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 .
Detailed Action
This action is in response to the papers filed September 17, 2025.
Election/Restrictions
Applicant’s reply filed 09/17/2025 to the Requirement for Restriction/Election mailed 07/18/2025 is acknowledged.
Applicant elected without traverse:
the invention of Group 1, drawn to a method of making a cell having a genetically modified CCR5 locus;
mucopolysaccharidosis type 1, as the lysosomal storage disorder;
iduronidase, as the therapeutic protein;
a phosphoglycerate kinase (PGK) promoter, as the promoter; and
hematopoietic stem and progenitor cell (HSPC), as the cell type.
Claims 20-27, 37, 43-44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 09/17/2025.
Claim Listing
The instant claim listing was filed on 08/18/2023.
Claims 1, 3, 6-9, 14, 16-27, 37, 43-44 are pending.
Claims 20-27, 37, 43-44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Claims 1, 3, 6-9, 14, 16-19 are under examination.
Priority
The instant application 18/010,773 was filed on 12/15/2022. This application is a national stage of international application PCT/US21/39206 filed 06/25/2021, claiming priority based on U.S. Provisional Patent Application 63/044,951 filed 06/26/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 09/17/2025 has been considered.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, or by applicant in an information disclosure statement (IDS), they have not been considered.
Sequence Compliance
The instant application was filed on or after 07/01/2022, and therefore the instant application is subject to the requirements of 37 C.F.R. 1.831 through 1.835.
The sequence rules embrace all nucleotide and amino acid sequences defined by:
(1) An unbranched sequence or linear region of a branched sequence containing 4 or more specifically defined amino acids, wherein the amino acids form a single peptide backbone; or
(2) An unbranched sequence or linear region of a branched sequence of 10 or more specifically defined nucleotides, wherein adjacent nucleotides are joined by:
(i) A 3' to 5' (or 5' to 3') phosphodiester linkage; or
(ii) Any chemical bond that results in an arrangement of adjacent nucleobases that mimics the arrangement of nucleobases in naturally occurring nucleic acids (i.e., nucleotide analogs).
Disclosed nucleotide or amino acid sequences that do not meet this definition must not be included in the Sequence Listing.
See 37 C.F.R. 1.831, and see guidance of WIPO ST.26 and MPEP 2412-2419.
Appropriate action is required so that the sequences disclosed in the application comply with the sequence rules. Examples found in the application which fail to comply with the sequence rules include, for example, Table 1 on page 48, and paragraphs 159, 161, 165, 180, 181, 210, 212, 213 and 214.
Applicant is reminded that a sequence containing fewer than 10 specifically defined nucleotides or fewer than 4 specifically defined amino acids must not be included in the Sequence Listing. See 37 C.F.R. 1.831(j).
Applicant should carefully review the entire specification to ensure compliance with the sequence rules. Applicant should provide a corresponding sequence identifier (SEQ ID NO) with every appearance of a sequence embraced by the sequence rules. Where a sequence is presented in a drawing, reference must be made to the sequence by use of the sequence identifier, either in the drawing or in the Brief Description of the Drawings, where the correlation between multiple sequences in the drawing and their sequence identifiers in the Brief Description is clear. For guidance on amending the Sequence Listing, see MPEP 2414. Appropriate action is required in reply to this Office action. See attached PTO-2301.
Specification
The disclosure is objected to because of the following informalities:
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. See, for example, page 64, lines 9 and 19, and page 67, line 18. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Appropriate action is required.
Claim Objections
Claim 1 is objected to because of the following informalities:
In claim 1, line 7, the phrase “the genome” should be “the genome of the cell” to indicate that the “genome” refers to that of the cell recited in the preamble.
In claim 1, the phrase “the sequence shown as SEQ ID NO:3 or SEQ ID NO:4” should be “the sequence of SEQ ID NO:3” to indicate the sequence is that of SEQ ID NO:3 or SEQ ID NO:4 found in the Sequence Listing.
Appropriate action is required.
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.
Claim 14 is 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.
The limitation “the cells” in claim 14 lacks sufficient antecedent basis. In this case, the claims do not previously recite a plurality of cells. Rather, claim 1, only refers to a singular “cell.”
For these reasons, one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Changing the phrase “the cells” to “the cell” would be remedial.
