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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/28/2025 has been entered.
Response to Amendment/Status of Claims
Receipt of Arguments/Remarks filed on 11/28/2025 is acknowledged. Claims 3-5,14 and 17-19 stand cancelled. Claims 1,10,11,12,15 and 20-23 were amended. Claims 1,2,6-13,15,16 and 20-25 are pending and under examination.
Priority
This application is a 371 of PCT/EP2019/074131, filed 09/10/2019 and claims foreign priority to EP18306191.0, filed 09/11/2018 as reflected by the most recent filing receipt.
Response to Arguments
Applicant’s arguments and amendments, see pages 6-7, filed 11/28/2025, with respect to the rejection(s) of claim(s) 1,2,6-13,15,16 and 20-22 under 35 U.S.C. 103 as unpatentable over Martyn et al. and ‘705 have been fully considered but are not persuasive. While Applicant has amended the claims to recite that the DNA targeting endonuclease cleaves the genomic DNA of the cell between positions -198 and -197 in the HBG1 or HBG2 promoter, and has removed SEQ ID NO: 31 from claims 10,11 and 20-22 and replaced with SEQ ID NO: 2, ‘705 still teaches a guide RNA having 100% identity to instant SEQ ID NO: 2. Given the claim interpretation provided in the last office action in the final dated 07/03/2025 and applying the same claim interpretation, both Martyn et al. and ‘705 still provide a case of obviousness for the instant claims, and therefore Applicant’s arguments that ‘705 fails to teach a guide RNA having the sequence of SEQ ID NO: 2 is not found persuasive. Applicant did not provide any arguments regarding the examiner’s claim interpretation in the last action regarding that the structure of the guide RNA would carry out the recited functional limitations in the wherein clauses. See also the final rejection dated 08/21/2024 in which the examiner provided a 103 rejection using Martyn et al. and ‘705 on pages 15-20, pertaining to the claim limitations of the DNA-targeting endonuclease cleaving the genomic DNA of the cell between positions -198 and -197 in the HBG1 promoter, and the gRNA comprising SEQ ID NO: 2. Therefore, the examiner has already provided art rejections on the claims as amended regarding the cleavage between the specific positions and the guide RNA of SEQ ID NO: 2.
The examiner has also provided new art rejections using other references for the claims as amended based on the provided claim interpretation.
Specification
The use of the term “TALEN”, which is a trade name or a mark used in commerce, has been noted in this application (see pages 7-9, etc.). 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
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 1,2,6-13,15,16 and 20-25 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.
Claims 1,6,12 and 15 contain the trademark/trade name “TALEN”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a DNA-targeting nuclease which is a transcription activator-like effector nuclease and, accordingly, the identification/description is indefinite.
Claims 2,7-11,13,16 and 20-25 are included in the rejection as they depend on the above claims reciting the trademark “TALEN” and do not correct the issue.
35 U.S.C. 112(a) New Matter Rejection
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 1,2,6-11,16,20 and 23 introduce new matter as the claims recite the limitation: "wherein cleavage of the genomic DNA between positions -198 and -197 leads to an insertion or deletion (InDel) of at least 1 base pair at the positions -196 and -195 of the HBG1 or HBG2 promoter”. There is no support in the specification for this limitation that cleavage of the genomic DNA between positions -198 and -197 would lead to an InDel of at least one base pair at positions -196 and -195. The limitation of cleavage of the genomic DNA between the positions -198 and -197 would lead to an InDel of at least one base pair at positions -196 and -195 was not described in the specification as filed, and person skilled in the art would not recognize in the applicant' s disclosure a description of the invention as presently claimed. The specification does not describe the positions at which the InDel occurs when cleavage of the genomic DNA between positions -198 and -197 occurs, and does not describe that this leads to an InDel of at least one base pair at the positions -196 and -195 of the HBG1 or HBG2 promoter as instantly recited. While Applicant points to paragraphs [0040] and [0102] as describing deletions and insertions in -197-edited samples, and paragraphs [0082-0083] that InDel events were detected, this does not state the positions at which the InDels occur, much less that it occurs at positions -196 and -195. Applicant has not pointed to where the specification provides support that the InDel occurs at positions -196 and -195 as a result of cleavage of the genomic DNA between positions -198 and -197. MPEP 2163.06 states: “Applicant should therefore specifically point out the support for any amendments made to the disclosure.” Applicant has not directed the Examiner to the support in the specification for this limitation as recited. Therefore, it is the Examiner' s position that the disclosure does not reasonably convey that the inventor had possession of the subject matter of the amendment at the time of filing of the instant application. The examiner believes this may have been an oversight or mistake regarding leaving positions -196 and -195 as recited since the other positions were amended to positions -198 and -197, however is making the new matter rejection in the interest of compact prosecution.
