DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 04/18/2025 has been entered.
Claim Status
Claims 1-6, 8-13, 15-21 are pending and are examined here.
Priority
Application’s benefit of U.S. Provisional application 62/657,412, filed on 04/13/2018, is acknowledged. All examined claims enjoy the filing date of ‘412. Although corrected figures and additional sequence listing (submitted SEQ ID NO: 1129-1138) are submitted on 04/18/2025, Fig. 6F of ‘412 provides the sequence of SEQ ID NO: 1134, thus claim 8 enjoys the filing date of ‘412.
Nucleotide and/or Amino Acid Sequence Disclosures
The objection of sequence disclosure is withdrawn, claim 8 provides SEQ ID NO consistent with sequence listing.
Claim Objections
Claims are objected to because of the following informalities: some claims are indented (cl. 1-6, 8-11), while others are not (cl. 12 and greater). Appropriate correction is required.
Claim Rejections - 35 USC § 112
35 U.S.C. 112(a)-Written Description
Rejection of claims 1-6, 8-11, 13, 15-21 is withdrawn, the claim recites “mobilizing CD34+ HSPCs in an individual” is supported by the specification.
112a-Scope of Enablement
Rejection of claims 5 and 7 is withdrawn, claim 5 cancels C2c2 protein, while claim 7 is canceled.
35 U.S.C. 112(b):
Rejection of claim 8 is withdrawn as the claim recites SEQ ID NO: 1134.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 9-13, 15-21 are rejected under 35 U.S.C. 103 as being unpatentable over Cotta-Ramusino and Gori (US20160281111, pub. 09/29/2016, hereafter Cotta-Ramusino, in IDS) and Dewitt et al. (2016, Sc. Transl. Med., 9, pg. 1-9, along with Supplemental Material, pg. 1-30 referred as DeWitt, shares co-inventor but is outside the 102(b) exception grace period).
Regarding instant cl. 1, Cotta-Ramusino discloses methods and compositions for treating sickle cell disease (SCD) by correcting a target position (exon 6 is mutated from GAG codon encoding glutamate a.a. to a mutant GTG codon for valine due to the single substitution of A nt. to a T nt.) in the hemoglobin-beta gene (HBB) (par. 235) which comprises administering mobilization agent Plerixafor to a patient to mobilize peripheral blood CD34+ cells (par. 1062, relevant to instant cl. 1Aa); discloses a target cell CD34+ cells being removed from the subject (par. 1064, relevant to instant cl. 1Ab); manipulation of the cells ex vivo (par. 1063) with Cas9 molecule and gRNA delivered to target cell (par. 1062, Table 6 discloses various forms of formulation and delivery, either as a nucleic acid or RNP, relevant to instant cl. 1Bai and ii) and notes the alteration of target sequence occurs by HDR with an exogenously provided donor template (par. 214, 692). Further, Cotta-Ramusino discloses SEQ ID NO: 16259, an exogenous donor template of 2201 nt. comprising both 5’ (SEQ ID NO: 16257) and 3’ (SEQ ID NO: 16258) homologous arms, each flanking the SNP nt., of WT β-globin seq. with the correct nt. sequence of codon 6 (GAG) (see par. 961-963).
Cotta-Ramusino does not disclose the specific donor template of instant SEQ ID NO: 1126, a 168 nt. donor DNA template.