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.
Claims 1, 3, 6, 8-9, 14, 16-17, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2019) “Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I” Nature communications, 10:4045, 14 pages; in view of Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989.
Gomez-Ospina discloses a method of genetically modifying a CD34+ hematopoietic stem and progenitor cell (HSPC) from a subject, the method comprising:
introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, a Cas9 nuclease, and a homologous donor template comprising a transgene encoding iduronidase (IDUA);
wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome of the cell comprising the sequence 5′-GCAGCATAGTGAGCCCAGAAGGG-3′ (identical to SEQ ID NO: 3; comprises the sequence of SEQ ID NO: 4, nucleotides 1-20), whereupon the nuclease cleaves the CCR5 locus at the target sequence;
wherein the homologous donor template comprises 500 bp left and right homology arms for the CCR5 locus flanking the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus;
wherein the integrated transgene directs the expression of IDUA in the cell;
wherein the sgRNA comprises chemical modifications at one or more nucleotides;
wherein the sgRNA and Cas9 nuclease are introduced into the cell as a ribonucleoprotein (RNP);
wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector; and
wherein the transgene is part of an expression cassette comprising the coding sequence for IDUA operably linked to a phosphoglycerate kinase (PGK) promoter.
See Abstract, Figure 1, and pages 2 and 10.
Gomez-Ospina further discloses that the genetically modified CD34+ HSPCs would be useful for treating a subject having mucopolysaccharidosis type I (MPS I), which is a common lysosomal storage disease (LSD) caused by insufficient IDUA activity. To this end, a mouse model of MPS I was developed, and genetically-modified, human HSPCs were transplanted into the mice. For clinical application, Gomez-Ospina suggests that the HSPCs should be autologous to the subject. See Abstract, Figure 3, and pages 2, 10-11. For these reasons, Gomez-Ospina is found to teach or fairly suggest (1) that the subject has MPS I and (2) IDUA is absent or deficient in the subject, as instantly claimed.
Gomez-Ospina does not teach that the first homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and the second homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof, as instantly claimed.
Prior to the effective filing date of the instantly claimed invention, Hendel is relevant prior art for teaching a method genetically modifying a CD34+ hematopoietic stem and progenitor cell (HSPC) from a subject, the method comprising:
introducing into a cell isolated from the subject a sgRNA targeting the CCR5 locus, a Cas9 nuclease, and a homologous donor template comprising a transgene;
wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome of the cell comprising the sequence 5′- GGCAGCATAGTGAGCCCAGAAGG-3′ (nucleotides 2-23 identical to nucleotides 1-22 of SEQ ID NO: 3; comprises the sequence of SEQ ID NO: 4, nucleotides 2-21), whereupon the nuclease cleaves the CCR5 locus at the target sequence;
wherein the homologous donor template comprises left and right homology arms for the CCR5 locus flanking the transgene, and the transgene is integrated into the genome by HDR at the site of the cleaved CCR5 locus;
wherein the integrated transgene directs the expression of an encoded polypeptide in the cell;
wherein the sgRNA comprises chemical modifications at one or more nucleotides;
wherein the sgRNA and Cas9 nuclease are introduced into the cell as an RNP;
wherein the homologous donor template is introduced into the cell using a vector; and
wherein the transgene is part of an expression cassette comprising the coding sequence for a polypeptide operably linked to a promoter.
See Abstract; pages 985 and 987; first page of Online Methods; Supplementary Tables 1, 3 and 5; Supplementary Note 1: CCR5 targeting vector sequence.
Hendel further provides the sequences for the left and right homology arms for the CCR5 targeting vector sequence in Supplementary Note 1. The left homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and the right homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof (see alignments below).
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina by using the left and right homology arms of Hendel with a reasonable expectation of success because Gomez-Ospina teaches inserting a donor template having left and right arms of homology with the CCR5 locus, and Hendel provides sequences suitable for the left and right homology arms targeting the same insertion site. As shown above, the left and right homology arms of Hendel read on the first and second homology arms instantly claimed.
For these reasons, claims 1, 3, 6, 8-9, 14, 16-17, and 19 would have been prima facie obvious over the cited references.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2019) “Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I” Nature communications, 10:4045, 14 pages; and Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, as applied to claims 1, 3, 6, 8-9, 14, 16-17, and 19 above; in further view of WO 2018/195555 A1 to Bak et al.