Claim Interpretation
Regarding claims 1,2,6-13,15,16 and 20-25, which recite contacting the eukaryotic cell or population of eukaryotic cells with an effective amount of one or more guide RNAs and a DNA-targeting endonuclease whereby the DNA-targeting endonuclease cleaves the genomic DNA of the cell between positions -198 and -197 in the HBG1 or HBG2 promoter wherein positions -198 and -197 correspond to positions 13 and 14 in SEQ ID NO: 1; wherein cleavage of the genomic DNA between the positions -198 and -197 leads to an insertion or deletion (InDel) of at least 1 base pair at the positions -196 and -195 of the HBG1 or HBG2 promoter, and thereby disrupts a binding site for LRF in the HBG1 or HBG2 promoter, and wherein a resulting mutation caused by the InDel is not a substitution mutation, the examiner is interpreting all of the above recited “wherein clauses” to be functional limitations that would result by contacting the eukaryotic cell with the one or more guide RNAs having the sequence as set forth in SEQ ID NO: 2 (claims 10,11 and 20-22) and a DNA targeting endonuclease. Therefore, any guide RNA that has the identical sequence to that of instant SEQ ID NO: 2, would have the function of recruiting the DNA-targeting endonuclease to the HBG1 and HBG2 promoters and generate double-strand breaks between positions -198 and -197 which correspond to positions 13 and 14 in SEQ ID NO: 1, and would result in an InDel of at least 1 base pair at positions -198 and -197 of the HBG1 or HBG2 promoter, as well as the functional limitations of disrupting a binding site for LRF in the HBG1 or HBG2 promoter, and wherein a resulting mutation caused by the InDel is not a substitution mutation, as well as the InDel limitations in claims 23-25. Therefore, art that teaches contacting the recited eukaryotic cell(s) with an effective amount of one or more guide RNAs comprising a sequence as set forth in SEQ ID NO: 2 and a DNA-targeting endonuclease, would read on the instant claim limitations.
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 1,2,7-13,15 and 20-25 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Mickanin et al. (US 20200102561, effectively filed 06 Feb 2017).
See claim interpretation above.
Regarding claims 1,7-10 and 20, Mickanin et al. teach a gRNA molecule wherein the targeting domain comprises any one of SEQ ID NOs: 1-72 (Embodiment 2, paragraph 1004). SEQ ID NO: 40 of Mickanin et al. is 20 nucleotides in length and has 100% identity to the gRNA of instant SEQ ID NO: 2. See alignment below:
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Mickanin et al. teach a method of altering a cell (e.g. a population of cells) at or near a target sequence within said cell comprising contacting (e.g. introducing into) said cell with one or more gRNA molecules of any of the embodiments 1-68 and a Cas9 molecule (Embodiment 115, paragraphs 1334-1335), and teaches the method of any of embodiments 115-118, wherein the cell is an animal cell, mammalian cell, primate or human cell (Embodiments 119-120, paragraphs 1346-1347), and the method of any of embodiments 115-126 wherein altering results in an indel at or near a genomic DNA sequence complementary to the targeting domain of the one or more gRNA molecules (Embodiment 127, paragraph 1355).
Regarding claim 2, Mickanin et al. teach the method of embodiment 120, wherein the cell is a hematopoietic or progenitor cell (Embodiment 121, paragraph 1348).
Regarding claim 11, Mickanin et al. teach a gRNA molecule of any of embodiments 1-68 wherein when a CRISPR system (e.g., an RNP as described herein) comprising the gRNA molecule is introduced into a cell, an indel is formed at or near the target sequence complementary to the targeting domain of the gRNA molecule (Embodiment 69, paragraph 1253).