DeWitt discloses a method of modifying a hemoglobin gene in hematopoietic stem/progenitor cells (HSPCs) by obtaining both mobilized wild-type and sickle cell disease (SCD) CD34+ HSPCs (Fig. 1, supplemental material, last par. of pg. 4 and 1st par. of pg. 5) and editing the hemoglobin gene in HSPCs by electroporating a mixture of a ribonucleoprotein (RNP) complex, comprising Cas9 protein and unmodified single guide RNA (gRNA), and single-stranded DNA oligonucleotide (ssODN) template with the desired edit to demonstrate efficient replacement of the SCD mutation in vitro (abstract, Fig. 3a). To correct the single nucleotide mutation in SCD HSPCs, an appropriate donor template along with RNP complex is required. One of the three ssODNs template used to correct the SCD gene was T111-57S (supplemental material, pg. 27 for sequence; see Fig. 3, pg. 6 for results), which is identical to instant SEQ ID NO: 1126 (see alignment below, relevant to instant cl. 1Bb)). When the authors first attempted to identify an optimal donor template in culture cells (K562), they used a ssODN containing a GTA codon (encoding valine a.a.) that resulted in wild-type hemoglobin B gene being mutated to a sickle-cell mutation and demonstrated that “a [ssODN] template with a 111-nt 5′ arm and a 57-bp 3′ arm (T111-57) yielded an HDR frequency modestly higher than the original template T88-107 (33% versus 28.5% in the same experiment)” (pg. 3). Thus, once the optimal donor template structure was identified, the authors used the donor template (T111-57S) containing the GAA codon (encodes Glu a.a.) to correct the sickle-cell mutation (Fig 3, relevant to functional language of cl. 1, “at least 2% of the SCD-associated SNPs are corrected in the in vitro mixed population”).
Alignment between instant SEQ ID NO: 1126 (Qy) and T111-57S (Db):
Query Match 100.0%; Score 168; DB 1; Length 168;
Best Local Similarity 100.0%;
Matches 168; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 TCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAAC 60
Qy 61 CTCAAACAGACACCATGGTGCACCTGACTCCTGAAGAGAAGTCTGCGGTTACTGCCCTGT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CTCAAACAGACACCATGGTGCACCTGACTCCTGAAGAGAAGTCTGCGGTTACTGCCCTGT 120
Qy 121 GGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGT 168
||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGT 168
One of the KSR rationale that may be used to support a conclusion of obviousness is that there is some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the filing date of the claimed invention to have substituted one the donor template of Cotta-Ramusino in view of DeWitt and arrive at the claimed invention with a reasonable expectation of success. Based on the success of gene editing by Cotta-Ramusino using a ssODN and nickase with gRNAs, and success of DeWitt of correcting SCD-SNP using Cas9-gRNA RNP complex and T111-57S DNA template, one of ordinary skill in the art would reasonably expect success by substituting ssDNA of Cotta-Ramusino with T111-57S donor template with corresponding gRNA of DeWitt to correct E6V mutation of SCD. Thus, cl. 1 is obvious.
Regarding instant cl. 2, Cotta-Ramusino discloses practicing the invention using the Type II Cas system (par. 675).
Regarding instant cl. 3, Cotta-Ramusino discloses and claims a targeting domain sequence (i.e. gRNA) SEQ ID NO: 387 to use with Cas9 polypeptide; also discloses using a software tool to optimize the choice of potential targeting domains for Cas9 (par. Cl. 12, 128, 420, Table 3, par. 394, 395). Thus, SEQ ID NO: 387 is a Cas9 guide RNA that recruits Cas9 upon binding to genome target sequence.
Regarding instant cl. 9, Cotta-Ramusino discloses all bases of gRNA are modified (par. 1178).
Regarding instant cl. 10, Cotta-Ramusino discloses that the entire backbone is phosphorothioated (par. 1178); thus the first three nt. at the 5’ end of the guide RNA are modified.
Regarding instant cl. 11, Cotta-Ramusino discloses use of various nucleotide (nt.) modifications (par. 1169-1236), including LNA (1204), which is a modified sugar moiety; PNA (par. 1201); phosphorothioate (par. 1178); modification of nucleobase (par. 1209-1211).
Regarding instant cl. 12, Cotta-Ramusino discloses treating patient with mobilization agent Plerixafor to mobilize peripheral blood CD34+ (par. 1062).
Regarding instant cl. 13, Cotta-Ramusino discloses a mutation of GAG to GTG resulting in substitution of valine to glutamic acid at amino acid position 6 of exon 1 of the HBB gene for sickle cell disease (par. 179); discloses SEQ ID NO: 16259 comprising WT β-globin seq. with the correct nt. sequence of codon 6 (GAG) (see par. 961-963).
