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 .
Election/Restrictions
Applicant’s election without traverse of Group III (Claims 1-6, 9-10, 12, 15-16, 19-20, 23-24, 26, 28-33, 35-38, 41, and 52-53), drawn to a method of correcting a gene variant associated with Parkinson’s Disease, in the reply filed on 10/20/2025 is acknowledged. Claims 42, 49-51, 54-57, 62, 64-65, and 68 are withdrawn from consideration, as being directed to a non-elected invention. Claims 1-6, 9-10, 12, 15-16, 19-20, 23-24, 26, 28-33, 23-38, 41, and 52-53 have been examined on the merits.
Priority
Acknowledgement is made that the instant application is a National Stage of International application No. PCT/US2021/028256 (filed 04/20/2021), which claims the benefits of US Provisional Application No. 63/013449 (filed 04/21/2020).
Claim Rejections - 35 USC § 102
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 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 1-4, 16, 19, 23-24, 26, 28-30, 36-38, 41, and 52 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Laganiere et al (US20120214241A1).
Laganiere et al discloses a method of correcting a G2019S mutant encoding LRRK2 gene in a fibroblast or patient derived iPSCs (See claims 1 and 7 and ¶0146). The iPSCs of Laganiere et al are derived from fibroblasts isolated from patients with heterozygous and homozygous LRRK G2019S mutations (See ¶0157). The LRRK2 mutation G2019S has been suggested to play an important role in Parkinson’s disease (PD) (See ¶006). The SNP responsible for this missense mutation in patients is annotated as rs34637584 in the human genome, and is a G to A substitution at the genomic level (6055G>A) (See ¶006). The method of Laganiere et al comprises using zinc finger nucleases (ZFNs) or TALENs which bind a target site in the LRRK2 gene and cleave the gene within the coding region of the gene, a non-coding sequence, or adjacent to the gene (See ¶0011-0012). The ZFNs or TALENs are used to create a double-stranded break in the target sequence at a predetermined site, and a donor polynucleotide, having homology to the nucleotide sequence in the region of the break, can be introduced into the cell. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination ¶0054). The donor polynucleotide can be DNA or RNA, single-stranded or double-stranded (See ¶0115). The donor sequence corrects a known mutation, e.g., corrects the ‘G’ to a ‘A’ in a mutant LRRK2 gene to cause the correction of the G2019S mutation (See ¶0024). The donor sequence can contain sequences that are homologous, but not identical to genomic sequences in the region of interests. In certain embodiments portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% sequence identity with the genomic sequence that is replaced. In some embodiments the homology between the donor and genomic sequences is higher than 99% (See ¶0057). The donor sequence is preferably 200-500 nucleotides in length (See ¶0063). In an exemplary embodiment, the donor polynucleotide was designed with a silent mutation that introduced an AciI restriction site (See ¶0173). The AciI restriction site allows the detection of integrated donor; the presence of AciI site suggests cells have undergone HDR-mediated donor insertion (gene correction) (See ¶0041 and 0178). The detection method comprises lysing half of every single cell clone colony, performing PCR, digesting the PCR products with AciI (See ¶0176). Laganiere et al further disclose genotyping can be performed by sequencing to confirm G2019S corrected cells (See ¶0168 and 0176).
Regarding claim 1: Laganiere et al discloses a method of correcting a G2019S mutant encoding LRRK2 gene (reads on an LRRK2 comprising a SNP) in patient derived iPSCs (reads on the LRRK2 is human LRRK2). The G2019S mutation is associated with PD. The method of Laganiere et al comprises introducing a ZFN or TALEN into the iPSCs resulting in cleavage of the LRRK2 gene which reads on introducing into an iPSC one or more agents comprising a recombinant nuclease for inducing a DNA break within an endogenous target gene in the cell. The method of Laganiere et al further discloses introducing a donor polynucleotide, which can be single-stranded, and is used as a template for repair of the break via homologous recombination which results in integration of the donor and correction of the mutation at position 6055 of LRRK2 thereby correcting the G2019S mutation. Introducing a donor polynucleotide in the method of Laganiere et al therefore reads on introducing a single-stranded DNA oligonucleotide that is homologous to the target gene and comprises a corrected for of the SNP wherein the ssODN results in HDR and integration of the ssODN into the target gene and after integration of the ssODN the target gene comprises the corrected form of the SNP.