Regarding claims 12,13,15, 21 and 22, Mickanin et al. teach a method of increasing fetal hemoglobin expression in a mammal, comprising administering to a patient a cell of any of embodiments 134-156 (Embodiment 171, paragraph 1412). Embodiment 134 recites a cell altered by the method of any of embodiments 115-133 which as stated above includes using the gRNA of SEQ ID NO: 40. Mickanin et al. teach a method of treating a hemoglobinopathy comprising administering to a patient a cell of any of embodiments 134-156 (Embodiment 170, paragraph 1411), wherein the hemoglobinopathy is beta-thalassemia or sickle cell disease (Embodiment 172, paragraph 1413). Mickanin et al. teach a method for treating a hemoglobinopathy, e.g., sickle cell disease or beta-thalassemia, or a method for increasing fetal hemoglobin expression in a mammal, e.g., a human, in need thereof, the method comprising: contacting the population of HSPCs (e.g., CD34+ cells) with an effective amount of: a composition comprising at least one gRNA molecule comprising a targeting domain described herein, e.g., a targeting domain described in Table 1, or a nucleic acid encoding said gRNA molecule, and at least one cas9 molecule, e.g., described herein, or a nucleic acid encoding said cas9 molecule, e.g., one or more RNPs as described herein, e.g., with a CRISPR system described herein; f) returning a population of cells comprising said modified HSPCs (e.g., CD34+ cells) to the mammal (paragraphs 0823-0828).
Regarding claim 23-25, Mickanin et al. teach the method of embodiment 128 wherein the indel is a single nucleotide deletion (Embodiment 129, paragraph 1358).
See above claim interpretation regarding the wherein clauses. In addition, although the reference is silent about the cleavage of the genomic DNA between positions -198 and -197 in the HBG1 or HBG2 promoter wherein positions -198 and -197 correspond to positions 13 and 14 in SEQ ID NO: 1, and wherein cleavage between these positions leads to an InDel of at least one base pair at these positions of the HBG1 or HBG2 promoter and thereby disrupts a binding site for LRF and wherein a resulting mutation caused by the InDel is not a substitution mutation, it does not appear that the claim language or limitations result in a manipulative difference in the method steps when compared to the prior art disclosure. See Bristol-Myers Squibb Company v. Ben Venue Laboratories, 58 USPQ2d 1508 (CAFC 2001). “It is a general rule that merely discovering and claiming a new benefit of an old process cannot render the process again patentable.” In re Woodruff, 16 USPQ2d 1934, 1936 (Fed. Cir. 1990). Granting a patent on the discovery of an unknown but inherent function would remove from the public that which is in the public domain by virtue of its inclusion in, or obviousness from, the prior art. In re Baxter Travenol Labs, 21 USPQ2d 1281 (Fed. Cir. 1991). See M.P.E.P. 2145. On this record, it is reasonable to conclude that the same cells and the same subject is being administered the same agents by the same step of contacting the cells and administering to the subject the population of cells, in both the instant claims and the prior art reference. The fact that Applicant may have discovered yet another beneficial effect from the method set forth in the prior art does not mean that they are entitled to receive a patent on that method.
Thus, Mickanin et al teach, either expressly or inherently implied, each and every limitation of the instant claims above.
Claims 1,7-10,20 and 23 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by WO 2017160890, hereinafter “’890”, Published 21 Sept 2017, cited in a previous office action.
See claim interpretation above.
Regarding claims 1,7-10,20 and 23, ‘890 recites a method of altering a cell comprising contacting said cell with a gRNA molecule of any one of claims 1-33 and an RNA-guided nuclease (claim 194), further comprising contacting said cell with a second gRNA molecule comprising a targeting domain comprising a nucleotide sequence complementary or partially complementary with a target domain located wholly or partially within an HBG1 or HBG1 regulatory region (claim 195), wherein the RNA-guided nuclease is a Cas9 molecule (claim 196), wherein the second gRNA molecule is a gRNA molecule of any one of claims 1-33. ‘890 recites a gRNA molecule comprising a targeting domain comprising a nucleotide sequence complementary or partially complementary with a target domain located wholly or partially within an HGB1 or HBG2 regulatory region (claim 1), and claim 8 recites the transcriptional regulatory element is a promoter, and claim 12 recites the gRNA molecule wherein the targeting domain comprising a nucleotide sequence identical to the nucleotide sequence set forth in any of SEQ ID NOs: 251-901. The gRNA molecule comprising the targeting domain of SEQ ID NO: 354 of ‘890 is a 100% match to instant SEQ ID NO: 2.