Regarding instant cl. 15, Cotta-Ramusino discloses SEQ ID NO: 16030, which matches 100% with instant SEQ ID NO: 1128 and is a portion of a guide RNA. See alignment below.
Sequence 16030, US/15081456
Publication No. US20160281111A1
GENERAL INFORMATION
APPLICANT: EDITAS MEDICINE, INC.
APPLICANT: COTTA-RAMUSINO, Cecilia
APPLICANT: GORI, Jennifer L.
TITLE OF INVENTION: CRISPR/CAS-MEDIATED GENE CONVERSION
FILE REFERENCE: 126454-00406 / EM048US1
CURRENT APPLICATION NUMBER: US/15/081,456
CURRENT FILING DATE: 2016-03-25
PRIOR APPLICATION NUMBER: US 62/138,948
PRIOR FILING DATE: 2015-03-26
PRIOR APPLICATION NUMBER: US 62/182,416
PRIOR FILING DATE: 2015-06-19
PRIOR APPLICATION NUMBER: US 62/194,078
PRIOR FILING DATE: 2015-07-17
PRIOR APPLICATION NUMBER: US 62/220,660
PRIOR FILING DATE: 2015-09-18
PRIOR APPLICATION NUMBER: US 62/232,675
PRIOR FILING DATE: 2015-09-25
PRIOR APPLICATION NUMBER: US 62/232,683
PRIOR FILING DATE: 2015-09-25
PRIOR APPLICATION NUMBER: PCT/US2015/059782
PRIOR FILING DATE: 2015-11-09
NUMBER OF SEQ ID NOS: 16324
SEQ ID NO 16030
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
Query Match 100.0%; Score 20; Length 20;
Best Local Similarity 100.0%;
Matches 20; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 CUUGCCCCACAGGGCAGUAA 20
||||||||||||||||||||
Db 1 CUUGCCCCACAGGGCAGUAA 20
Further, a couple of gRNAs used by DeWitt to demonstrate correction of SCD-SNP mutation is G10 and TrG10; G10 comprises the SEQ ID NO: 1128 (see alignment below, pg. 24 of Supplemental Material, T=U, labeled “FwdVar-G10”), while TrG10 is a truncated version of G10, missing the initial 2 nt. from the 5’ end. The authors note that the truncated G10 (trG10) showed robust HDR and few off-target effects.
SEQ ID NO: 1128: 1 CUUGCCCCACAGGGCAGUAA 20
|::||||||||||||||:||
G10 25 CTTGCCCCACAGGGCAGTAA 44
One of the KSR rationale that may be used to support a conclusion of obviousness is that there is some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the filing date of the claimed invention to have substituted the appropriate guide RNA of Cotta-Ramusino in view of DeWitt and arrive at the claimed invention with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to substitute the gRNA SEQ ID NO: 16030 and donor template of Cotta-Ramusino with appropriate guide RNA (i.e. either G10 or patentably indistinct TrG10) and corresponding donor template of DeWitt and due to the success of editing the SCD site on the genome of DeWitt, a skilled artisan would expect reasonable success in achieving gene correction with appropriate Cas9-RNP mixture with ssODN and G10/TrG10 of DeWitt. Thus, cl. 15 is obvious.
Regarding instant cl. 16-19, Cotta-Ramusino discloses various results demonstrating ranges of % modification from ~60% to ~80% at HBB locus with use of WT or variants of Cas9 (see Fig. 10C, par. 13B and C, par. 118; Fig. 14, par. 120). The graphs noted have at least have 2% gene correction (see Fig. 10C). Further, fig. 32 demonstrates HDR gene editing, which uses exogenous nucleic acid, i.e. a donor template (par. 7), of CD34+ cells with various doses of RNP and % editing ranging from ~20% to ~50% of overall editing (fig. 32B).
