Amino acids 1-45 of SEQ ID NOs: 920 or 921”; and “Amino acids 1-35 of SEQ ID NOs: 922 or 923”; however, these are nucleic acid sequences, not amino acid sequences.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification – New objection(s)
Abstract
The abstract of the disclosure is objected to because the abstract and title read the same without more, i.e., the abstract is 10 words. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
Embedded Hyperlink
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Embedded hyperlink can be found in para [00293], line 3.
Trademarks/Tradenames
The use of the following terms, which is/are a trade name or a mark used in commerce, has been noted in this application.
Lipofectamine [00268], [00281], [00297], [00299]
GeneJet [00268]
MiSeq [00269], title of table 3B, [00282], [00301], [00324], [00331]
CRISPResso2 [00269], [00282]
Promega [00281], [00297], [00299], [00307]
Maxwell [00281], [00291], [00297], [00299], [00307], [0331]
Nucleofector [00289], [00305]
Nucleocuvette [00289]
ImageXpress [00290], [00307]
Genewiz [00292]
QuBiT [00301], [00317], [00321], [00324], [00331]
Tapestation [00301], [00331]
PhiX [00301], [00326], [00331], [00333]
Lonza [00305]
Qiagen [00313], [00317], [00321], [00324]
AllPrep [00313], [00317], [00321], [00324]
Triton [00315]
GenScript (table 12)
Diagenode (table 12)
QuantStudio [00319], [00322]
AMPure [00324], [00331]
Illumnia [00333]
The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Minor informalities
The disclosure is objected to because of the following informalities:
At [00333] lines 2-3, the words “using” and “Trimmomatic” are repeated.
Appropriate correction is required.
Starting on page 5, the specification refers to “of claim/claims [N]” when describing the embodiments. This goes until page 27. Claim(s) get amended to recite different limitations as well as depend from other claims. It would be remedial to amend every occurrence that an embodiment is referring to “claim/claims” and amend it to recite “of embodiment/embodiments [N]”.
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.
Claim(s) 1, 11, 14, 25, 42, 161, 164, and 171 are rejected under 35 U.S.C. 103 as being unpatentable over Hongmei et al (US 11,680,262 B2; Published June 20th, 2023; Filing date of March 30th, 2017) in view of the teachings of Hongmei et al (supra) in further view of Lau et al (In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease, F1000Research, Vol 6, Issue 2153, pages 1-21; published December 20th, 2017; IDS filed 08/03/2022 as #3 under NPL).
Regarding claim(s) 1, 25, and 171, Hongmei et al teaches in Figure(s) 9-11 and 15 using two guide RNAs to target exon 45 within at least 250 base pairs of each other in conjunction with SpCas9.
More specifically, Figure 9 teaches the target nucleotide sequences of sgRNA-DMD 1-5. Figure 10 teaches the combination of sgRNAs: 5 and 1, 5 and 2, 5 and 3, and 5 and 4. Figure 11 teaches the deletion frequency with the combination of sgRNAs targeting DMD as well as Hongmei et al states, “As a result, as shown in FIG. 10, any of five kinds of gRNAs exhibited a high DNA cleavage activity. Further, it was found that, in the double nicking method, the cleavage activity is low when two gRNAs are overlapping, and that induction of efficient DNA cleavage requires the presence of a certain distance. . . As a result, when two gRNAs were designed such that they were arranged at an appropriate distance in the double nicking method, DNA cleavage patterns with occurrence of deletion between the nicking induction sites of the gRNAs were frequently observed. Thus, it was discovered that, in cases where a splice acceptor sequence, especially the “AG” acceptor sequence, is included in this region, efficient induction of exon skipping is possible.”, (col 39, lines 26-32 and lines 36-44).
Figure 15 teaches combining sgRNAs to yield a higher exon skipping activity percentage, e.g., DMD sgRNA-8 had a 0.4% exon skipping activity and DMD sgRNA-23 had 0.94%, however when combined, DMD sgRNA 8+23 had a 45.6% skipping activity (see figure 15). DMD sgRNA-8 is located 15 bp away from the cleavage position and DMD sgRNA-23 is located 171 bp away, thus making them within 156 nucleotides of each other (also see col 13, lines 4-8). Hongmei et al teaches, “Further, seven kinds of gRNAs that individually showed exon skipping activity (DMD #1, 2, 4, 8, 9, 20, and 23) were selected, and arbitrary combinations of two sgRNAs among these were subjected to measurement of the exon skipping efficiency in 293T cells using the Luc2 (G967A)+hEx45 (0.7 kb) reporter. As a result, it was found that the exon skipping efficiency can be further increased by simultaneous introduction of two kinds of gRNAs (FIG. 15).”, (col 40, lines 10-18).
Regarding claim(s) 11, 42 and 164, Hongmei et al teaches SEQ ID NO: 142 which comprises 100% identity of SEQ ID NOs: 10 and 1016 (see alignment below), i.e., in one nucleic acid.
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Regarding claim 161, Hongmei et al teaches Scaffold sequences are linked to the spacer sequences corresponding to the gene of interest, e.g., a first guide has its own scaffold included, and a second guide has its own scaffold included (col 10, lines 49-51).
