DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status and Election
Applicant’s amendment filed August 7, 2025 is acknowledged. Claims 1-3, 5-6, 17-19, 21, 33-34, 36, 39, 44, 47, 54, 57, 59, 63, 65, 71, 77-78, 80, 82, 85-86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 160-161, 178, 180, 190, and 201 are pending.
Applicant’s election without traverse of CasX with SEQ ID NO 3505 (PmCasX with L379R, A708K and [P793]) and gNA with SEQ ID NO 2238 in the reply filed on August 7, 2025 is acknowledged. It is noted that a CasX with SEQ ID NO 3505 and a gNA with SEQ ID NO 2238 with the recited functions is free of the prior art. The search for CasX variants was widened to other variant proteins of SEQ ID NO 1-3 and now includes deletions of the NTSB domain. The species election for the gNA is withdrawn.
Claims 36, 57, 59, 63 and 65 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected species, there being no allowable generic or linking claim.
Claims 1-3, 5-6, 17-19, 21, 33-34, 39, 44, 47, 54, 71, 77-78, 80, 82, 85-86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 160-161, 178, 180, 190, and 201 are under examination
Drawings
The drawings are objected to because the view numbers for the partial views are followed by "(Cont.)” instead of a capital letter such as FIG. 1A, FIG. 1B, etc. 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.” Appropriate correction is required.
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
The use of the term Lipofectamine®, Q5®, EmulsiflexTM, and ZymocleanTM, which are trade names or marks used in commerce, have been noted in this application. 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.
Claim Rejections - 35 USC § 112(b)
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.
Claims 17 and 34 are 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.
Claim 17 recites “The CasX variant of claim 5, wherein the modification results in an increase ability to edit the target DNA”. There is insufficient antecedent basis for “the target DNA”. Target DNA is not recited in either claim 5 or claim 1 from which claim 17 depends. Although CasX proteins are known to bind to DNA in a site-specific manner, the site-specificity is provided by a guide nucleic acid, which is also not recited in claims 1, 5 and 17. Because claims 17 is only directed to the CasX protein, there is no inherent DNA-binding site function. Therefore, it is not clear what “the target DNA” is referring to.
Claim 34 recites The CasX variant of claim 1, wherein the CasX variant has a sequence selected form the group consisting of the sequences of Tables 3, 8, 9, 10 and 12…” Reference to tables in claims renders the claim indefinite when there is are practical ways to define the invention in words. See MPEP 2173.05(s). Here, the tables contain exclusively text, and refer to each CasX construct/variant in terms of SEQ ID NOs. Thus, the appropriate SEQ ID NOs could simply be imported into the claim. Merely pasting the SEQ ID NOs into the claim, however, would not overcome this rejection because the tables themselves contain SEQ ID NOs that refer to both DNA and to proteins. A SEQ ID NO that recites a nucleic acid protein would be confusing since claim 34 is directed to a CasX variant, i.e., a protein.
To overcome this rejection, applicants must amend claim 34 such that it clearly sets forth a closed list of SEQ ID NOs for the variant CasX proteins.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—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 or joint inventor of carrying out the invention.
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 1-3, 5-6, 17-19, 21, 33-34, 39, 44, 47, 54, 71, 77-78, 80, 82, 85-86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 160-161, 178, 180, 190, and 201 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.
MPEP 2163.II.A3.(a).(i) states, “whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention.”
For claims drawn to a genus, MPEP 2163.II.A3.(a).(ii) states, “written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species” where “representative number of species' means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.”
The claims recite a CasX variant, an gNA variant, or both. Because each of the CasX variants (i.e., proteins) and gNA variants (i.e., nucleic acids) are mutually exclusively, an analysis of their genus and predictability of having the recited function is addressed separately below. For claims that recite both a CasX variant and a gNA variant, both analyses apply.