Regarding dependent claim 7, Gomez-Ospina and Hendel does not teach the sgRNA comprises a sequence having 95% or greater identity to SEQ ID NO: 5, as instantly claimed.
Prior to the effective filing date of the instantly claimed invention, Bak is relevant prior art for teaching a system for CRISPR/Cas9-mediated integration of a donor construct into a genomic locus of a cell, wherein the system comprises a sgRNA targeting the CCR5 locus. See, e.g., paragraphs 1-7.
Bak’s CCR5-targeting sgRNA according to SEQ ID NO: 1 comprises a sequence having 95% or greater identity to SEQ ID NO: 5, as instantly claimed. See alignment below.
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina to using the CCR5-targeting sgRNA of Bak with a reasonable expectation of success because Gomez-Ospina teaches a sgRNA targeting the CCR5 locus, and Bak provides a sgRNA sequence suitable for targeting the same genomic site. In particular, while Gomez-Ospina and Bak provide the same spacer sequence, Bak provides the spacer sequence and scaffold sequence. As shown above, the CCR5-targeting sgRNA of Bak reads on the sgRNA instantly claimed.
Claims 1, 3, 6, 8-9, 14, 16-17, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2019) “Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I” Nature communications, 10:4045, 14 pages; in view of WO 2020/227637 A1 to Patterson et al.
Gomez-Ospina discloses a method of genetically modifying a CD34+ hematopoietic stem and progenitor cell (HSPC) from a subject, the method comprising:
introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, a Cas9 nuclease, and a homologous donor template comprising a transgene encoding iduronidase (IDUA);
wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome of the cell comprising the sequence 5′-GCAGCATAGTGAGCCCAGAAGGG-3′ (identical to SEQ ID NO: 3; comprises the sequence of SEQ ID NO: 4, nucleotides 1-20), whereupon the nuclease cleaves the CCR5 locus at the target sequence;
wherein the homologous donor template comprises 500 bp left and right homology arms for the CCR5 locus flanking the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus;
wherein the integrated transgene directs the expression of IDUA in the cell;
wherein the sgRNA comprises chemical modifications at one or more nucleotides;
wherein the sgRNA and Cas9 nuclease are introduced into the cell as a ribonucleoprotein (RNP);
wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector; and
wherein the transgene is part of an expression cassette comprising the coding sequence for IDUA operably linked to a phosphoglycerate kinase (PGK) promoter.
See Abstract, Figure 1, and pages 2 and 10.
Gomez-Ospina further discloses that the genetically modified CD34+ HSPCs would be useful for treating a subject having mucopolysaccharidosis type I (MPS I), which is a common lysosomal storage disease (LSD) caused by insufficient IDUA activity. To this end, a mouse model of MPS I was developed, and genetically-modified, human HSPCs were transplanted into the mice. For clinical application, Gomez-Ospina suggests that the HSPCs should be autologous to the subject. See Abstract, Figure 3, and pages 2, 10-11. For these reasons, Gomez-Ospina is found to teach or fairly suggest (1) that the subject has MPS I and (2) IDUA is absent or deficient in the subject, as instantly claimed.
Gomez-Ospina does not teach that the first homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and the second homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof, as instantly claimed.
Prior to the effective filing date of the instantly claimed invention, Patterson is relevant prior art for teaching a method of genetically modifying a plurality of progenitor cells with a CRISPR/Cas system comprising a guide RNA (gRNA) targeting a genomic locus and a donor construct, wherein the donor construct is inserted by homologous recombination and the genomic locus is CCR5. See, e.g., paragraphs 8, 25.
Patterson further discloses that the donor construct comprises left and right homology arms for targeting the CCR5 locus. See, e.g., paragraphs 213-214. Patterson’s left homology arm according to SEQ ID NO: 14 comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and Patterson’s right homology arm according to SEQ ID NO: 15 comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof (see alignments below).
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina by using the left and right homology arms of Patterson with a reasonable expectation of success because Gomez-Ospina teaches inserting a donor template having left and right arms of homology with the CCR5 locus, and Patterson provides sequences suitable for the left and right homology arms targeting the same insertion site. As shown above, the left and right homology arms of Patterson read on the first and second homology arms instantly claimed.