Despite Hongmei et al disclosing multiple guide RNA use with spCas9 for targeting regions of the dystrophin gene withing 250 nucleotides of each other, Hongmei et al does not disclose (1) multiple guide RNA use with saCas9 in a single nucleic acid/vector or (2) at least 3 guides.
Hongmei et al teaches using saCas9 in figures 13a and 13d. Hongmei et al teaches, “As a CRISPR-Cas9 system, class 2 type II Cas9 derived from Streptococcus pyogenes, which is widely used as a genome editing tool, may be used. Class 2 type II Cas9 systems derived from other bacteria have also been reported, and, for example, Cas9 derived from Staphylococcus aureus (Sa), Cas9 derived from Neisseria meningitidis (Nm), or Cas9 derived from Streptococcus thermophilus (St) may also be used.”, (col 8, lines 44-51).
Hongmei et al teaches constructing the saCas9 expression vector combined with sgRNA (col 19, lines 3-31).
Hongmei et al does not teach multiple guide use with saCas9 in a single nucleic acid/vector and/or at least three guides.
Regarding claim(s) 1 and 14, Lau et al teaches, “Owing to a small AAV viral genome-packaging capacity (~4.8 kb), it has been technically challenging to co-package Streptococcus pyogenes-derived Cas9 (SpCas9) (4.1 kb) and multiple sgRNAs into all-in-one AAV vectors for multiplex genome editing.”, (page 5, col 1 para 3). Moreover, “To overcome these drawbacks, other recently discovered small Cas9 orthologues, including Staphylococcus aureus-derived Cas9 (SaCas9, 3.16 kb) and Campylobacter jejuni-derived Cas9 (CjCas9, 2.95 kb), have been used to package the Cas9 and its gRNA into a single AAV delivery vehicle for in vivo genome editing. To date, at least 11 independent in vivo studies have used the AAV-SaCas9 system to edit disease-associated genes in a variety of tissues, including brain, muscle, retina, heart, and liver. More recently, the quadruplex gRNAs/SaCas9 vector consisting of SaCas9 and multiplex sgRNAs was successfully delivered using AAV-DJ/8 for in vivo excision of HIV-1 proviral DNA in various solid tissues/organs via a single intravenous injection in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection.”, (page 5, col 1, para 4 to col 2 para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the spCas9 of Hongmei et al with saCas9, as taught by Hongmei et al. The substituted components, i.e., saCas9 for spCas9, and their functions were known in the art, as taught by Hongmei et al. One of skill in the art could have looked to the disclosure of Hongmei et al and substituted one Cas9 for the other and the result of the substitution would have been predictable. Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Hongmei et al in view of Hongmei et al (i.e., saCas9 in combination with two guide RNAs targeting the dystrophin gene within at least 200 base pairs of each other) with the teachings of Lau et al, i.e., using a single vector with saCas9 and multiple sgRNAs for in vivo gene editing, to yield the predictable results of a single nucleic acid comprising an saCas9 and two or more sgRNAs targeting the dystrophin gene within 200 base pairs of each other. One of skill in the art would be motivated to so do because Lau et al teaches that it is technically challenging to get spCas9 and multiple sgRNAs into one vector and that to overcome such draw back, swapping spCas9 for saCas9 allows genome editing with multiple guides and Cas9 in the same vector/nucleic acid.
Accordingly, claim(s) 1, 11, 14, 25, 42, 161, 164, and 171 are unpatentable over Hongmei et al in view of the teachings of Hongmei et al in further view of Lau et al.
Claim(s) 1, 11, 12, 44, 162, 163, and 172 are rejected under 35 U.S.C. 103 as being unpatentable over Hongmei et al (US 11,680,262 B2; Published June 20th, 2023; Filing date of March 30th, 2017) in view of the teachings of Hongmei et al (supra) in further view of Lau et al (In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease, F1000Research, Vol 6, Issue 2153, pages 1-21; published December 20th, 2017; IDS filed 08/03/2022 as #3 under NPLs) as applied to claim(s) 1, 11, 14, 25, 42, 161, 164, and 171 above, in view of Cohnen et al (WO 2019/118935 A1; Published June 20th, 2019; cited on IDS filed 12/16/2021 as Foreign Doc #5) and in further view of Dang et al (Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency, Genome Biology, vol 16, issue 280, pages 1-10; published December 15th, 2015).
Regarding claim 172, Hongmei et al in view of the teachings of Hongmei et al and in further view of Lau et al, teach saCas9 in combination with two guide RNAs targeting the dystrophin gene within at least 200 base pairs of each other
Hongmei et al teaches Scaffold sequences are linked to the spacer sequences corresponding to the gene of interest, e.g., a first guide has its own scaffold included, and a second guide has its own scaffold included (col 10, lines 49-51).
Hongmei et al also teaches, “In this case, the distance between the guide RNA-binding site (spacer sequence) in the sense strand and the guide RNA-binding site (spacer sequence) in the antisense strand is preferably −10 to 200 bases, more preferably 0 to 100 bases.”, (col 13, lines 4-8).