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Regarding the eukaryotic cell, claim 212 recites the cell is from a subject suffering from a beta-hemoglobinopathy, and claim 214 recites the cell is an erythroid cell, and therefore recites the cell being contacted is a eukaryotic cell. Claim 218 of ‘890 recites the method comprises introducing an indel into an HBG target position.
See above claim interpretation regarding the wherein clauses. In addition, although the reference is silent about the cleavage of the genomic DNA between positions -198 and -197 in the HBG1 or HBG2 promoter wherein positions -198 and -197 correspond to positions 13 and 14 in SEQ ID NO: 1, and wherein cleavage between these positions leads to an InDel of at least one base pair at these positions of the HBG1 or HBG2 promoter and thereby disrupts a binding site for LRF and wherein a resulting mutation caused by the InDel is not a substitution mutation, it does not appear that the claim language or limitations result in a manipulative difference in the method steps when compared to the prior art disclosure. See Bristol-Myers Squibb Company v. Ben Venue Laboratories, 58 USPQ2d 1508 (CAFC 2001). “It is a general rule that merely discovering and claiming a new benefit of an old process cannot render the process again patentable.” In re Woodruff, 16 USPQ2d 1934, 1936 (Fed. Cir. 1990). Granting a patent on the discovery of an unknown but inherent function would remove from the public that which is in the public domain by virtue of its inclusion in, or obviousness from, the prior art. In re Baxter Travenol Labs, 21 USPQ2d 1281 (Fed. Cir. 1991). See M.P.E.P. 2145. On this record, it is reasonable to conclude that the same cells are being contacted with the same agents by the same step of contacting the cells with said agents, in both the instant claims and the prior art reference. The fact that Applicant may have discovered yet another beneficial effect from the method set forth in the prior art does not mean that they are entitled to receive a patent on that method.
Thus, ‘890 teaches, either expressly or inherently implied, each and every limitation of the instant claims above.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 2,11-13,15,16,21,22,24 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over ‘890 as applied to claims 1,7-10,20 and 23 above.
See above claim interpretation.
The teachings of ‘890 as applicable to claims 1,7-10,20 and 23 have been described above.
‘890 does not explicitly teach contacting hematopoietic progenitor cells, hematopoietic stem cells or pluripotent cells with the guide RNA of SEQ ID NO: 354, or that the CRISPR-associated endonuclease is pre-complexed with one or more guide RNA to form a ribonucleoprotein (RNP) complex.
However, ‘890 teaches Cas9 molecules and gRNA molecules can be used to alter a target nucleic acid (e.g. HBG1, HBG2) regulatory region in a wide variety of cells, and alteration of a target nucleic acid in a target cell may be performed in vitro, ex vivo, or in vivo (page 125). ‘890 teaches target cells include hematopoietic stem/progenitor cell (page 126), an embryonic stem cell or induced pluripotent stem cell (page 127).
‘890 teaches a Cas9 RNP containing gRNA targeting HPFH mutation which supports gene editing in human hematopoietic stem/progenitor cells (Example 2, page 158).
‘890 recites a method of treating a beta-hemoglobinopathy selected from SCD and beta-Thal in a subject in need thereof, comprising contacting the subject or cell from the subject with a gRNA molecule of any one of claims 1-33 and an RNA-guided nuclease which is Cas9 (claims 257-259). ‘890 teaches the target cell is manipulated ex vivo by editing a gamma-globin gene regulatory region, then the target cell is administered to the subject (page 126).