Regarding claims 2-3: Following the discussion of claim 1 above, Laganiere et al discloses using a ZFN or TALEN (read on recombinant nucleases capable of cleaving both strands of double stranded DNA) to create double stranded breaks in LRRK2 which reads on creating a double stranded bread at a cleavage site within the endogenous target gene.
Regarding claim 4: Following the discussion of claim 1 above, the method of Laganiere et al comprises a ZFN or TALEN.
Regarding claim 16: Following the discussion of claim 1 above, Laganiere discloses a donor polynucleotide (reads on ssODN) comprising a sequence homologous to LRRK2 which can be used to correct the 6055G>A mutation of LRRK2 (reads on the ssODN comprises a nucleic acid sequence homologous to a targeting sequence in the target gene wherein the targeting sequence comprises the SNP and is not homologous to the targeting sequence at the nucleotide of the SNP. Laganiere et al further discloses in some embodiments the donor polynucleotide comprises a nucleotide sequence that is greater than 99% homologous to the region of interest (reads on at least 80% homologous).
Regarding claim 19: Following the discussion of claims 1 and 16 above, Laganiere et al discloses a donor polynucleotide comprising a sequence homologous to LRRK2 (reads on targeting sequence) which can be used to correct the 6055G>A mutation of LRRK2. The donor polynucleotide is preferably between 200-500 nucleotides in length (reads on between about 50 and about 500 nucleotides in length). Given that the donor polynucleotide of Laganiere et al is between 200-500 nucleotides in length and the donor polynucleotide comprises a region homologous to the targeting sequence, the targeting sequence in the method of Laganiere et al is between 200-500 nucleotides in length.
Regarding claim 23: Following the discussion of claims 1 and 16 above, Laganiere et al discloses an exemplary embodiment in which the donor polynucleotide comprises a silent mutation which introduces an AciI restriction site which reads on the ssODN comprises a nucleic acid sequence that comprise one or more nucleotides that are not homologous to the corresponding nucleotides of the targeting sequence and the one or more nucleotide introduce a restriction site that is recognized by a restriction enzyme.
Regarding claim 24: Following the discussion of claim 1 above, the method of Laganiere et al corrects the 6055G>A of LRRK2 which reads on the corrected form of the SNP is not associated with PD and is a wildtype form of the SNP.
Regarding claim 26: Following the discussion of claim 1 above, Laganiere et al discloses correcting a G2019S mutation in LRRK2 which is known as rs34637584 in the human genome, and is a ‘G’ to ‘A’ substitution at the genomic level (6055G>A).
Regarding claim 28: Following the discussion of claim 1 above, Laganiere et al discloses correcting a G2019S mutation in LRRK2 which reads on an LRRK2 comprising a SNP that encodes a serine rather than a glycine at position 2019.
Regarding claims 29 and 30: Following the discussion of claim 1 above, the method of Laganiere et al discloses replacing the ‘A’ at position 6055 of a mutant LRRK2 with a ‘G’ thereby correcting the G2019S mutation which reads on the corrected form of the SNP is a guanine wildtype variant and after integration of the ssODN the LRRK2 comprises the corrected form of the NSP and encodes a glycine at position 2019.
Regarding claims 36 and 37: Following the discussion of claim 1 above, Laganiere et al discloses generating iPSCs from fibroblasts isolated from patients with LRRK2 G2019S mutations which reads on the iPSCs are artificially derived from non-pluripotent cells from a subject who has Parkinson’s Disease.
Regarding claims 38, 41, and 52: Following the discussion of claims 1, 16, and 23 above, the method of Laganiere et al can comprise using a donor polynucleotide encoding a silent mutation which produces an AciI restriction site to allow for detection of the integrated donor. The detection method comprises lysing half of every single cell clone colony, performing PCR, digesting the PCR products with AciI (reads on contacting DNA isolated from the cell with a restriction enzyme). Digestion with AciI demonstrates that the donor has been inserted and that the G2019S mutation has been corrected in the iPSC which reads on determining whether the DNA isolated from the cell has been cleaved at the restriction site and wherein if the DNA has been cleaved the cell is identified as comprising an integrated ssODN. Thus the detection method of Laganiere et al further reads on a method for selecting a cell comprising an integrated ssODN.
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.
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 1-6, 9-10, 12, 16, 19-20, 23-24, 26, 28-32, 36-38, 41, and 52-53 are rejected under 35 U.S.C. 103 as being unpatentable over Laganiere et al (US20120214241A1).
The teachings of Laganiere et al are set forth above.