Alternatively, it would also be obvious based on the results of DeWitt demonstrating at least 2% of SCD-associated SNPs are corrected in vitro mixed population (see Fig. 3).
Regarding instant cl. 20, Cotta-Ramusino discloses using Cas RNP editing in SCD patient cells using Cas9 D10A nickase RNP with two different gRNA pairs to target the sequence specific to the HBB gDNA of SCD patients (par. 1315, 1348); discloses that ~75% of the cells were viable after electroporation; discloses 57% editing after DNA sequencing (par. 1315). Cotta-Ramusino discloses gene edited CD34+ cells maintained ex vivo hematopoietic activity and multipotency as indicated by their ability to give rise to erythroid and myeloid (par. 1332). Thus, inherently by differentiating and survivability, the cells inherently expressed or will express two alleles, a homozygote dominant alleles (i.e. two separate alleles) of repaired GAG genotype. Further, Cotta-Ramusino discloses target gene repaired by gene conversion in at least 4 -6 % of the cells in CD34+ HSCs population (par. 58, 88), which includes creating double stranded 5’ overhangs and an endogenous homologous region repair event (par. 86). Also Cotta-Ramusino discloses use of dsDNA donor, upper strand donor or lower strand donor and their percent contributions on HDR (see Fig. 13C), thus using either strands illustrates repairing each separate SNP mutant allele of different strands.
Alternatively, it would also be obvious based on the results of DeWitt demonstrating at least 2% of SCD-associated SNPs are corrected in vitro mixed population (see Fig. 3).
Regarding instant cl. 21, Cotta-Ramusino discloses as noted above regarding SCD treatment in an individual, modifying a globin gene using CRISPR/Cas9 system and manipulating it ex vivo, therefore creating an in vitro mixed population (since basically even one SNP editing would create a mixed population), and then the ex vivo manipulated cell is returned to a subject (par. 2238-2239). Again as noted above, results described in Fig. 32B demonstrate at least 2% of overall editing at HBB locus.
Claims 4-6 are rejected under 35 U.S.C. 103 as being unpatentable over Cotta-Ramusino and Gori (US20160281111, pub. 09/29/2016, referred as Cotta-Ramusino, in IDS) and Dewitt et al. (2016, Sc. Transl. Med., 9, pg. 1-9, referred as DeWitt, shares co-inventor but is outside the 102(b) exception grace period), as applied to claims 1-3, 9-13, 15-21 above, and further in view of Cebrian-Serrano and Davies (2017, Mamm. Genome 28, 247–261, hereinafter referred as Cebrian-Serrano).
The rejection of claims 1-3, 9-13, 15-21 are disclosed above.
As noted above, Cotta-Ramusino discloses various types of Cas effector polypeptides of Type II Cas polypeptides, but does not disclose type V or Cas12 enzymes.
Cebrian-Serrano discloses Cas12a, also known as Cpf1, as an alternative genome editing RNA-guided, highlights the interesting traits, including requiring only the crRNA and not the tracrRNA, as required by Cas9, and cleaving DNA via a staggered DSB, thus leaving overhangs, a feature that may facilitate the introduction of specific sequences into the genome (pg. 257). Cas9 creates blunt ends.
One of the KSR rationale that may be used to support a conclusion of obviousness is that there is some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the filing date of the claimed invention to have simply substituted Cas9, one type of Class 2 CRISPR/Cas polypeptide, of Cotta-Ramusino in view of Cebrian-Serrano and arrive at the claimed invention with a reasonable expectation of success. Based on the improved feature of Cpf1 leaving overhangs for improved homologous recombination as taught by Cebrian-Serrano and success of Cotta-Ramusino utilizing D10A or WT Cas9 and an donor template for editing HBB gene, one of ordinary skill in the art would expect reasonable success to substitute Cas9 of Cotta-Ramusino with a Cas12a (or Cpf1) for genome editing that leaves overhang to introduce specific sequences as taught by Cebrian-Serrano that would introduce a specific donor template for editing.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Cotta-Ramusino and Gori (US20160281111, pub. 09/29/2016, hereafter Cotta-Ramusino, in IDS) and Dewitt et al. (2016, Sc. Transl. Med., 9, pg. 1-9, referred as DeWitt, shares co-inventor but is outside the 102(b) exception grace period), as applied to claims 1-3, 9-13, 15-21 above, and further in view of Kleinstiver et al. (2016, Nature, 529, pg. 490-495).