Therefore, it would be obvious to one of ordinary skill in the art to apply the method of ‘890 using a gRNA of SEQ ID NO: 354 and the Cas9 endonuclease to contact hematopoietic progenitor cells, hematopoietic stem cells, pluripotent cells including embryonic stem cells or induced pluripotent stem cells for the purpose of modifying the cells ex vivo for later administration for treatment. One of ordinary skill in the art would be motivated to contact the gRNA of SEQ ID NO: 354 and Cas9 endonuclease with hematopoietic progenitor cells, hematopoietic stem cells, pluripotent cells including embryonic stem cells or induced pluripotent stem cells because ‘890 teaches that Cas9 molecules and gRNA molecules can be used to altered a target nucleic acid (HBG1 or HBG2 regulatory region) in a wide variety of cells, including a hematopoietic stem/progenitor cell (page 126), an embryonic stem cell or induced pluripotent stem cell (page 127) and that these modified cells generated ex vivo can be administered to a subject (page 127), and would make obvious the limitations of claims 2 and 16.
It would be obvious to one of ordinary skill in the art at the time of the effective filing date, that the CRISPR-associated nuclease is pre-complexed with one or more gRNA to form a ribonucleoprotein (RNP) complex for the purpose of increasing thermostability. One of ordinary skill in the art would be motivated to provide the CRISPR-associated endonuclease and guide RNA of SEQ ID NO: 354 in the form of an RNP complex, as ’890 teaches that Cas9 RNP containing gRNA targeting the HPFH mutation supports gene editing in cells (Example 2 page 158), and would make obvious the limitations of claim 11.
It would be obvious to one of ordinary skill in the art at the time of the effective filing date, to use the Cas9 endonuclease and gRNA of SEQ ID NO: 354 of ‘890 to contact a population of hematopoietic progenitor cells, HSCs or pluripotent cells and administer to the subject a therapeutically effective amount of the population of cells, because ‘890 teaches the alteration of a target nucleic acid in a target cell may be performed ex vivo, or in vivo (page 125), and recites a method of treating a beta-hemoglobinopathy in a subject, including sickle-cell disease or beta-thalassemia by contacting the subject or a cell from the subject with a gRNA molecule and an RNA-guided nuclease which is a Cas9 molecule (claim 259), and would make obvious the limitations of claims 12,13,15,21,22,24 and 25.
Therefore, the invention as a whole would have been prima facie obvious at the time of the effective filing date.
Claims 1,2,6-13,15,16 and 20-25 are rejected under 35 U.S.C. 103 as being unpatentable over Martyn et al. (Nature Genetics, Vol. 50, 2018, pages 498-503) and US 20150056705 (‘705), as cited in the previous Office Action mailed 07/03/2025.
See above claim interpretation.
Regarding claim 1, Martyn et al. teach that some forms of hereditary persistence of fetal hemoglobin (HPFH) in which individuals express the gamma-globin gene throughout adulthood, are caused by point mutations in the gamma-globin gene promoter at regions residing -115 and 200 bp upstream of the transcription start site and that the major fetal globin gene repressor, ZBTB7A (also known as LRF) directly bound to the sites at -115 and 200 bp (Abstract). Martyn et al. teach that HPFH-associated point mutations are located in the proximal promoters of the duplicated gamma-globin genes, HBG1 and HBG2 (page 498, left column). Regarding the step in claim 1 of contacting the eukaryotic cell with an effective amount of a DNA-targeting endonuclease selected from the group consisting of a TALEN, ZNF or CRISPR-associated endonuclease whereby the DNA-targeting endonuclease cleaves the genomic DNA of the cell between positions -196 and -195 in the HBG1 or HBG2 promoter, as well as claims 7 and 8, Martyn et al. teach the introduction of hereditary persistence of fetal hemoglobin (HPFH)-associated mutations in a human erythroid cell line HUDEP2, using CRISPR-Cas9 genome editing, by introducing homozygous HPFH-associated mutation c.-195 C>G (page 498, right column; Fig 3A) and that these positions are located upstream of the gamma-globin gene in a region bound by LRF (Figure 5). Martyn et al. found that gamma-globin mRNA and HbF levels were elevated in the clonal populations with this mutation (page 498, right column; Fig 3A). Martyn et al. teach the CRISPR-Cas9 sgRNA target sites for introducing the c.-195C>G mutation into HUDEP-2 cells (Fig 3A). Martyn et al. teach that the cluster at -200bp of HPFH-associated mutations disrupts the binding of the zinc fingers of ZBTB7A (Figure 1e), and that the mutations fall into approximately 115 and 200bp upstream of the gamma-globin gene transcriptional start site (page 498, left column, Figure 1A).