Laganiere et al anticipates claims 1-4, 16, 19, 23-24, 26, 28-30, 36-38, 41, and 52.
Regarding claim 53: Laganiere et al discloses a method for correcting a SNP comprising editing an LRRK2 G2019S mutation in a patient derived iPSC (reads on the cell of claim 1). Laganiere et al further discloses sequencing analysis can be used to confirm the presence of G2019S corrected cells which reads on a method for selecting for a cell comprising a corrected SNP.
Laganiere et al does not disclose sequencing DNA isolated from a cell of claim 1.
Although Laganiere et al does not disclose sequencing DNA isolated from a cell of claim 1, it would have been prima facie obvious to sequence DNA isolated from a cell of claim 1 to verify the presence of a G2019S correction. One would have been motivated to sequence the DNA isolated from a cell of claim 1 in order to verify the presence of a G2019S correction. There is a reasonable expectation of success because Laganiere et al teaches sequencing can be used to verify a G2019S correction.
Claims 1-6, 9-10, 12, 16, 19-20, 23-24, 26, 28-32, 36-38, 41, and 52-53 are rejected under 35 U.S.C. 103 as being unpatentable over Laganiere et al (US20120214241A1) in view of Offen et al (WO2019111258A1).
The teachings of Laganiere et al are set forth above.
Laganiere et al anticipates claims 1-4, 16, 19, 23-24, 26, 28-30, 36-38, 41, and 52 and renders claim 53 obvious.
Regarding claims 5, 6, 9, and 10: Following the discussion of claim 1 above, Laganiere et al discloses a method of correcting a G2019S mutation in LRRK2 comprising administration of a ZFN or TALEN (read on recombinant nucleases) and a donor polynucleotide. The ZFN or TALEN induce a double strand break in LRRK2 and the donor polynucleotide is used as a template for homology directed repair of the G2019 mutation.
Laganiere et al does not disclose a method of correcting the mutation in which a Cas nuclease and a single guide RNA (sgRNA) are used.
Offen et al discloses a method of treating PD, characterized by a G2019S mutation, comprising administering to a subject a CRISPR-Cas system guide RNA (gRNA)(reads on a sgRNA) and a CRISPR endonuclease (See claims 1-2). The gRNA of Offen et al utilizes a PAM sequence comprising the G2019S mutation (See claim 6). The CRISPR endonuclease of Offen et al can comprise a Cpf1 or Cas9 (See claim 3 and pg. 10, ln 28). Additionally Offen teaches genome editing using engineered endonucleases such as ZFNs, TALENs, and CRISPR-Cas systems can be used to create site-specific single or double stranded breaks at desired locations in the genome which are then repaired by HDR and NHEJ (See pg. 6, lns 14-28).
Given that Laganiere et al and Offen et al both teach methods of correcting a G2019S mutation in LRRK2 comprising cleaving LRRK2 with an endonuclease capable of making a double stranded break, it would have been prima facie obvious, to a person of ordinary skill in the art, to substitute the ZFN of the method of Laganiere et al for the gRNA and Cpf1 or Cas9 (read on Cas nucleases) of Offen et al in the method of Laganiere et al. One would have expected the sgRNA and Cpf1 or Cas9 to work equivocally with a ZFN in the method of Laganiere et al because Offen et al teaches ZFNs and CRISPR-Cas systems are endonucleases that can be used to make site-specific double stranded breaks at desired locations. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Regarding claim 15: Following the discussion of claims 1 and 2 above, Laganiere et al teaches a method of correcting a G2019S mutation in LRRK2 comprising administering a ZFN and a donor polynucleotide comprising a correction of the point mutation to be used as a template for HDR. It would have been prima facie obvious to substitute the ZFN of Laganiere et al for the gRNA and CRISPR endonuclease of Offen et al (See rejections of claims 5, 6, 9, and 10 above). The gRNA of Offen et al uses a PAM that comprises the SNP in G2019S mutation. Figs. 1-5 of Offen et al show exemplary gRNAs comprising a cleavage site within the 20 nucleotides of the gRNA. Additionally the gRNA is immediately upstream of the PAM/SNP. Therefore, the cleavage site in exemplary embodiments of Laganiere et al in view of Offen et al is less than 200 nucleotides from the SNP.
Regarding claim 20: Following the discussion of claims 1 and 16 above, Laganiere et al teaches a method of correcting a G2019S mutation in LRRK2 comprising administering a ZFN and a donor polynucleotide comprising a correction of the point mutation to be used as a template for HDR.