Disclosure pertaining to rejection of claims 1-3, 9-13, 15-21 is noted above. Cotta-Ramusino discloses use of Cas9 D10A nickase that results in a single DNA nick in the target strand and provides results noting improved gene conversion with the D10A nickase than WT Cas9 (e.g. see Fig. 7A). Gene conversion is believed to be the homologous dependent repair of HBB gene from the endogenous homologous HBD gene (par. 1266).
Further, DeWitt also tested RNP complexes comprising variants of Cas9, eSpCas9-1.1 and HF1 variants, and G10 sgRNA to edit WT CD34+ HSPCs in the absence of ssODN, and both had reduced on-target indel formation, but both modified enzymes “also completely eliminated all previously observed off-target events. . . (Fig. 2C)” (pg. 5). As noted below, as evidenced by Kleinstiver, HF1 variant has the following substitutions: N497A, R66A, Q695A, Q926A substitutions, but not the D10 substitution of Cas9 D10A nickase.
Cotta-Ramusino and DeWitt do not disclose the high-fidelity variant of SEQ ID NO: 1134, a Cas9 variant that comprises D10A, N497A, R66A, Q695A, Q926A substitutions (see alignment below comparing instant SEQ ID NO: 1134 and instant SEQ ID NO: 1129, a SpCas9 WT).
Kleinstiver demonstrates that by introducing specific alanine mutations at specific sites (N497A, R66A, Q695A, Q926A, SpCas9 with all the mutations was designated “SpCas9-HF1”) where SpCas9 makes contact with the target DNA site resulted in reduced cleavage (almost to “undetectable levels”) of mismatched off-target sites but did not reduce the on-target cleavage efficiency when compared to WT Cas9 (pg. 491, see Fig. 1b). Even some high-fidelity variants with only 2 or 3 (e.g. Q695A and Q926A and R661A) substitutions noted provided reduced cleavage of mismatched off-target sites (see Fig. 1b). Kleinstiver also attempted to refine SpCas9-HF1 by introducing other known substitutions (see pg. 493-494, Fig. 5). Alignment of Cas9 of Fig. 6 and instant SEQ ID NO: 1134 is noted below to highlight the mutated positions, noted as bolded/underlined letter. (SEQ ID NO: 1134 is missing a methionine at pos. 1, thus the numbering is off by 1 between the two polypeptides)
One of the KSR rationale that may be used to support a conclusion of obviousness is that there is some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the filing date of the claimed invention to have modified the D10A Cas9 variant of Cotta-Ramusino in view of Kleinstiver and arrive at the claimed invention with a reasonable expectation of success. Based on the success of Cotta-Ramusino in utilizing D10A Cas9 variant for gene editing and success of Kleinstiver of reducing off-target cleavage of a few high-fidelity variants of SpCas9 (one includes , one of ordinary skill in the art would expect reasonable success by combining the substitutions of D10A-Cas9 of Cotta-Ramusino and a few high-fidelity variants of Kleinstiver to improve gene editing by reducing off-target effects. Thus, cl. 8 is obvious.
Response to Arguments
Applicant's arguments filed 04/18/2025 (hereinafter referred as the Remarks) have been fully considered but they are not persuasive.