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Regarding claim 9, Martyn et al. teach CRISPR-Cas9 sgRNA target sites for introducing the c.-195C>G mutation into HUDEP-2 cells (Fig 3A).
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Martyn et al. does not teach that the DNA-targeting endonuclease cleaves the genomic DNA of the cell between positions -198 and -197 in the HBG1 or HBG2 promoter wherein positions -198 and -197 correspond to positions 13 and 14 in SEQ ID NO: 1, the guide RNA sequence as set forth in SEQ ID NO: 2, that the eukaryotic cell is a hematopoietic progenitor cell, hematopoietic stem cell and pluripotent cell, or that the pluripotent cells are embryonic stem cells or induced pluripotent stem cells, or increasing fetal hemoglobin or treating a hemoglobinopathy in a subject.
However, before the effective filing date, ‘705 teaches genome engineering by targeted nuclease-mediated genome cleavage at a desired location such as a double-stranded break created by a site-specific nuclease such as a zinc-finger nuclease (ZFN) or TAL effector domain nuclease (TALEN) ([0009]) as well as a CRISPR/Cas system that binds to a target site in a region of interest in an endogenous gene [0016,0017]. ‘705 teaches a method of modifying an endogenous gene which may be a beta globin gene (HBB) or gamma globin gene (HBG1) [0022], and methods for genome engineering to treat a disease including hemoglobinopathies [0041]. ‘705 teaches the target genes include genes involved in hemoglobinopathies and that genetic defects in sequences encoding hemoglobin chains can be responsible for hemoglobinopathies including sickle cell anemia and thalassemia [0042,0043]. ‘705 teaches correction of the human HBB gene that encodes beta globin can be accomplished with the CRISPR/Cas system of the invention [0046]. ‘705 teaches the methods in eukaryotic cells which are embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells [0023].
‘705 also teaches an approach for treating SCD and beta thalassemia is to increase expression of the gamma-globin with the aim to have HbF functionality replace aberrant adult hemoglobin ([0047]) as well as an approach to increase expression of HbF by identifying genes whose products play a role in regulation of gamma-globin expression [0048]. ‘705 teaches sgRNAs used with the CRISPR/Cas system [0228].
‘705 teaches Cas9 [0229] and an sgRNA of SEQ ID NO: 157 (Table 1, page 31), having a length of 23 nucleotides and which is 100% identical to the guide RNA of instant SEQ ID NO: 2. See alignment below wherein Qy is SEQ ID NO: 2 and Db is SEQ ID NO: 157:
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Martyn et al. with the teachings of ‘705 using a gRNA of SEQ ID NO: 157 of ‘705 in order to increase HbF levels in embryonic stem cells, induced pluripotent stem cells or hematopoietic stem cells. There would be a reasonable expectation of success because the structure of the guide RNA of SEQ ID NO: 157 of ‘705 is identical to the guide RNA of instant SEQ ID NO: 2, and would therefore perform the same function of recruiting the CRISPR associated endonuclease to the HBG1 or HBG2 promoters and generating double-strand breaks between positions -198 and -197 which correspond to positions 13 and 14 of instant SEQ ID NO: 1, and leading to an Indel of at least 1 bp at the positions -198 and -197 of the HBG1 or HBG2 promoter, thereby disrupting a binding site for LRF in the HBG1 or HBG2 promoter and wherein a resulting mutation caused by the Indel is not a substitution mutation. One of ordinary skill in the art would be motivated to use a gRNA of SEQ ID NO: 157 to cleave the genomic DNA between positions -198 and -197 in the HBG1 or HBG2 promoter in embryonic stem cells, induced pluripotent stem cells or hematopoietic stem cells because Martyn et al. teach that hereditary persistence of fetal hemoglobin (HPFH)-associated mutations at positions located upstream of the gamma-globin gene in the HBG1 and HBG2 promoter region bound by LRF (Figure 5a) were associated with elevated HbF in cell populations with the HPFH-associated mutations, and that the cluster at -200bp of HPFH-associated mutations disrupts the binding of the zinc fingers of ZBTB7A (LRF) (Figure 1e). One of ordinary skill in the art would be motivated to provide the CRISPR-associated endonuclease and guide RNA of SEQ ID NO: 157 in the form of an RNP complex, as ’705 teaches Crispr-associated endonuclease with gRNA targeting an HBG1 gene (pages 73-74). Modifying the method of Martyn et al. with the teachings of ’705 using a gRNA of SEQ ID NO: 157 would make obvious the limitations of claims 1,2,7-11,16, 20 and 23.