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Regarding claim(s) 11, 12, 44, and 163, Hongmei et al teaches SEQ ID NO: 142 which comprises 100% identity of SEQ ID NO: 146 and 148 (see alignment below), i.e., in the same nucleic acid. As well as Hongmei et al teaches SEQ ID NO: 131, which comprises 100% identity to SEQ ID NO: 148 (see Figure 13a).
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Hongmei et al and Lau et al do no teach (a) sluCas9 and/or (b) a sluCas9 scaffold sequence.
Cohnen et al characterizes and teaches sluCas9.
Regarding claim 1, Cohnen et al teaches, “The present invention provides a novel CRISPR-Cas endonuclease of the CRISPR-Cas9 family from Staphylococcus lugdunensis (SluCas9) and variants thereof having different and advantageous characteristics and functionalities from known CRISPR-Cas endonucleases and thus provides further opportunities for genome editing that previously did not exist. The present invention further provides suitable PAM sequences and suitable guide RNAs (gRNAs), such as single-guide-RNAs (sgRNAs), for use in prokaryotic, eukaryotic, and in vitro environments.”, (p.2, lines 21-26).
Moreover, Cohnen et al teaches existing disadvantages with spCas and/or saCas9 such as the following:
“a) Their size is too large to be carried inside the genome of established therapeutically-suitable viral transfection systems such as adeno associated viruses (AAVs).
b) Their activity in non-host environments is generally too low for use in these environments, for example, too low for efficient use in eukaryotic, and in particular in mammalian environments.
c) Their nuclease action lacks fidelity, leading to unwanted off target effects that would for example make them unsuitable for gene therapeutic uses or other applications requiring high precision.
d) They may trigger an immune response that can limit their use for in vivo applications in mammals.
e) They require complex and/or long PAMs that restrict target selection for the DNA-targeting segments.
The novel SluCas9 CRISPR-Cas system provided herein exhibits advantageous characteristics over the already existing CRISPR-Cas systems. In some embodiments, the SluCas9 CRISPR-Cas system exhibits a higher activity in prokaryotic, eukaryotic, and/or in vitro environments, and/or greater expression of the Cas endonuclease from a nucleic acid in eukaryotic environments, such as, e.g., a human host cell.”, (p. 2 to p.3).
Lastly, Cohnen et al teaches, “Most existing type II CRISPR Cas systems are based on the enzyme from Streptococcus pyogenes, which has the particular disadvantage of being too large for packaging into viral vectors as AAV (1638 amino acids). There is an alternative type II CRISPR Cas system based on the nuclease from Staphylococcus aureus (EP 2 898 075) which is significantly smaller in size. However, this nuclease requires a rather complex PAM which greatly restricts is usability for gene editing applications.”, (p.7 lines 23-27).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the saCas9 as taught through the combined teachings of Hongmei et al and Lau et al above, i.e., a single nucleic acid/vector with two guides and saCas9 for targeting the dystrophin gene within 200 base pairs of each other, with sluCas9, as taught by Cohnen et al. The substituted components, i.e., sluCas9 for saCas9, and their functions were known in the art, as taught by Hongmei et al, Lau et al, and Cohnen et al. One of skill in the art could have looked to the teachings of Cohnen et al and substituted one Cas9 for the other and the result of the substitution would have been predictable as to providing (a) higher activity, (b) greater expression, and (c) a more suitable PAM sequence.
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Regarding claim(s) 162, Cohnen et al teaches a nucleic acid (SEQ ID NO: 8) comprising 97.9% homology to instant SEQ ID NO: 901; which differ by one nucleotide at position 5 (see alignment and highlighted mismatch below).
Despite Cohnen et al teaching a sluCas9 scaffold sequence, Cohnen et al does not teach a sequence comprising 100% identity to instant SEQ ID NO: 901.
Dang et al teaches substituting a C or G for the fourth T at the 5’ T-stretch in the scaffold. More specifically, Dang et al teaches, “In this study, we systematically investigated the effect of extending the duplex and mutating the continuous sequence of Ts, providing guidance for optimizing sgRNA structure. Our results clearly show that extending the duplex and mutating the continuous sequence of Ts at position 4 to C or G significantly increases knockout efficiency in most cases, and the extent of the improvement in knockout efficiency is striking (Figs. 3 and 4). The general optimized sgRNA structure is illustrated in Fig. 7.”, (p. 7, col 1, para 2 to col 2).
Further, Dang et al teaches, “Mutating the continuous sequence of Ts significantly increased sgRNA production (Fig. 5b), which is likely to be the result of increased transcription efficiency due to the disrupted pause signal [11]. The results with in vitro transcribed sgRNAs suggest that extending the duplex by itself also increases Cas9 functionality because of the structural change (Fig. 5d, e), since any effect of the RNA level was excluded in this experiment. When sgRNA is expressed inside cells, both effects contribute to increase the functionality. It is possible that the modified sgRNA structure might enhance binding to Cas9 or increase its stability.”, (p.7, col 2, para 2 to p.8, col 1, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the fourth U/T of the sluCas9 scaffold sequence taught by Cohnen et al with the teachings of Dang et al, i.e., substituting a C or G, to yield the predictable results of a 5’ scaffold sequence of GTTTCAG (reading on the 5’ portion of the instant SEQ ID NO: 901). One would be motivated to make such a modification because Dang et al teaches that by changing the fourth U/T to a C or G, that is significantly increases knockout efficiency and increased sgRNA production likely due to disrupting the polyT pause signal from the original sequence of TTTT (or UUUU).