CasX Variants – Genus size and Species encompassed
Claims 1-3, 5-6, 17-19, 21, 33-34, 39, 44, 47, 54, 71, 120, 137, 160-161, 178, 180, 190, and 201 recite and/or encompass at least a CasX variant comprising at least one modification compared to a reference CasX that is SEQ ID NO 1-3, wherein the CasX variant exhibits at least on improved characteristic. The variations can include one or more amino acid substitutions, deletions, insertions, domain substitutions and deletions, or combinations thereof. SEQ ID NOs 1-3 are between 855 and 986 amino acids in length and share only between 20-70% identity (See OA Appendix). There is no minimum identity to SEQ ID NOs 1-3, for which the CasX variant needs to be considered a variant. Therefore, the genus of CasX variants which comprise at least one substitution, addition or deletion is phenomenally huge. The genus of just SEQ ID NO 1 comprising a single substitution is 18,734 different structures. The genus of just SEQ ID NO 1 comprising two substitutions is 1.8 x 108 different structures. The number of variations increases exponentially with increasing number of allowed variants, and considering that unlimited additional amino acids and domains can be added to the SEQ ID NOs, the number of CasX structures encompassed by at least claim 1 is infinite. The claims also recite “at least one improved characteristic” including protein stability and solubility and activities of binding and/or cleaving DNA. For the reasons below, Applicant has not described the genus of CasX variants that correspond to each of the improved characteristics that are encompassed and/or explicitly stated in the claims.
CasX Variants – Specification disclosure
Applicants undertake a Deep Mutagenesis Evolution (DME) screen to create and identify changes in CasX proteins that improve target site binding and/or cleaving (See e.g., Examples 4-10). In each DME, the reference CasX used was Planctomycetes CasX (PmCasX, SEQ ID NO 2), which has only 70% and 20% sequence identity to SEQ ID NOs 1 and 3, respectively. Applicants discovered ~ 100 combinations of point substitutions and/or single amino acid deletions that had improved editing in HEK293T cells (Table 7). Other combinations had decreased editing in HEK293T cells (Table 7), and there is no evidence of those variants having an improved function at another target site or some other characteristic. Therefore, it is not predictable that any substitution/deletion/additional will necessarily improve a CasX functional characteristic. Importantly, Applicants do not attempt to recapitulate the amino acid substitutions or deletions at corresponding residues in SEQ ID NOs 1 and 3. Thus it is unknown whether the improved characteristics from substitutions/deletions described in Tables 7 and 12 would result in an improved function of the CasX proteins of SEQ ID NO 1 or 3. Lastly, Applicants have described one set of “domain exchange” variants in which the NTSB and helical Ib domains in CasX 2 (PmCasX) are replaced with the NTSB and helical 1b domains from CasX1 (i.e., SEQ ID NO 1, DpbCasX), which demonstrated improved editing activity with sgRNA variants with a modified scaffold region ([0902]). It is noted that no other domain swap experiments between SEQ ID NOs 1-3 were disclosed in the Specification.
In conclusion, Applicant has described approximately 100 amino acid substitutions and/or deletions combinations which have at least improved editing functions, but no other improvement described, and a single domain swap CasX variants. These 100 CasX variants do not represent the large number of diverse structures in the claimed genus. Additionally, the Specification does not provide a structure-function relationship between the variants and the improved characteristics such that the skilled artisan could predict what additional substitutions/insertions/deletions, if any, would result in an improved function. Thus, based on the Specification it would have been unpredictable what CasX variant structures from the genus of claimed CasX variants would possess the claimed functional characteristics.
CasX Variants – Knowledge in the Prior Art
The art was searched for any variant CasX polypeptides having at least a single substitution, deletion or replacement of one or more amino acids in the entire length of the protein. Liu describes the crystal structure of CasX from Deltaproteobacteria DpbCasX in February 2025, 4 months before the effective filing date of the claimed invention (Liu et al., Nature (2019), 566: 218-223). DpbCasX, whose native sequence is SEQ ID NO 1, was first described in Burstein, along with CasX from Planctomycetes, whose native sequence is SEQ ID NO 2 (Burstein et al., Nature (2017), 542: 237-243). Neither Burstein or Liu attempt to mutate CasX from Planctomycetes. Liu created a single DpbCasX variant in which the NTSB domain was deleted (Figs 3, 5). The DpbCasX DNTSB mutant had reduced DNA unwinding and target cleavage, but did have improved trans-cleavage activity (Fig 5b). Therefore, the prior art describes a single CasX variant that comprises both the structural and functional requirements of the incredibly large and diverse genus. Liu teaches that the only conserved portion of the CasX and other Type V Cas nucleases like Cpf1/Cas12a is the RuvC domain, although even the RuvC domains of Cpf1/Cas12a and CasX/Cas12e share less than 16% identity each other (page 218, ¶2). Therefore, it is highly unlikely that any mutations that render a Cpf1/Cas12a with improved characteristics would also generate the same or different improved characteristics in CasX.
Thus, based on the Specification and the prior art it would have been unpredictable what CasX variant structures from the genus of claimed CasX variants would possess the claimed functional characteristics.