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Martyn et al. with the teachings of ‘705 using a TALEN or ZFN as the DNA targeting nuclease and gRNA of SEQ ID NO: 157 with a reasonable expectation of success. There would be a reasonable expectation of success because the structure of the guide RNA of SEQ ID NO: 157 of ‘705 is identical to the guide RNA of instant SEQ ID NO: 2, and would therefore perform the same function of recruiting the TALEN or ZFN to the HBG1 and HBG2 promoters and cleave the genomic DNA of the cell between positions -198 and -197 which correspond to positions 13 and 14 of instant SEQ ID NO: 1, and forming an Indel of at least 1 bp, thereby disrupting a binding site for LRF in the HBG1 or HBG2 promoter and wherein a resulting mutation caused by the Indel is not a substitution mutation. One of ordinary skill in the art would have been motivated to use a TALEN or ZFN as the DNA-targeting endonuclease in the method, because ‘705 teaches genome engineering by targeted nuclease-mediated genome cleavage at a desired location such as a double-stranded break created by a site-specific nuclease such as a zinc-finger nuclease (ZFN) or TAL effector domain nuclease (TALEN), and is therefore a known and obvious way to use TALEN and ZNFs, and would make obvious the limitations of claim 6.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Martyn et al. with the teachings of ‘705 using a gRNA of SEQ ID NO: 157 and contact the embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells of ‘705 with the gRNA of SEQ ID NO: 157 and the CRISPR-associated nuclease and administering said cells to a subject, for the purpose of treating a hemoglobinopathy in a subject by increasing HbF levels. There would be a reasonable expectation of success because the structure of the guide RNA of SEQ ID NO: 157 of ‘705 is identical to the guide RNA of instant SEQ ID NO: 2, and would therefore perform the same function of recruiting the CRISPR associated endonuclease to the HBG1 and HBG2 promoters and generating double-strand breaks between positions -198 and -197 which correspond to positions 13 and 14 of instant SEQ ID NO: 1, as well as resulting in forming an insertion or deletion of at least 1 base pair, including a 1-bp insertion, a 1-bp deletion or a 2-bp deletion, and would also result in disrupting a binding site for LRF in the HBG1 or HBG2 promoter, and the resulting mutation caused by the Indel would not be a substitution mutation. One of ordinary skill in the art would have been motivated to do so because Martyn et al. teach that hereditary persistence of fetal hemoglobin (HPFH)-associated mutations at positions located upstream of the gamma-globin gene in the HBG1 and HBG2 promoter region bound by LRF (Figure 5a) were associated with elevated HbF in cell populations with the HPFH-associated mutations, and that the cluster at -200bp of HPFH-associated mutations disrupts the binding of the zinc fingers of ZBTB7A (LRF) (Figure 1e) and ‘705 teaches genome editing using CRISPR-Cas systems in the HBG1 gene for treatment of hemoglobinopathies including sickle cell anemia and thalassemia [0042,0043] and ways to increase expression of HbF. Modifying the method of Martyn et al. with the teachings of ‘705 using a gRNA of SEQ ID NO: 157 would make obvious the limitations of claims 12,13,15,21,22,24 and 25.
Therefore, the invention as a whole would have been prima facie obvious before the time of the effective filing date.
Conclusion
Claims 1,2,6-13,15,16 and 20-25 are rejected.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636