Accordingly, claim(s) 1, 11, 12, 44, 162, 163, and 172 are unpatentable over Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al and in further view of Dang et al.
Claim(s) 95 and 102 are rejected under 35 U.S.C. 103 as being unpatentable over Hongmei et al (US 11,680,262 B2; Published June 20th, 2023; Filing date of March 30th, 2017) in view of the teachings of Hongmei et al (supra) in further view of Lau et al (In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease, F1000Research, Vol 6, Issue 2153, pages 1-21; published December 20th, 2017; IDS filed 08/03/2022 as #3 under NPLs) in view of Cohnen et al (WO 2019/118935 A1; Published June 20th, 2019; cited on IDS filed 12/16/2021 as Foreign Doc #5) and in further view of Dang et al (Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency, Genome Biology, vol 16, issue 280, pages 1-10; published December 15th, 2015) as applied to claim(s) 1, 11, 12, 44, 162, 163, and 172 above, in view of Fry et al (Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene editing Efficiency, The CRISPR Journal, Vol 3, Issue 4, pages 276 to 283; published August 24th, 2020).
The combination of Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al and in further view of Dang et al teach a nucleic acid encoding sluCas9 with multiple guide RNAs targeting the dystrophin gene within 200 base pairs of one another.
Hongmei et al teaches that the scaffolds are in combination with the sgRNAs, thus, sgRNAs read on the combination of a targeting nucleic acid (i.e., spacer) and scaffold for the purpose of this rejection.
Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al and in further view of Dang et al do not teach a specific orientation of the nucleic acid molecule, i.e., 5’ to 3’ with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding the Staphylococcus aureus Cas9 (SaCas9) or the Staphylococcus lugdunensis Cas9 (SluCas9), a nucleotide sequence encoding the SaCas9 or SluCas9, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the SaCas9 or the SluCas9, a nucleotide sequence encoding the second guide RNA sequence, and a nucleotide sequence encoding the second guide RNA scaffold sequence.
Fry et al teaches that for clinical translation, packaging both saCas9 and guideRNA in a single AAV-CRISPR vector is ideal (p. 276, col 1, para 2).
Regarding claim 95, Fry et al teaches, “Some authors have noted that the orientation of the U6–gRNA transcriptional unit within a vector can affect the efficiency of gene editing. To investigate this further, we compared the expression and gene editing efficiency from two single AAV-CRISPR vector designs where Cas9 is driven by the cytomegalovirus intermediate-early enhancer and promoter (CMV-IE) and the gRNA is expressed under the human U6 (hU6) promoter. . . In this system, orientation of the gRNA relative to the Cas9 affects both Cas9 and gRNA expression and gene editing efficiency when delivered via AAV.”, (p. 276, col 1, para 3 to col 2 para 1).
Moreover, Fry et al teaches, “We cloned plasmid constructs that contained the hU6–gRNA construct in either the forward or reverse direction relative to an upstream SaCas9 driven by the ubiquitous RNA polymerase II-mediated CMV-IE promoter (Fig. 1).”, (p. 278, col 2, para 1). “In cells transduced with AAV, however, editing rates with constructs containing the U6–gRNA in the reverse direction compared to constructs with the gRNA in the forward direction were reduced by 28.7% p = 0.0009) for the VEGF guide (Fig. 2).”, (p. 278, col 2, para 2).
Further, Fry et al teaches, “We found that compared to the forward-oriented vector, the reverse-oriented U6 construct resulted in a significant reduction in SaCas9 expression in a guide independent manner. . . A similar significant reduction in gRNA expression was also found.”, (p. 279, col 1, para 1 to p.280, col 1, para 1).
Fry et al teaches, “As gene editing using AAV-delivered CRISPR-Cas9
moves toward the clinic, it is important to optimize the design of AAV transgenes to enable therapeutic efficacy. Here, we show that a small change to the transgene design, reversing the direction of the U6 promoter and gRNA complex, has a marked impact on Cas9 and gRNA expression and subsequent editing rates in a vector with SaCas9 driven by the CMV-IE promoter. . . Most recently, Levy et al. engineered a dual-AAV split-intein SaCas9 base editor and found that moving the U6–gRNA complex from upstream of the SaCas9 in the forward orientation to downstream in the reverse orientation substantially improved editing rates.”, (p. 280, col 1 para 3 to col 2 para 1, and p. 281, col 1, para 1).
Fry et al teaches the three possible arragments of dual-transgene containing Cas9 and gRNA in Figure 4.
Lastly, Fry et al teaches that there is a bovine growth hormone poly-A tail present in all vectors (p. 277, col 1, under Viral vector production and titration).