CasX Variants – Dependent Claims
Claim 2 does not limit the possible claimed modifications and does not meaningfully limit the types of improvements since the recited characteristics are nearly every characteristic known for a Cas nuclease. As such the genus of CasX variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 1.
Claims 3, 5, 19 and 21 do not meaningfully limit the possible claimed modifications, since the modifications and domains listed encompass nearly every possible modification and domain. As such the genus of CasX variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 1.
Claim 6 recites at least one modification at specific residues, that the Specification teaches can be modified to improve CasX characteristics. However, claim 6 does not recite the specific substitution at the recited residues and the genus of CasX proteins is not limited to only a modification at the recited residues. Additionally, it is evident that some combinations of mutations do not improve CasX characteristics, as indicated in Table 7 of the Specification. As such the genus of CasX variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 1.
Claims 17-18, 39, 44, 47, 54, 120, 137, 160-161, 178, 180, 190 and 201 do not limit the possible claimed modifications. As such the genus of CasX variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 1.
Claim 33 recites at least one modification at specific residues with a specific amino acid substitution or residue deletion. However, the genus of CasX proteins is not limited to only a modification at the recited residues. Claim 33 does not require a minimum percent identity to the reference CasX (i.e., SEQ ID NO 2), and therefore the genus of claimed CasX variants is still massive. Additionally, the claimed function of “improved characteristics” is still not limited. As such the genus of CasX variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 1.
Claim 34 recites the CasX has a sequence from those recited in Tables 3, 8, 9, 10, 12, SEQ ID NOs 258-327, 3508-3520 and 4412-4415 or a sequence having at least 50% identity thereto. Claim 34 lacks sufficient written description for several reason. First, many of the SEQ ID NOs recited in the tables have reduced editing efficiency. Since editing efficiency was the only characteristic many of the variants were screened for it is not predictable what improved characteristic they would have. Second, while the genus of CasX variants is reduced, it is still vast with varied structures since 50% identity allows at least half of the residues to be varied independently. A protein with only 50% identity to SEQ ID NO 258, which is 977 residues is ~10917 different variants. As such, for the reasons described above for claim 1, the genus of CasX variants with the genus of improved characteristics is not sufficiently described.
Claim 71 recites the CasX comprises a sequence selected from the groups of ~300 SEQ ID NOs. The editing efficiency of many of the SEQ ID NOs are found in Table 7. According to Table 7, at least 90 of the SEQ ID NOs do not show improved editing efficiency. It is noted that the only characteristic that was directly tested was editing efficiency. However, because editing efficiency requires the CasX to be soluble, stable, bind to a gNA to form an RNP and have nuclease activity, editing efficiency is an indirect measure of the many of the characteristics. If the editing efficiency using a CasX is merely maintained or even reduced it is not predictable that a CasX solubility, stability, gNA-binding, or DNA-binding would be improved. For instance, SEQ ID NOs 3493, with a L212P substitution compared to SEQ ID NO 2, has 2/3 of the editing capability as SEQ ID NO 2 (Table 7), and as such is not predicted to have any of the claimed improved characteristics.
gNA Variants – Genus size and Species encompassed
Claims 77-78, 80, 82, 85-86, 89-9, 93-94, 96-97, 103-104, 108, 120, 137, 160-161, 178, 180, 190, and 201 recite and/or encompass a variant of a gNA scaffold, where in the gNA variant has at least one modification relative to SEQ ID NOs 4-16 and at least one improved characteristic compared to SEQ ID NOs 4-16. SEQ ID NOs 4-16 range in size from 20 to 108 nucleotides and share very little if any identity. The variations can include one or more nucleotide substitutions, deletions, insertions, or combinations thereof and addition of known domains that improve stability like hairpins and G-quadruplexes. Therefore, the genus of gNA variants which comprise at least one substitution, addition or deletion is huge. The genus of just SEQ ID NO 4 that is at least 50% identical to SEQ ID NO 4 is over 1060 different structures that are 108 nucleotides in length. The number of variations increases exponentially with each option of adding different domains to the gNAs which may or may not interfere with gNA function. The claims also recite “at least one improved characteristic” including improved stability and solubility, improved transcription, improved biding to a CasX protein, which also represents a very large genus. For the reasons below, Applicant has not described the genus of CasX variants that correspond to each of the improved characteristics that are encompassed and/or explicitly stated in the claims.