For these reasons, claims 1, 3, 6, 8-9, 14, 16-17, and 19 would have been prima facie obvious over the cited references.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2019) “Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I” Nature communications, 10:4045, 14 pages; and WO 2020/227637 A1 to Patterson et al., as applied to claims 1, 3, 6, 8-9, 14, 16-17, and 19 above; in further view of US 2013/0045539 A1 to Delenda et al.; US 2012/0308544 A1 to Steinfeld, Robert; and US 2011/0312029 A1 to Enenkel, Barbara.
Regarding dependent claim 18, Gomez-Ospina does not teach the homologous donor template comprises a sequence having 95% or greater identity to SEQ ID NO: 7, as instantly claimed.
As defined on page 99 of the specification, SEQ ID NO: 7 is a 3711 bp construct comprising a left homology arm (1-500 bp; identical to SEQ ID NO: 1), a PGK promoter (507-995 bp), an IDUA cDNA (1002-2960 bp), a BgH Poly A (2987-3194 bp), and a right homology arm (3212-3711 bp; identical to SEQ ID NO: 2). Accordingly, the number of filler nucleotides is 55 nucleotides in the construct. A sequence having as little as 95% identity to SEQ ID NO: 7, as instantly claimed, may have up to 185 nucleotide changes relative to SEQ ID NO: 7.
As discussed above, Gomez-Ospina provides a donor construct comprising a PGK promoter operably linked to an IDUA cDNA and flanked by left and right homology arms, as instantly claimed. The difference between Gomez-Ospina’s disclosure and the invention claimed in claim 18 is, accordingly, a disclosure of the particular sequences constituting the donor construct. However, as shown below, one of ordinary skill in the could have constructed a donor construct comprising a sequence having as little as 95% identity to SEQ ID NO: 7, as instantly claimed, using sequences known in the art prior to the effective filing date of the instantly claimed invention.
First, Delenda is prior art and discloses a 507 bp murine PGK promoter (paragraph 59; SEQ ID NO: 14) that comprises the PGK promoter of SEQ ID NO: 7 (507-995 bp). See alignment below. The difference between Delenda’s PGK protomer and that of SEQ ID NO: 7 is an additional 18 nucleotides.
Second, Steinfeld is prior art and discloses an IDUA cDNA (Table 7 on pages 23-24; SEQ ID NO: 59) that is identical to the IDUA cDNA of SEQ ID NO: 7 (1002-2960 bp). See alignment below. There is no difference between Steinfeld’s IDUA cDNA and that of SEQ ID NO: 7.
Third, Enenkel is prior art and discloses an BgH Poly A (paragraph 56; SEQ ID NO: 12) that is identical to the BgH Poly A of SEQ ID NO: 7 (2987-3194 bp). See alignment below. There is no difference between Enenkel’s BgH Poly A and that of SEQ ID NO: 7.
Finally, as discussed above, Patterson provides a left homology arm (SEQ ID NO: 14, 552 bp) and right homology arm (SEQ ID NO: 15, 541 bp) that comprises a sequence identical to the left homology arm (1-500 bp) and right homology arm (3212-3711 bp) of SEQ ID NO: 7, respectively. The difference between the Patterson’s homology arms and those of SEQ ID NO: 7 is an additional 93 nucleotides.
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina by using the sequences for the PGK promoter, IDUA cDNA, BgH Poly A and homology arms, as taught by Delenda, Steinfeld, Enenkel and Patterson, respectively, with a reasonable expectation of success because Gomez-Ospina teaches a donor construct comprising an expression cassette having a PGK promoter operably linked to an IDUA cDNA and flanked by left and right homology arms, and the cited secondary references provide the sequences for constructing such a donor construct. This combination reads on a sequence having as little as 95% identity to SEQ ID NO: 7, as broadly claimed, because the total number of changes relative to SEQ ID NO: 7 would be at most 166 nucleotides (55 filler nucleotides + 18 additional nucleotides from PGK promoter + 93 additional nucleotides from homology arms), which is less than the maximum of 185 changes allowed with a sequence having as little as 95% identity to SEQ ID NO: 7.