Regarding claim 102, the combination of Cohnen et al and Dang et al teach the SEQ ID NO: 901 (see rejection above).
Therefore, it would have been obvious to try to one of ordinary skill in the art before the effective filing date of the claimed invention to orient the single nucleic acid encoding two sgRNAs including scaffolds and the cas9 nuclease – as taught by the combination of Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al and in further view of Dang et al – with the teachings of Fry et al, to yield the predictable results of one of the promoter/sgRNA/scaffold combinations in the reverse orientation relative the promoter/nuclease and second promoter/sgRNA/scaffold combination. One would have been motivated to do so because Fry et al teaches (1) rotating orientation allows for fine-tuning of mRNA and protein expression of the nucleic acid, and (2) when moving at least one sgRNA complex from upstream of saCas9 in the forward orientation to downstream in the reverse (in a circular vector, downstream in the reverse still reads on 5’ of the nuclease), this substantially improved editing rates. One could have looked to the teachings of Fry et al, which taught a finite number of orientations of sgRNAs relative to nucleases, and could have arrived at the claimed invention with a high likelihood of success.
Accordingly, claim(s) 95 and 102 are rejected as being unpatentable over of Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al in view of Dang et al and in further view of Fry et al.
Claim(s) 165 and 170 are rejected under 35 U.S.C. 103 as being unpatentable over Hongmei et al (US 11,680,262 B2; Published June 20th, 2023; Filing date of March 30th, 2017) in view of the teachings of Hongmei et al (supra), in view of Lau et al (In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease, F1000Research, Vol 6, Issue 2153, pages 1-21; published December 20th, 2017; IDS filed 08/03/2022 as #3 under NPLs) in further view of Cohnen et al (WO 2019/118935 A1; Published June 20th, 2019; cited on IDS filed 12/16/2021 as Foreign Doc #5).
Regarding claim 165, Hongmei et al teaches in Figure(s) 9-11 and 15 using two guide RNAs to target exon 45 within at least 250 base pairs of each other in conjunction with SpCas9.
More specifically, Figure 9 teaches the target nucleotide sequences of sgRNA-DMD 1-5. Figure 10 teaches the combination of sgRNAs: 5 and 1, 5 and 2, 5 and 3, and 5 and 4. Figure 11 teaches the deletion frequency with the combination of sgRNAs targeting DMD as well as Hongmei et al states, “As a result, as shown in FIG. 10, any of five kinds of gRNAs exhibited a high DNA cleavage activity. Further, it was found that, in the double nicking method, the cleavage activity is low when two gRNAs are overlapping, and that induction of efficient DNA cleavage requires the presence of a certain distance. . . As a result, when two gRNAs were designed such that they were arranged at an appropriate distance in the double nicking method, DNA cleavage patterns with occurrence of deletion between the nicking induction sites of the gRNAs were frequently observed. Thus, it was discovered that, in cases where a splice acceptor sequence, especially the “AG” acceptor sequence, is included in this region, efficient induction of exon skipping is possible.”, (col 39, lines 26-32 and lines 36-44).
Figure 15 teaches combining sgRNAs to yield a higher exon skipping activity percentage, e.g., DMD sgRNA-8 had a 0.4% exon skipping activity and DMD sgRNA-23 had 0.94%, however when combined, DMD sgRNA 8+23 had a 45.6% skipping activity (see figure 15). DMD sgRNA-8 is located 15 bp away from the cleavage position and DMD sgRNA-23 is located 171 bp away, thus making them within 156 nucleotides of each other (also see col 13, lines 4-8). Hongmei et al teaches, “Further, seven kinds of gRNAs that individually showed exon skipping activity (DMD #1, 2, 4, 8, 9, 20, and 23) were selected, and arbitrary combinations of two sgRNAs among these were subjected to measurement of the exon skipping efficiency in 293T cells using the Luc2 (G967A)+hEx45 (0.7 kb) reporter. As a result, it was found that the exon skipping efficiency can be further increased by simultaneous introduction of two kinds of gRNAs (FIG. 15).”, (col 40, lines 10-18).
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Hongmei et al teaches SEQ ID NO: 142 which comprises 100% identity of SEQ ID NO: 146 and 148 (see alignment below), i.e., in the same nucleic acid. As well as Hongmei et al teaches SEQ ID NO: 131, which comprises 100% identity to SEQ ID NO: 148 (see Figure 13a).
Despite Hongmei et al disclosing multiple guide RNA use with spCas9 for targeting regions of the dystrophin gene withing 250 nucleotides of each other, Hongmei et al does not disclose (1) multiple guide RNA use with sluCas9 in a single nucleic acid/vector.