gNA Variants – Specification disclosure
In an initial screen SEQ ID NO 4 (the sgRNA version of the endogenous crRNA/tracrRNA from Deltaproteobacteria) was compared with SEQ ID NO 5 (the sgRNA version of the endogenous crRNA/tracrRNA Planctomycetes). Applicants tested replacing the known extended loop in SEQ ID NO 5 (see Liu above) with known stability enhancers, and found some replacements, but not all, improved editing in vivo (FIG 5B). Applicant also appended known ribozyme sequences to the 5’ end and found just 5 out of 26 that improved editing efficiency in vivo (Fig 5D). Applicant also used Deep Mutagenesis Evolution (DME) screen to create and identify changes in SEQ ID NO 5 was used as the reference CasX (FIG 5). Applicants found approximately 20 changes that increased editing in HEK293 cells, but did not test any other characteristic like solubility, folding kinetics, binding to CasX variants. Finally, Applicant combined some of the substitutions/deletions found in the DME screen with known guide RNA appendages and found mixed results for whether the combinations improved gene editing over a single change (FIG 5E), underscoring the unpredictability of combining modifications and their overall result on improved characteristics. It is also noted that all editing assays were presumably performed in combination with the CasX protein from Planctomycetes. Given the claimed CasX variants in the gene editing pairs and kits, it is also unpredictable whether the gNA variants that produced improved editing rates with SEQ ID NO 2, would also increase editing with the extremely large genus of CasX variants. Applicant does test some CasX/gNA variant combinations, some of which can improve overall editing, but some of which do not show an improvement (FIG. 12). Finally, it is noted that SEQ ID NOs 10-15 appear to be derived from tracrRNA sequences for C. Sunbacteria, which is a species in which a third CasX locus was discovered ([0150]). Applicants do not attempt to modify the SEQ ID NOS 10-13 or test any modifications.
In conclusion, Applicant has described approximately 34 gNA variants with single modifications and only 5 gNA with multiple variations that have at least improved editing functions, but no other improvement described. These gNA variants tested do not represent the large number of diverse structures in the claimed genus. Additionally, the Specification does not provide a structure-function relationship between the variants and the improved characteristics such that the skilled artisan could predict what additional substitutions/insertions/deletions, if any, would result in improved characteristics of the gNA. Thus, based on the Specification it would have been unpredictable what gNA variant structures from the large genus of claimed gNA variants would possess the claimed functional characteristics.
gNA Variants – Specification disclosure
The prior art search was limited to guide RNAs known to form a complex with CasX polypeptide. Liu describes the crystal structure of the complex between CasX and a sgRNA form of the endogenous crRNA/tracrRNA from Deltaproteobacteria DpbCasX in February 2025, 4 months before the effective filing date of the claimed invention (Liu et al., Nature (2019), 566: 218-223). Liu recites the DpbCasX sgRNA, whose native sequence is 4 nucleotides shorter than SEQ ID NO 4. Liu demonstrates that the sgRNA from Deltaproteobacteria comprises and triplet forming section, a scaffold stem that binds to DpbCasX and an “extended stem” that juts away from center of mass of the complex (Fig 3). Liu also makes small substitutions and deletions in the sgRNA, but none of the modifications improve target DNA binding of the complex in bacteria, which is the only characteristic measured (Ext Data Fig 6). Liu did not attempt to extend or replace the extended stem so it is not predictable from the art how one could modify at least SEQ ID NO 4 to increase its DNA biding or other characteristic. Notably, there is no teaching in the art about the structure of CasX/sgRNA for Planctomycetes or any deletion/mutation studies that may lend predictability about modifying SEQ ID NO 5. Finally, it is noted that SEQ ID NOs 10-13 appear to be derived from tracrRNA sequences for C. Sunbacteria, which is a species in which a third CasX locus was discovered ([0150]). There is no teaching in the prior art of the crystal structure or structure-function studies for the C. Sunbacteria guide RNAs, so it is completely unknown how one could modify SEQ ID NOs 10-13 to render an improved characteristic. Finally, the origins of SEQ ID NOs 14-16 are unknown, so it is unpredictable how one could modify SEQ ID NOs 14-16 to render an improved characteristic.
Thus, based on the Prior Art it would have been unpredictable what gNA variant structures from the large genus of claimed gNA variants would possess the claimed functional characteristics.