The Remarks argue that “presently claimed methods are the result of rigorous and careful optimization of many parameters” associated with editing b-globin gene using CRISPR platform (pg. 9). First, noting differences in donor template of cited reference and instant SEQ ID NO: 1126, the Remarks provide parameters that would need to be considered: sense or antisense strand of the β-globin gene, the length of 5’ and 3’ homology arms (2, 3); location (5’ or 3’) and distance of targeting site of guide strand relative to the mutation to be corrected (4, 5); and whether the targeting site of the guide strand is on the sense strand or antisense strand (6); and parameters associated with PAM sequence (7) (pg. 9). Then the Remarks further details arriving at the specific instant donor DNA template SEQ ID NO: 1126 requires consideration of factors noted above and that a skilled artisan would not arrive to instant SEQ ID NO: 1126, including differences in the modified PAM sequence, based on the Cotta-Ramusino’s SEQ ID NO: 16259 (pg. 9-10). Further, Cotta-Ramusino discloses “at least 415 guide RNAs” and each targeting a different strand of the dsDNA of HBB gene (pg. 10). The argument is that there are a very high number of possible options disclosed in Cotta-Ramusino that a skilled artisan could not arrive to the claimed SEQ ID NO: 1126 (pg. 11). Further, there is an issue of editing efficacy, since Cotta-Ramusino demonstrates using nickase (i.e. D10A-Cas9) requires 2 guide RNAs to modify a target gene and of the 415 disclosed guide RNA, only 4 are tested, and none of which would lead the skilled person to modify the sequence of SEQ ID NO: 16259. Also Cotta-Ramusino has different efficacy depending on Cas9 wild type v. D10A with dual guide RNAs and based on donor strand type (dsDNA, upper strand, lower strand) (pg. X).
The argument is not persuasive.
The DeWitt reference addresses the routine procedural steps and reagents used to identify optimal products for gene editing. DeWitt tests various single-stranded oligonucleotide DNA templates and several gRNAs targeting both the sense and antisense strands of the genome (Fig. 1A,B) to identify optimal combination that correct SCD-SNP mutation (e.g. see supplemental figures S1 noting that to prevent re-cutting, one donor template introduced silent mutation at all PAMs of all sgRNAs tested). DeWitt demonstrates successful correction with T111-57 (i.e. same as instant SEQ ID NO: 1126) and RNP complex comprising G10/trG10 (either identical or patentably indistinct to instant SEQ ID NO: 1128 guide RNA, respectively; see Fig. 3).
Thus rejection of claims 1-6 , 8-13, 15-21 is maintained.
RESULT 1: Alignment between SpCas9 WT and instant SEQ ID NO; 1134 (QY – SEQ ID NO: 1129, a SpCas9; Db – SEQ ID NO: 1134, both from Fig. 6)
Query Match 99.4%; Score 6966; DB 1; Length 1367;
Best Local Similarity 99.6%;
Matches 1362; Conservative 0; Mismatches 5; Indels 0; Gaps 0;
Qy 2 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 61
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60
Qy 62 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 121
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 120
Qy 122 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 181
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 180
Qy 182 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 241
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 240
Qy 242 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 301
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 300
Qy 302 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 361
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 360
Qy 362 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 421
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 420
Qy 422 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 481
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 480
Qy 482 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 541
||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||
Db 481 VDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 540
Qy 542 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 601
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 600
Qy 602 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 661
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGA 660
Qy 662 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 721
||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||
Db 661 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKEDIQKAQVSGQGDSLH 720
Qy 722 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 781
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 780
Qy 782 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 841
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 840
Qy 842 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 901
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 900
Qy 902 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 961
|||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||
Db 901 KAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 960
Qy 962 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1021
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1020
Qy 1022 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1081
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1080
Qy 1082 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1141
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1140
Qy 1142 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1201
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1200
Qy 1202 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1261
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1260
Qy 1262 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1321
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1261 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1320
Qy 1322 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1368
|||||||||||||||||||||||||||||||||||||||||||||||
Db 1321 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1367
Double Patenting
The rejection of claims 1-6, 15 on the grounds of nonstatutory double patenting is withdrawn due to amendment to claim 1.
Conclusion
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/KEYUR A VYAS/Examiner, Art Unit 1637
/Soren Harward/Primary Examiner, TC 1600