Lau et al teaches, “Owing to a small AAV viral genome-packaging capacity (~4.8 kb), it has been technically challenging to co-package Streptococcus pyogenes-derived Cas9 (SpCas9) (4.1 kb) and multiple sgRNAs into all-in-one AAV vectors for multiplex genome editing.”, (page 5, col 1 para 3). Moreover, , “To overcome these drawbacks, other recently discovered small Cas9 orthologues, including Staphylococcus aureus-derived Cas9 (SaCas9, 3.16 kb) and Campylobacter jejuni-derived Cas9 (CjCas9, 2.95 kb), have been used to package the Cas9 and its gRNA into a single AAV delivery vehicle for in vivo genome editing. To date, at least 11 independent in vivo studies have used the AAV-SaCas9 system to edit disease-associated genes in a variety of tissues, including brain, muscle, retina, heart, and liver. More recently, the quadruplex gRNAs/SaCas9 vector consisting of SaCas9 and multiplex sgRNAs was successfully delivered using AAV-DJ/8 for in vivo excision of HIV-1 proviral DNA in various solid tissues/organs via a single intravenous injection in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection.”, (page 5, col 1, para 4 to col 2 para 1).
Hongmei et al and Lau et al do no teach (a) sluCas9.
Cohnen et al characterizes and teaches sluCas9.
Cohnen et al teaches, “The present invention provides a novel CRISPR-Cas endonuclease of the CRISPR-Cas9 family from Staphylococcus lugdunensis (SluCas9) and variants thereof having different and advantageous characteristics and functionalities from known CRISPR-Cas endonucleases and thus provides further opportunities for genome editing that previously did not exist. The present invention further provides suitable PAM sequences and suitable guide RNAs (gRNAs), such as single-guide-RNAs (sgRNAs), for use in prokaryotic, eukaryotic, and in vitro environments.”, (p.2, lines 21-26).
Moreover, Cohnen et al teaches existing disadvantages with spCas and/or saCas9 such as the following:
“a) Their size is too large to be carried inside the genome of established therapeutically-suitable viral transfection systems such as adeno associated viruses (AAVs).
b) Their activity in non-host environments is generally too low for use in these environments, for example, too low for efficient use in eukaryotic, and in particular in mammalian environments.
c) Their nuclease action lacks fidelity, leading to unwanted off target effects that would for example make them unsuitable for gene therapeutic uses or other applications requiring high precision.
d) They may trigger an immune response that can limit their use for in vivo applications in mammals.
e) They require complex and/or long PAMs that restrict target selection for the DNA-targeting segments.
The novel SluCas9 CRISPR-Cas system provided herein exhibits advantageous characteristics over the already existing CRISPR-Cas systems. In some embodiments, the SluCas9 CRISPR-Cas system exhibits a higher activity in prokaryotic, eukaryotic, and/or in vitro environments, and/or greater expression of the Cas endonuclease from a nucleic acid in eukaryotic environments, such as, e.g., a human host cell.”, (p. 2 to p.3).
Lastly, Cohnen et al teaches, “Most existing type II CRISPR Cas systems are based on the enzyme from Streptococcus pyogenes, which has the particular disadvantage of being too large for packaging into viral vectors as AAV (1638 amino acids). There is an alternative type II CRISPR Cas system based on the nuclease from Staphylococcus aureus (EP 2 898 075) which is significantly smaller in size. However, this nuclease requires a rather complex PAM which greatly restricts is usability for gene editing applications.”, (p.7 lines 23-27).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the spCas9 as taught by Hongmei et al and Lau et al above, with sluCas9, as taught by Cohnen et al. The substituted components, i.e., sluCas9 for spCas9, and their functions were known in the art, as taught by Hongmei et al, Lau et al, and Cohnen et al. One of skill in the art could have looked to the teachings of Cohnen et al and substituted one Cas9 for the other and the result of the substitution would have been predictable as to providing (a) higher activity, (b) greater expression, and (c) a more suitable PAM sequence.
Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Hongmei et al in view of Cohnen et al (i.e., sluCas9 in combination with two guide RNAs targeting the dystrophin gene within at least 200 base pairs of each other) with the teachings of Lau et al, i.e., using a single vector with sluCas9 and multiple sgRNAs for in vivo gene editing, to yield the predictable results of a single nucleic acid comprising an sluCas9 and two gRNAs targeting the dystrophin gene within 200 base pairs of each other. One of skill in the art would be motivated to so do because Lau et al teaches that it is technically challenging to get spCas9 and multiple sgRNAs into one vector and that to overcome such draw back, swapping spCas9 for other recently discovered small Cas9 orthologues allows genome editing with multiple guides and Cas9 in the same vector/nucleic acid.
Accordingly, claim(s) 165 and 170 are unpatentable over Hongmei et al in view of the teachings of Hongmei et al in view of Lau et al in view of Cohnen et al.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of claim 13 under 35 U.S.C 103 for obviousness is moot in view of Applicant’s cancellation of the claim filed 08/15/2025.