CasX Variants – Dependent Claims
Claim 78 does not limit the possible claimed modifications and does not meaningfully limit the types of improvements since the recited characteristics are nearly every characteristic known for a Cas guide RNA. As such the genus of gNA variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 77.
Claims 80, 82 and 85 do not meaningfully limit the possible claimed modifications, since the modifications and domains listed encompass nearly every possible modification and domain and adding a region does not limit possible substitutions and/or deletions. As such the genus of gNA variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 77.
Claims 86, 89-90 and 108 recite at least type of modification; however, the modifications are not limited to the recited modifications as additional modifications are still comprised. Thus, the size and diversity of the genus is still vast and improved characteristics of the genus is unpredictable for the reasons recited above for claim 77.
Claims 93-94, 96-97, 120, 137, 160-161, 178, 180, 190 and 201 do not limit the possible claimed modifications. As such the genus of gNA variants with the genus of improved characteristics is not sufficiently described for the reasons recited above for claim 77.
Claim 103 recites the gNA has a sequence selected from SEQ ID NOs 2101-2280 or a sequence having at least 80% identity thereto. Claim 103 lacks sufficient written description because while the genus of CasX variants is reduced, it still vast with varied structures since 80% identity allows a substantial portion of residues to be varied independently. The longest SEQ ID NO listed in the recited group is SEQ ID NO 2180 at 197 nucleotides. The genus of gNA variants with at least 80 % identity to just SEQ ID NO 2180 is over 1065 different sequences. As indicated above for claim 77, Applicants have described less than 40 gNA structures that have improved properties and there does not seem to be sufficient structure-function relationship such that the skilled artisan could predict which of the other 1065 variants of SEQ ID NO 2180 would also have the improved characteristics.
Claim 104 recites the scaffold of the gNA variant sequence consists of a SEQ ID NO 2101-2280. While the genus of gRNA variants is much smaller, the Specification teaches that gNAs with many of the scaffolds with the recited SEQ ID NOs do not have improved editing efficiency (Table 5). It is noted that the only characteristic that was directly tested was editing efficiency. However, because editing efficiency requires the gNA to be soluble, stable, bind to CasX and bind DNA, editing efficiency is an indirect measure of the many of the characteristics. If the editing efficiency using a gNA is reduced it is not predictable that a gNA’s solubility, stability, CasX-binding or DNA-binding would be improved. For instance, SEQ ID NOs 2133, with an A14C substitution compared to SEQ ID NO 5, has only a third of the editing capability as SEQ ID NO 5 (Table 2), and as such is not predicted to have any of the claimed improved characteristic.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-3, 5, 18-19, 21, 34, 44 and 54 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Liu (Liu et al., Nature (2019), 566: 218-223, and online content).
Regarding claims 1-2, Liu describes the domain structure, in vitro assays, and crystal structure of DpbCasX, a CasX protein from Deltaproteobacteria (Figs 1-3). Liu teaches the length of DpbCasX is 986 amino acids and has an Asp at residues 672 and 935, Glu at residue 769, an Arg at residue 917 and a Gln at residue 920 (Fig 3, Ext Fig 1), which are the same amino acid residues and length of SEQ ID NO 1, which is described in the sequence listing as Deltaproteobacteria CasX. Therefore, Liu’s DpbCasX is a reference CasX with SEQ ID NO 1. Liu teaches deleting the NTSB domain from residues 101-190 from DpbCasX (i.e., a CasX variant with at least one modification) (Fig 5). Liu teaches that the DpbCasX D101-190 variant has improved trans-cleavage activity (i.e., increased nuclease activity) (Fig 5b; page 223, ¶1).
Regarding claim 3, Liu teaches the DpbCasX D101-190 variant has a deletion of all of the NTSB domain (Figs 3, 5).
Regarding claim 5, Liu teaches the deletion (i.e., modification) is in the NTSB domain (Figs 3, 5).
Regarding claim 18, Liu teaches the DpbCasX D101-190 variant is able to form a complex with the guide RNA (Ext Data Fig 9).
Regarding claim 19, Liu teaches the DpbCasX D101-190 variant has a deletion of 90 consecutive amino acids (Figs 3, 5).
Regarding claim 21, “two or more modifications in one domain” is interpreted as encompassing deletions of two or more amino acids in the same domain. Liu teaches the DpbCasX D101-190 variant has a deletion of 90 amino acids (i.e., two or more modifications in the NTSB domain) (Figs 3, 5).
Regarding claim 34, Liu’s DpbCasX D101-190 variant has 73% identity with SEQ ID NO 329, which is recited in Table 3 (see OA Appendix).