Laganiere et al does not disclose the targeting sequence comprise a PAM sequence.
Offen et al discloses a method of treating PD comprising administering a gRNA and a CRISPR endonuclease. The gRNA of Offen et al utilizes a PAM sequence comprising the G2019S mutation (reads on within the targeting sequence). It would have been prima facie obvious to substitute the ZFN of Laganiere et al for the gRNA and CRISPR endonuclease of Offen et al (See rejection of claims 5, 6, 9, and 10 above). Therefore, the targeting sequence of Laganiere et al in view of Offen et al comprises a PAM.
Regarding claim 31: Following the discussion of claims 1 and 6 above Laganiere et al teaches a method of correcting a G2019S mutation in LRRK2 comprising administering a ZFN and a donor polynucleotide comprising a correction of the point mutation to be used as a template for HDR. It would have been prima facie obvious to substitute the ZFN of Laganiere et al for the gRNA and CRISPR endonuclease of Offen et al (See rejections of claims 5, 6, 9, and 10 above). Offen et al further teaches a gRNA encodes a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas endonuclease in a single chimeric transcript. Additionally, Offen teaches five exemplary gRNAs that include a cleavage site (See Figs. 1-5). Therefore, the gRNA of Laganiere et al in view of Offen et al comprises a crRNA sequence that is homologous to a sequenc3e in the target gene that includes the cleavage site.
Regarding claim 32: Following the discussion of claims 1, 6, and 31 above, Laganiere et al in view of Offen et al teach a method of correcting a G2019S mutation in LRRK2 comprising a gRNA that is immediately upstream of a PAM sequence (See Figs. 1-5). The gRNA of Offen et al comprises the cleavage site. Therefore, the cleavage site is immediately upstream of the PAM sequence.
Off-target
Claims 1-6, 9-10, 12, 15-16, 19-20, 23-24, 26, 28-32, 36-38, 41, and 52-53 are rejected under 35 U.S.C. 103 as being unpatentable over Laganiere et al (US20120214241A1) in view of Offen et al (WO2019111258A1) and AddGene (CRISPR Guide, 2019).
The teachings of Laganiere et al are set forth above.
Laganiere et al anticipates claims 1-4, 16, 19, 23-24, 26, 28-30, 36-38, 41, and 52 and renders claim 53 obvious.
Laganiere et al in view of Offen et al render claims 1-6, 9-10, 12, 15-16, 19-20, 23-24, 26, 28-32, 36-38, 41, and 52-53 obvious.
Regarding claims 12, 33, and 35: Following the discussion of claims 1, 5, 9, and 10 above Laganiere et al teaches a method of correcting a G2019S mutation in LRRK2 comprising administering a ZFN and a donor polynucleotide comprising a correction of the point mutation to be used as a template for HDR. It would have been prima facie obvious to substitute the ZFN of Laganiere et al for the gRNA and CRISPR endonuclease of Offen et al (See rejections of claims 5, 6, 9, and 10 above).
Laganiere et al in view of Offen et al do not teach the Cas9 is a variant that reduces off-target effector activity or lacks the ability to induce a DSB.
AddGene teaches gRNA targeting sequences will have additional sites throughout the genome where partial homology exists known as off-targets. AddGene further teaches using dual Cas9 nickases, which are D10A mutants of SpCas9 and can only cleave one strand of DNA, or using high fidelity Cas9s e.g., eSpCas9 and SpCas9-HF1 have mutations that generate less off-target editing than wildtype Cas9 (See Sec. Enhancing Specificity with Nickases and High Fidelity Enzymes).
Given that Laganiere et al in view of Offen et al teach a method of editing a G2019S mutation in LRRK2 which can comprise editing with a CRISPR endonuclease and a gRNA and AddGene teaches gRNAs have homologous off-target sites throughout the genome it would have been prima facie obvious to modify the method of Laganiere et al in view of Offen et al to use dual Cas9 nickases or high fidelity Cas9 as the CRISPR endonuclease in the gene editing method of Laganiere et al. One would have been motivated to use dual Cas9 nickases or a high fidelity Cas9 because AddGene teaches dual nickases and high fidelity Cas9s generate less off target editing. There is a reasonable expectation of success because Offen discloses a method which can comprise a Cas9 and dual Cas9 nickases and high fidelity Cas9 are variants of Cas9.
Conclusion
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/MARISOL ANN O'NEILL/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633