The previous rejection(s) of claim 1, 11-14, 14, 25, 42, 44, 95, and 102 under 35 U.S.C 103 for obviousness has been withdrawn in view of Applicant’s amendments of claim 1 to recite, “A composition comprising a single nucleic acid molecule comprising a nucleic acid encoding a Staphylococcus aureus Cas9 (SaCas9) or a Staphylococcus lugdunensis Cas9 (SluCas9) and at least two guide RNAs; wherein the SaCas9 or the SluCas9, and the at least two guide RNAs, are capable of excising a DNA fragment from the dystrophin gene; wherein the DNA fragment is between 5 and 250 nucleotides in length; and wherein the DNA fragment does not comprise an entire exon of the dystrophin gene.”, filed 08/15/2025.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claim(s) 1, 11-12, 14, 25, 42, 44, 95, 102, 161-165, and 170-172 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim(s) 5, 30, and 32 of copending Application No. 18/262,808 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because of the following:
Claim 5 of ‘808 recites, “The composition of “A composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of Table 6; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Table 6; iii) a guide sequence that is at least 90% identical to a guide sequence of Table 6; or iv) any one of the guide sequence pairs shown in Tables 1B, 1D, 3B, 3D, 5B, and 5D, optionally further comprising a SaCas9 or a nucleic acid encoding a SaCas9 (for SEQ ID NOs: 1-159) or a SluCas9 or a nucleic acid encoding a SluCas9 (for SEQ ID NOs: 200-292, 924-938, or 950-955).”, comprising a pair of guide RNAs, wherein the pair of guide RNAs is capable of excising a portion of the DMD gene; wherein the portion of the DMD gene DNA fragment is between 5-250 nucleotides in length.”
Wherein SEQ ID NO: 4, 143, 225, and 227, read on instant SEQ ID NOs: 10, 1016, 146, and 148, respectively with 100% identity.
Claim 5 of ‘808 reads on instant claim(s) 1, 11, 12, 14, 25, 42, 44, 163, 164, 165, 170, and 171.
Claim 30 of ‘808 recites, “The composition of claim l (see above in the claim 5 rejection), wherein (a the one or more guide RNAs is a guide RNA for SaCas9, and the one or more guide RNAs comprise a scaffold comprising the sequence of SEQ ID NO: 504; or (b) the one or more guide RNAs is a guide RNA for SluCas9, and the one or more guide RNAs comprise a scaffold comprising the sequence of SEQ ID NO: 901.
Wherein SEQ ID NO: 901 reads on instant SEQ ID NO: 901 with 100% identity.
Claim 30 of ‘808 reads on instant claim(s) 102, 161, 162, and 172.
Claim 32 of ‘808 recites, “The composition of claim 1,wherein the one or more guide RNAs is in an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence; the reverse complement of a nucleic acid encoding a first guide RNA sequence; the reverse complement of a promoter for expression of the nucleic acid encoding the first guide RNA sequence; a promoter for expression of a nucleic acid encoding aSaCas9,a SluCas9, or a sRGN (e.g., CK8e); a nucleic acid encoding the SaCas9, the SluCas9, or the sRGN (e.g., sRGN3.1, sRGN3,3, or sRGN4); a polyadenylation sequence; a promoter for expression of a second guide RNA sequence in the same direction as the promoter for the SaCas9, the SluCas9, or the sRGN; a nucleic acid encoding a second RNA guide sequence; and a second guide RNA scaffold sequence.”
Claim 32 of ‘808 reads on instant claim 95.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim(s) 1, 11-12, 14, 25, 42, 44, 95, 102, 161-165, and 170-172 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim(s) 5, 20, and 22 of copending Application No. 18/826,876 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because of the following:
Claim 5 of the ‘876 recites, “The composition of “A composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of Table lA or Table 1B; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Table lA or Table 1B;_or iii) a guide sequence that is at least 90% identical to a guide sequence of Table lA or Table 1B; optionally further comprising a SaCas9 or a nucleic acid encoding a SaCas9 (for SEQ ID NOs: 1000-3081 in Table lA) or a SluCas9 or a nucleic acid encoding a SluCas9 (for SEQ ID NOs 4000-5226 in Table 1B).”, comprising a pair of guide RNAs, wherein the pair of guide RNAs is capable of excising a portion of the DMD gene; wherein the portion of the DMD gene is between 5-250 nucleotides in length.”
Claim 5 of ‘876 reads on instant claim(s) 1, 11, 12, 14, 25, 42, 44, 163, 164, 165, 170, and 171.
Claim 20 of ‘876 recites, “The composition of claim 1, wherein (a if the one or more guide RNAs is a guide RNA for SaCas9, the one or more guide RNAs comprise a scaffold comprising the sequence of SEQ ID NO: 504; or (b) if the one or more guide RNAs is a guide RNA for SluCas9, the one or more guide RNAs comprise a scaffold comprising the sequence of SEQ ID NO: 901.”
Claim 20 of ‘876 reads on instant claim(s) 102, 161, 162, and 172.
Claim 22 of ‘876 recites, “The composition of claim 1, wherein the one or more guide RNAs are in an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence; the reverse complement of a nucleic acid encoding a first sgRNA guide sequence; the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA guide sequence; a promoter (e.g., CK8e) for expression of a nucleic acid encoding an SaCas9, a SluCas9, or a sRGN; a nucleic acid encoding the SaCas9, the SluCas9 or the sRGN; a polyadenylation sequence; a promoter for expression of a second sgRNA guide sequence in the same direction as the promoter for theSaCas9, the SluCas9, or the sRGN; a nucleic acid encoding a second sgRNA guide sequence; and a second sgRNA scaffold sequence.”