Regarding claim 44, Liu teaches the DpbCasX D101-190 variant has about 2-fold improved trans-cleavage than the DpbCasX (Fig 5b).
Regarding claim 54, Liu teaches the DpbCasX D101-190 variant has trans-cleavage of ssDNA, indicating that it at least comprises ssDNA cleavage activity (i.e., nickase activity) (Fig 5b).
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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Claims 1-3, 5, 17-19, 21, 34, 37, 39, 44, 47, 54, 71, 77-78, 80, 82, 85, 93-94, 96-97, 103-104, 120, 137, 160-161, 178, 180, 190 and 201 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-26 of U.S. Patent No. 11535835. Claim 39 and is rejected in view of Doudna (US 20180346927 A1).
Patented claim 1 recites a composition comprising a CasX variant comprising SEQ ID NO 138 and a gRNA. The patented Specification indicates that SEQ ID NO 138 is also called CasX 491 which is derived from SEQ ID NO 2 and comprises the NTSB and Helical 1B from SEQ ID 1 (Table 3). SEQ ID NO 3548 of the present application comprises a sequence that is 100% identical to SEQ ID NO 138 with an additional NLS sequence. Patented claim 15 recites wherein the CasX variant protein further comprises an NLS. Although the patented claims do not recite an improved characteristic of the CasX variant, because the structure of the patented and instant CasX are identical, the patented CasX protein must have an improved characteristic compared to SEQ ID NO 2. Patented claim 14 recites wherein the gRNA has a scaffold sequence of SEQ ID NO 2238, which is identical to SEQ ID NO 2238 of the instant application. Although the patented claims do not recite an improved characteristic of the gNA variant, because the structure of the patented and instant gNA are identical, the patented gNA must have an improved characteristic to a reference gNA. Patented claim 23 recites a nucleic acid vector comprising CasX with SEQ ID NO 138 and a gRNA. Patented claims 25 recites wherein the nucleic acid vector is an AAV vector. Patented claim 26 recites a method comprising contacting cells of the eye (i.e., eukaryotic cells) with the nucleic acid vector of claim 25. Therefore, the patented claims anticipate instant claims 1-3, 5, 17-19, 21, 34, 37, 44, 47, 54, 71, 77-78, 80, 82, 85, 93-94, 96-97, 103-104, 120, 137, 160-161, 178, 180, 190 and 201.
Regarding claim 39, the patented claims do not recite a specific sequence for the NLS. However, Doudna teaches appending NLS sequences at the N-terminus of CasX domains including the NLS from SV40 virus with the sequence PKKKRKV. It would have been obvious to specifically choose the NLS sequence with PKKKRKV to append onto the patented CasX because Doudna suggests it.
Claims 1-3, 5, 17-19, 21, 34, 37, 39, 44, 47, 54 and 71 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 11560555.
Patented claim 1 recites An engineered protein comprising a RuvC cleavage domain, wherein the RuvC cleavage domain comprises the sequence of amino acids 648-812 of SEQ ID NO: 2 with one or more amino acid modifications relative to said RuvC cleavage domain sequence (i.e., a variant of a CasX with SEQ ID NO 2), wherein the engineered protein exhibits one or more improved characteristics compared to SEQ ID NO: 2 selected from the group consisting of improved editing of target DNA, improved target DNA cleavage rate, increased formation of cleavage-competent ribonucleoprotein (RNP) complexes, improved protospacer adjacent motif (PAM) utilization, and improved solubility. SEQ ID NO 2 of the patented and instant application are identical. Patented claims 3-5 recites wherein the one or more amino acid modifications comprise an amino acid substitution, including A708K. Patented claims 7-8 recite wherein the one or more amino acid modifications comprise an amino acid deletion, including at amino acid position P793. Patented claim 10 recites the engineered protein of claim 1, further comprising one or more of (a) a non-target strand binding (NTSB) domain; (b) a target strand loading (TSL) domain; (c) a helical I domain; (d) a helical II domain; and (e) an oligonucleotide binding domain (OBD) (i.e., all the remaining domains of CasX). Patented claim 11 recites comprising a NTSB domain, wherein the NTSB domain comprises amino acids 101-191 of SEQ ID NO: 1 (i.e., having an NTSB domain of a different CasX variant). Patented claims 21/22 recite wherein the engineered proteins comprise all [the domains of a CasX protein] comprising amino acid substitutions at A708 and L379. Patented claim 25 recites wherein the protein is selected from SEQ ID NOs 3505, 3506, 3507 and 3548, which are identical to the corresponding SEQ ID NOs in the instant application. It is noted that SEQ ID NO 3548 comprises “PKKKRKV”, an NLS sequence. Therefore, the patented claims anticipate instant claims 1-3, 5, 17-19, 21, 34, 37, 44, 47, 54 and 71.