Claims 1, 3, 6, 8-9, 14, 16-17, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2018, May) “Engineering the Hematopoietic System for Lysosomal Storage Disorders: Correction of Mucopolysaccharidosis Type I Using Genome-Edited, Human Hematopoietic Stem and Progenitor Cells” In MOLECULAR THERAPY, Vol. 26, No. 5S1, pp. 310-311, Abstract #673, 50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA: CELL PRESS; in view of Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989.
Gomez-Ospina discloses a method of genetically modifying a CD34+ hematopoietic stem and progenitor cell (HSPC) from a subject, the method comprising:
introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, a Cas9 nuclease, and a homologous donor template comprising a transgene encoding iduronidase (IDUA);
wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome of the cell, whereupon the nuclease cleaves the CCR5 locus at the target sequence;
wherein the homologous donor template comprises left and right homology arms for the CCR5 locus flanking the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus;
wherein the integrated transgene directs the expression of IDUA in the cell;
wherein the sgRNA and Cas9 nuclease are introduced into the cell as a ribonucleoprotein (RNP);
wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector; and
wherein the transgene is part of an expression cassette comprising the coding sequence for IDUA operably linked to a phosphoglycerate kinase (PGK) promoter.
See pages 310-311, bridging paragraph.
Gomez-Ospina further discloses that the genetically modified CD34+ HSPCs would be useful for treating a subject having mucopolysaccharidosis type I (MPS I), which is a common lysosomal storage disease (LSD) caused by insufficient IDUA activity. To this end, a mouse model of MPS I was developed, and genetically-modified, human HSPCs were transplanted into the mice. For clinical application, Gomez-Ospina suggests that the transplanted HSPCs should be autologous to the subject. See pages 310-311, bridging paragraph. For these reasons, Gomez-Ospina is found to teach or fairly suggest (1) that the subject has MPS I and (2) IDUA is absent or deficient in the subject, as instantly claimed.
Gomez-Ospina does not teach that the sgRNA target sequence comprises the sequence of SEQ ID NO: 3 or 4, the first homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and the second homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof, as instantly claimed.
Prior to the effective filing date of the instantly claimed invention, Hendel is relevant prior art for teaching a method genetically modifying a CD34+ hematopoietic stem and progenitor cell (HSPC) from a subject, the method comprising:
introducing into a cell isolated from the subject a sgRNA targeting the CCR5 locus, a Cas9 nuclease, and a homologous donor template comprising a transgene;
wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome of the cell, whereupon the nuclease cleaves the CCR5 locus at the target sequence;
wherein the homologous donor template comprises left and right homology arms for the CCR5 locus flanking the transgene, and the transgene is integrated into the genome by HDR at the site of the cleaved CCR5 locus;
wherein the integrated transgene directs the expression of an encoded polypeptide in the cell;
wherein the sgRNA comprises chemical modifications at one or more nucleotides;
wherein the sgRNA and Cas9 nuclease are introduced into the cell as an RNP;
wherein the homologous donor template is introduced into the cell using a vector; and
wherein the transgene is part of an expression cassette comprising the coding sequence for a polypeptide operably linked to a promoter.
See Abstract; pages 985 and 987; first page of Online Methods; Supplementary Tables 1, 3 and 5; Supplementary Note 1: CCR5 targeting vector sequence.
Hendel further provides the sequence of sgRNA target sequence in Supplementary Tables 1, 3 and 5 (5′- GGCAGCATAGTGAGCCCAGAAGG-3′). The sgRNA target sequence comprises the sequence of SEQ ID NO: 4, as instantly claimed (see alignment below).
Hendel also provides the sequences for the left and right homology arms for the CCR5 targeting vector sequence in Supplementary Note 1. The left homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 1 or a fragment thereof, and the right homology arm comprises a sequence having 95% or greater identity to SEQ ID NO: 2 or a fragment thereof, as instantly claimed (see alignments below).
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina by using the sgRNA target sequence and homology arms of Hendel with a reasonable expectation of success because Gomez-Ospina teaches a sgRNA for inserting a homologous donor construct into the CCR5 locus, and Hendel provides suitable sgRNA target and homology arm sequences for modifying the same insertion site. As shown above, the sgRNA target and homology arm sequences of Hendel read on those instantly claimed.