Claim 22 of ‘876 reads on instant claim 95.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim(s) 1, 14, 25, 95, 102, 161-162, and 171-172 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim(s) 6, 29, and 31 of copending Application No. 18/598,647 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because of the following:
Claim 6 of ‘647 recites, “The composition of “The composition of “A composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the guide RNAs comprise i) a guide sequence of Table 2; ii) at least 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Table 2; iii) a guide sequence that is at least 90% identical to a guide sequence of Table 2; or iv) any one of the guide sequence pairs shown in Tables 1B, and 1D, optionally further comprising a SaCas9 or a nucleic acid encoding a SaCas9 (for SEQ ID NOs: 11-15 or 27- 69) or a SluCas9 or a nucleic acid encoding a SluCas9 (for SEQ ID NOs 243-269).”, comprising a pair of guide RNAs, wherein the pair of guide RNAs is capable of excising a DNA fragment from the DMD gene; wherein the DNA fragment is between 5 and 250 nucleotides in length.”, wherein the excised DNA fragment does not comprise an entire exon of the DMD gene.”
Claim 6 of ‘647 reads on instant claim(s) 1, 14, 25, and 171.
Claim 29 of ‘647 recites, “The composition of claim l, wherein the one or more guide RNAs is a guide RNA for SluCas9, and the one or more guide RNAs comprises a scaffold comprising the sequence of SEQ ID NO: 901.
Claim 29 of ‘647 reads on instant claim(s) 102, 161, 162, and 172.
Claim 31 of ‘647 recites, “The composition of claim 1, wherein the one or more guide RNAs is in an AAV vector, wherein the vector comprises from 5' to 3' with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence, the reverse complement of a nucleic acid molecule encoding a first guide RNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid molecule encoding the first guide RNA, a promoter for expression of a nucleic acid molecule encoding SaCas9 or SluCas9 (e.g., CK8e), a nucleic acid molecule encoding a SaCas9 or SluCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA in the same direction as the promoter for SaCas9 or SluCas9, a second sgRNA guide sequence, and a second sgRNA scaffold sequence.”
Claim 31 of ‘647 reads on instant claim 95.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim(s) 1, 14, 25, and 171.provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 56 of copending Application No. 19/025,012 in view of Lau et al (In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease, F1000Research, Vol 6, Issue 2153, pages 1-21; published December 20th, 2017; IDS filed 08/03/2022 as #3 under NPL).
Claim 56 of ‘012 recites, “The nucleic acid of “A nucleic acid comprising a first sgRNA and a second sgRNA, wherein the first sgRNA and the second sgRNA are linked by a linker, and wherein the linker has a guanine and cytosine (GC) content of 5-37%, 5-30%, 5-25%, 5-20%, 10-37%, 10-35%, 10-30%, 10-25%, 10- 20%, 15-40%, 15-35%, 15-30%, or 15-25%.”, wherein a) the first sgRNA and the second sgRNA sgRNAs target any of exons 2, 3, 6, 9, 44, 45, 47,48, 50, 51 or 53 of human DMD; b ) the first sgRNA and the second sgRNA are capable of excising a DNA fragment from the DMD gene, wherein the DNA fragment is between 5 and 250 nucleotides in length; and/orc)the excised DNA fragment does not comprise an entire exon of the DMD gene.”
Claim 56 of ‘012 does not require the nucleic acid to comprise a sequence encoding an SaCas9 nucleic acid.
Lau et al teaches “Owing to a small AAV viral genome-packaging capacity (~4.8 kb), it has been technically challenging to co-package Streptococcus pyogenes-derived Cas9 (SpCas9) (4.1 kb) and multiple sgRNAs into all-in-one AAV vectors for multiplex genome editing.”, (page 5, col 1 para 3). Moreover, “To overcome these drawbacks, other recently discovered small Cas9 orthologues, including Staphylococcus aureus-derived Cas9 (SaCas9, 3.16 kb) and Campylobacter jejuni-derived Cas9 (CjCas9, 2.95 kb), have been used to package the Cas9 and its gRNA into a single AAV delivery vehicle for in vivo genome editing. To date, at least 11 independent in vivo studies have used the AAV-SaCas9 system to edit disease-associated genes in a variety of tissues, including brain, muscle, retina, heart, and liver. More recently, the quadruplex gRNAs/SaCas9 vector consisting of SaCas9 and multiplex sgRNAs was successfully delivered using AAV-DJ/8 for in vivo excision of HIV-1 proviral DNA in various solid tissues/organs via a single intravenous injection in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection.”, (page 5, col 1, para 4 to col 2 para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the nucleic acid of claim 56 (and in turn claim 1) to also comprise the sequence of an saCas9 to yield the predictable results of instant claim 1. One would be motivated to do so because Lau et al teaches that it is possibly to package sgRNA and small cas9 orthologues such as saCas9 into a single vector for multiple genome editing.
Accordingly, claim 56 of ‘012 reads instant claim(s) 1, 14, 25, and 171.
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims allowed.
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/L.M.T./Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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