Claims 178 and 180 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 11976277 in view of Doudna (US 20180346927 A1).
Patented claim 1 recites a delivery particle (XDP) system comprising five nucleic acids, wherein the five nucleic acids comprise, from 5′ to 3′, sequences encoding… (c) a CRISPR nuclease… (e) a CRISPR guide RNA. Patented claims 6-7 recite herein the CRISPR nuclease comprises a CasX CRISPR nuclease, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, or 1901. SEQ ID NO 3548 of the present application comprises a sequence that is 100% identical to SEQ ID NO 189 with an additional NLS sequence. Although the patented claims do not recite an improved characteristic of the CasX variant, because the structure of the patented and instant CasX are identical, the patented CasX protein must have an improved characteristic compared to SEQ ID NO 2. Patented claims 11/13 recite wherein the CRISPR guide RNA (gRNA) is a single-molecule guide RNA (sgRNA) comprising a scaffold sequence and a targeting sequence…, wherein the gRNA comprises a scaffold sequence selected from the group consisting of SEQ ID NOS:1959-2010 and SEQ ID NOS: 2238-2377. SEQ ID NO 2238 is of the patented application is identical to SEQ ID NO 2238 of the instant application. Although the patented claims do not recite an improved characteristic of the gNA variant, because the structure of the patented and instant gNA are identical, the patented gNA must have an improved characteristic to a reference gNA.
The patented claims do not recite a specific sequence for the NLS. However, Doudna teaches appending NLS sequences at the N-terminus of CasX domains including the NLS from SV40 virus with the sequence PKKKRKV. It would have been obvious to append the NLS sequence with PKKKRKV onto the patented CasX because Doudna suggests it.
Claims 77-78, 80, 82, 85, 93-94, 96-97, 103-104 and 108 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-38 of U.S. Patent No. 12084692.
Patented claim 1 recites a variant of a reference guide ribonucleic acid (gRNA) (gRNA variant) capable of binding an engineered protein comprising a RuvC cleavage domain, wherein the RuvC cleavage domain comprises the sequence of amino acids 648-812 of SEQ ID NO: 2 with one or more amino acid modifications relative to the RuvC cleavage domain sequence, wherein: a) the gRNA variant comprises at least one modification compared to the reference guide ribonucleic acid scaffold sequence, wherein the at least one modification is in a scaffold stem loop region, wherein the scaffold stem loop region comprises the sequence of SEQ ID NO: 245, or the sequence of SEQ ID NO: 245 with at least 1, 2, 3, 4, or 5 mismatches thereto; and wherein b) the gRNA variant exhibits one or more improved characteristics compared to the reference guide ribonucleic acid. SEQ ID NO 245 is comprised within SEQ ID NO 4 of the examined application. The patented claim 2 recites wherein the reference gRNA comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, which are identical to SEQ ID NOs 4 and 5 of the examined application. Patented claim 3 recites wherein the one or more improved characteristics of the gRNA variant is at least about 1.1 to about 100-fold or more improved relative to the reference gRNA. Patented claim 4 recites wherein the one or more improved characteristics of the gRNA variant is selected from the group consisting of: improved stability, among many other characteristics. Patented claims 5-6 recite further comprising at least one modification in a region of the gRNA variant, wherein the region is selected from the group consisting of an extended stem loop region, a 5′ unstructured region, a triplex region, a scaffold stem loop region, a triplex loop region, and a pseudoknot region, including at least one nucleotide substitution, deletion, insertion. Patetned claim 7 recites a) a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; b) a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; c) an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; d) any combination of (a)-(c). Patented claim 8 recites wherein the gRNA variant comprises a modification relative to the reference guide sequence of SEQ ID NO: 5, and is selected from one or more of: a) a C18G substitution in the triplex loop region; b) a G55 insertion in the scaffold stem loop region; c) a U1 deletion; and d) a modification of the extended stem loop region wherein: i. a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and ii. a deletion of A99 and a substitution of G64U that results in a loop-distal base that is fully base-paired. Copending claim 11 recites comprising an extended stem loop region comprising at least 10 or at least 100 nucleotides. Copending claims 12 and 13 recite comprising a heterologous stem loop capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule, wherein the heterologous stem loop is selected from the group consisting of MS2, Qβ, U1 hairpin II, PP7, phage replication loop, Kissing loop a, Kissing loop_b1, Kissing loop_b2, G quadruplex M3q, G quadruplex telomere basket, Sarcin-ricin loop, and Pseudoknot stem loops. Patented claim 14 recites wherein the gRNA variant further comprises a targeting sequence, wherein the targeting sequence is complementary to a target DNA sequence. Patented claim 15 recites, wherein the gRNA is a single-guide ribonucleic acid (sgRNA). Patented claim 18 recites wherein the gRNA variant sequence comprises the sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280, which are 100% identical to the corresponding SEQ ID NOs in the examined application. Therefore, the patented claims anticipate instant claims 77-78, 80, 82, 85, 93-94, 96-97, 103-104 and 108.