Regarding dependent claim 3, Hendel teaches the sgRNA comprises chemical modifications at one or more nucleotides. See, e.g., page 985; and Supplementary Table 1.
For these reasons, claims 1, 3, 6, 8-9, 14, 16-17, and 19 would have been prima facie obvious over the cited references.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2018, May) “Engineering the Hematopoietic System for Lysosomal Storage Disorders: Correction of Mucopolysaccharidosis Type I Using Genome-Edited, Human Hematopoietic Stem and Progenitor Cells” In MOLECULAR THERAPY, Vol. 26, No. 5S1, pp. 310-311, Abstract #673, 50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA: CELL PRESS; and Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, as applied to claims 1, 3, 6, 8-9, 14, 16-17, and 19 above; in further view of WO 2018/195555 A1 to Bak et al.
Regarding dependent claim 7, Gomez-Ospina and Hendel does not teach the sgRNA comprises a sequence having 95% or greater identity to SEQ ID NO: 5, as instantly claimed.
Prior to the effective filing date of the instantly claimed invention, Bak is relevant prior art for teaching a system for CRISPR/Cas9-mediated integration of a donor construct into a genomic locus of a cell, wherein the system comprises a sgRNA targeting the CCR5 locus. See, e.g., paragraphs 1-7.
Bak’s CCR5-targeting sgRNA according to SEQ ID NO: 1 comprises a sequence having 95% or greater identity to SEQ ID NO: 5, as instantly claimed. See alignment below.
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina to using the CCR5-targeting sgRNA of Bak with a reasonable expectation of success because Gomez-Ospina teaches a sgRNA targeting the CCR5 locus, and Bak provides a sgRNA sequence suitable for targeting the same genomic site. As shown above, the CCR5-targeting sgRNA of Bak reads on the sgRNA instantly claimed.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Gomez-Ospina et al. (2018, May) “Engineering the Hematopoietic System for Lysosomal Storage Disorders: Correction of Mucopolysaccharidosis Type I Using Genome-Edited, Human Hematopoietic Stem and Progenitor Cells” In MOLECULAR THERAPY, Vol. 26, No. 5S1, pp. 310-311, Abstract #673, 50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA: CELL PRESS; and Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, as applied to claims 1, 3, 6, 8-9, 14, 16-17, and 19 above; in further view of US 2013/0045539 A1 to Delenda et al.; US 2012/0308544 A1 to Steinfeld, Robert; US 2011/0312029 A1 to Enenkel, Barbara; and WO 2020/227637 A1 to Patterson et al.
Regarding dependent claim 18, Gomez-Ospina does not teach the homologous donor template comprises a sequence having 95% or greater identity to SEQ ID NO: 7, as instantly claimed.
As defined on page 99 of the specification, SEQ ID NO: 7 is a 3711 bp construct comprising a left homology arm (1-500 bp; identical to SEQ ID NO: 1), a PGK promoter (507-995 bp), an IDUA cDNA (1002-2960 bp), a BgH Poly A (2987-3194 bp), and a right homology arm (3212-3711 bp; identical to SEQ ID NO: 2). Accordingly, the number of filler nucleotides is 55 nucleotides in the construct. A sequence having as little as 95% identity to SEQ ID NO: 7, as instantly claimed, may have up to 185 nucleotide changes relative to SEQ ID NO: 7.
As discussed above, Gomez-Ospina provides a donor construct comprising a PGK promoter operably linked to an IDUA coding sequence and flanked by homology arms, as instantly claimed. The difference between Gomez-Ospina’s disclosure and the invention claimed in claim 18 is, accordingly, a disclosure of the particular sequences constituting the donor construct. However, as shown below, one of ordinary skill in the could have constructed a donor construct comprising a sequence having as little as 95% identity to SEQ ID NO: 7, as instantly claimed, using sequences known in the art prior to the effective filing date of the instantly claimed invention.
First, Delenda is prior art and discloses a 507 bp murine PGK promoter (paragraph 59; SEQ ID NO: 14) that comprises the PGK promoter of SEQ ID NO: 7 (507-995 bp). See alignment below. The difference between Delenda’s PGK protomer and that of SEQ ID NO: 7 is an additional 18 nucleotides.