Claims 1-3, 5, 18-19, 21, 34, 39, 44, 47, 54, 77-78, 80, 82, 85, 86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 178, 180, 190 and 201 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4, 14, 21, 27, 31-33, 35, 40, 46, 55, 58, 60, 64, 73, 75, 77-78, 80, 97-98, 100-102, 105-106, 108, 117-118, 120 of copending Application No. 17641404.
Copending claim 1 recites A system comprising a CasX protein and a first guide nucleic acid (gNA), wherein the gNA comprises a targeting sequence complementary to a target nucleic acid sequence of a gene encoding a first protein involved in antigen processing, antigen presentation, antigen recognition, and/or antigen response. Copending claim 27 recites wherein the gNA has a scaffold comprising the sequence of SEQ ID NO: 2238, or a sequence having at least about 70% sequence identity thereto. SEQ ID NO 2238 is identical to the corresponding SEQ ID NO of the present application. Copending claim 31 recites wherein the CasX protein comprises a sequence of SEQ ID NO: 138, or a sequence having at least about 70% sequence identity thereto. SEQ ID NO 138 is 86% identical to SEQ ID NO 4413 of the instant application, and comprises most of SEQ DI NO 2 and the NTSB Helical 1B swap from SEQ ID NO 1. Copending claim 33 recites wherein the CasX protein comprises at least one modification relative to a reference CasX protein having a sequence selected from SEQ ID NOS: 1-3, wherein the at least one modification comprises at least one amino acid substitution, deletion, or substitution in a domain of the CasX variant protein relative to the reference CasX protein, and wherein the domain is selected from the group consisting of a non- target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain. Copending claim 35 recites wherein the CasX protein further comprises one or more nuclear localization signals (NLS). Copending claim 40 recites wherein the CasX protein is capable of forming a ribonuclear protein complex (RNP) with the variant gNA and wherein an RNP of the CasX variant protein and the gNA variant exhibit at least one or more improved characteristics as compared to an RNP of a reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and/or a gNA comprising a sequence of any one of SEQ ID NOS: 4-16. Copending claim 46 recites wherein the RNP comprising the CasX variant and the gNA variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein of SEQ ID NO:2 and the gNA comprising the sequence of any one of SEQ ID NOS: 4-16 in a comparable assay system. Copending claim 58 recites further comprising a donor template nucleic acid. Copending claim 60 recites A polynucleotide comprising a sequence that encodes the CasX and/or gNA of claim 1. Copending claim 64 recites A vector comprising the polynucleotide of claim 60. Copending claim 73 recites a method of modifying a target nucleic acid sequence of a gene in a population of cells… comprising introducing into each cell of the population the system of claim 1. Therefore, the copending claims anticipate examined claims 1-3, 5, 18-19, 21, 34, 39, 44, 47, 54, 77-78, 80, 82, 85, 86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 178, 180, 190 and 201.
This is a provisional nonstatutory double patenting rejection.
Claims 1-3, 5-6, 17-19, 21, 33-34, 39, 44, 47, 54, 77-78, 80, 82, 85, 86, 89-90, 93-94, 96-97, 103-104, 108, 120, 137, 178, 180, 190 and 201 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 193-220 of copending Application No. 17791130. Claim 39 is rejected in view of Doudna (US 20180346927 A1).
Copending claim 193 recites A system comprising a CasX variant protein and a guide ribonucleic acid (gRNA), wherein the gRNA comprises a targe