Second, Steinfeld is prior art and discloses an IDUA cDNA (Table 7 on pages 23-24; SEQ ID NO: 59) that is identical to the IDUA cDNA of SEQ ID NO: 7 (1002-2960 bp). See alignment below. There is no difference between Steinfeld’s IDUA cDNA and that of SEQ ID NO: 7.
Third, Enenkel is prior art and discloses an BgH Poly A (paragraph 56; SEQ ID NO: 12) that is identical to the BgH Poly A of SEQ ID NO: 7 (2987-3194 bp). See alignment below. There is no difference between Enenkel’s BgH Poly A and that of SEQ ID NO: 7.
Finally, Patterson is prior art and teaches a method of genetically modifying a plurality of progenitor cells with a CRISPR/Cas system comprising a guide RNA (gRNA) targeting a genomic locus and a donor construct, wherein the donor construct is inserted by homologous recombination and the genomic locus is CCR5. See, e.g., paragraphs 8, 25. Patterson provides a left homology arm (SEQ ID NO: 14, 552 bp) and right homology arm (SEQ ID NO: 15, 541 bp) that comprises a sequence identical to the left homology arm (1-500 bp) and right homology arm (3212-3711 bp) of SEQ ID NO: 7, respectively. The difference between the Patterson’s homology arms and those of SEQ ID NO: 7 is an additional 93 nucleotides.
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the invention of Gomez-Ospina by using the sequences for the PGK promoter, IDUA cDNA, BgH Poly A and homology arms, as taught by Delenda, Steinfeld, Enenkel and Patterson, respectively, with a reasonable expectation of success because Gomez-Ospina teaches a donor construct comprising an expression cassette having a PGK promoter operably linked to an IDUA cDNA and flanked by left and right homology arms, and the cited secondary references provide the sequences for constructing such a donor construct. This combination reads on a sequence having as little as 95% identity to SEQ ID NO: 7, as broadly claimed, because the total number of changes relative to SEQ ID NO: 7 would be at most 166 nucleotides (55 filler nucleotides + 18 additional nucleotides from PGK promoter + 93 additional nucleotides from homology arms), which is less than the maximum of 185 changes allowed with a sequence having as little as 95% identity to SEQ ID NO: 7.
Duplicate Claims
Applicant is advised that should claim 1 be found allowable, claim 6 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Sequence Alignments
Alignment between SEQ ID NO: 1 of the claimed invention and the left homology arm found in Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, Supplementary Note 1: CCR5 targeting vector sequence.
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351
581
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Alignment between SEQ ID NO: 2 of the claimed invention and the right homology arm found in Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, Supplementary Note 1: CCR5 targeting vector sequence.
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584
605
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Alignment between SEQ ID NO: 4 of the claimed invention and the sgRNA target sequence found in Hendel et al. (2015) “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells” Nature biotechnology, 33(9), 985-989, Supplementary Tables 1, 3 and 5.
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118
584
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Alignment between SEQ ID NO: 5 of the claimed invention and SEQ ID NO: 1 of WO 2018/195555 A1 to Bak et al.
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209
704
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Alignment between SEQ ID NO: 1 of the claimed invention and SEQ ID NO: 14 of WO 2020/227637 A1 to Patterson et al.
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588
584
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Alignment between SEQ ID NO: 2 of the claimed invention and SEQ ID NO: 15 of WO 2020/227637 A1 to Patterson et al.
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587
585
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Alignment between nucleotides 507-995 of SEQ ID NO: 7 and SEQ ID NO: 14 of US 2013/0045539 A1 to Delenda et al.
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748
699
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Alignment between nucleotides 1002-2960 of SEQ ID NO: 7 and SEQ ID NO: 59 of US 2012/0308544 A1 to Steinfeld, Robert.
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898
688
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906
708
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751
717
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Alignment between nucleotides 2987-3194 of SEQ ID NO: 7 and SEQ ID NO: 12 of US 2011/0312029 A1 to Enenkel, Barbara.
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367
702
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Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAMES J GRABER whose telephone number is (571)270-3988. The examiner can normally be reached Monday-Thursday: 9:00 am - 4:00 pm.
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/JAMES JOSEPH GRABER/Examiner, Art Unit 1631