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 .
Claim Status
Claims 1-5, 10, 13-16, 18-20 are cancelled.
Claims 6-9, 11-12, 17, are amended.
Claims 26-32 are new.
Claims 6-9, 11-12, 17, 21-32 are examined on the merits.
Election/Restrictions
Applicant’s election without traverse of Group II corresponding to claims 6-9, 11-12, 17 and 21-25 in the reply filed on 10/15/2025 is acknowledged.
Priority
The applicant’s application is a U.S. National Stage application of PCT International Patent Application Serial No. PCT/EP2020/084489, filed December 03, 2020, which itself claims the benefit of European Patent Application Serial No. EP19214152.1, filed December 06, 2019 is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. See Figs. 1, 10, 11.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 112
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 7 and 24 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 7 is drawn to a product a fusion protein comprising a linker. The rejected claim comprising the fusion protein of claim 6, wherein said linker comprises or consists of an oligopeptide with an amino acid sequence selected from formula 1, formula 2 and formula 3: X1- X2- X3- X4- X5-X6- (G2S)4 - X7- X8- X9- Xio- X11-X12 (formula 1); (G2S)2 - X1- X2- X3- X4- X5-X6- X7- X8- X9- Xio- X11-X12 - (G2S)2 (formula 2); and X1- X2- X3- X4- X5-X6- X7- X8- X9- Xio- X11-X12- X13- X14- X15- X16- X17-X18- X19- X20- X21- X22- X23-X24 (formula 3); wherein G is glycine; S is serine; and each of Xi to X24 is independently selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glycine, lysine, serine and threonine; wherein said oligopeptide of formula 1, formula 2 or formula 3 does not consist of glycine and serine residues only.
The critical essential elements are a first and second recombinases and the linker. However, the negative limitation “wherein said oligopeptide of formula 1, formula 2 or formula 3 does not consist of glycine and serine residues only”, does not indicate the number, positions of other amino acids in the 3 oligopeptide formulas 1-3.
The specification envisions fusion of the recombinases with a linker comprising (G2S) repeats resulted in some cases in a decrease of recombination activity compared to the non-linked recombinases. Further experiments have shown that the length of the linker plays a significant role on the activity of the recombinases. Good results were achieved with a linker comprising eight (G2S) repeats. A further increase of the linker length did not improve recombination efficacy, however, decreased the specificity as the obtained fusion proteins showed activity on the symmetric sites of the recombinase target sites, which is not desired. Accordingly, in a preferred embodiment, the linker comprised in the fusion protein of the invention is most preferably an oligopeptide consisting of 24 amino acids (e.g., lane 10, page 26). The specification envisions further embodiment a fusion protein, wherein said linker comprises or consists of an oligopeptide selected from the group consisting of AEATSEGGSGGSGGSGGSNGARRT (SEQ ID NO: 8);
AGTTARGGSGGSGGSGGSGRRGAK (SEQ ID NO: 9);
KNGRGRGGSGGSGGSGGSRTKRET (SEQ ID NO: 10);
GGSGGSTAAKEGAASSASGGSGGS (SEQ ID NO: 11);
GGSGGSNSRSNTENSDKGGGSGGS (SEQ ID NO: 12);
GGSGGSNGEEGTERGKATGGSGGS (SEQ ID NO: 13);
GGSGGSTTKANRAKGGRGGGSGGS (SEQ ID NO: 14);
GANEDTNTEAAGSEGNEKTGTNSA (SEQ ID NO: 15); and
GESRAEDGAKGNGRGKGEATAGAA (SEQ ID NO: 16) (e.g., lane 1, page 32).
The working examples teach different linkers for fusing the F8 recombinase heterodimer were designed and tested. Flexible linkers with sequences (Gly-Gly-Ser)n and number of repeats (n) 2, 4, 6 and 8 were designed; showing from the results of the fusion with (G2S)2-s linkers, the length of the linker influences the recombination activity (e.g., lane 20, page 66; Example 3).
The application does not disclose any discussion identifying all-glycine/serine linkers as undesirable or excluded; and teaching that the linker must contain at least one amino acid other than glycine or serine; any examples demonstrating linkers containing non-glycine/serine residues; or any rationale linked to the invention indicating that glycine/serine are outside of the intended scope.
Additionally, the formulas in the claim encompass a number of possible linker sequences given 12 or 24 independently variable positions selected from 9 different amino acids, yet the disclosure contains no representative species demonstrating the inclusion of residues other than glycine and serine; the specification fails to show possession of the claimed negative limitation.
As such, the claim encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 7 and 24.
Claim 22 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 22 is drawn to a product a fusion protein. The rejected claim comprising at least a first and a second recombinase attached via a linker, wherein the first and second recombinase are engineered from the same naturally occurring source recombinase, wherein said first recombinase specifically recognizes a first genomic nucleic acid sequence target site of the recombinase and said second recombinase specifically recognizes a second genomic nucleic acid sequence target site of the recombinase. The critical essential elements are a first and second recombinases and the linker; however, the claim is not limited to any linker, the claim does not specify what type of linker.
The term “linker” is extremely broad, and encompass any type of linker, including flexible linkers, rigid linkers, peptide linkers of any length or composition, or non-peptide linkers, cleavable sequence, etc.
The specification envisions fusion protein for efficient and specific genome editing, comprising a complex of recombinases comprising at least a first recombinase enzyme, a second recombinase enzyme and at least one linker, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half site and a second half-site of an upstream target site and/or a downstream target site of a recombinase; wherein said first recombinase enzyme and said second recombinase enzyme are interconnected via a linker; and wherein said linker comprises or consists of an oligopeptide comprising 4 to 50 amino acids (e.g., lane 3, page 8). The specification envisions fusion of two heterospecific recombinases with specific linkers prevent the molecule from recombining the symmetric target sites, making the system obligate (e.g., lane 29, page 7). The specification envisions linker selection on the asymmetric loxF8 site. (A) Initial recombination activity of the heterodimer fused with three different linker libraries (libl-3) on the loxF8 site. 200 μg/ml L-arabinose was used for induction (e.g. lane 21, page 11; Fig. 14). The specification envisions a comparison of the recombination efficacy of the D7 heterodimer fused with (G2S)s and LS linkers on the loxF8 target site. Recombination activity of the recombinase heterodimer fused with (G2S)s or the LS linker on the loxF8 site (e.g., lane 25; Fig. 18). The specification envisions linker comprised in a fusion protein of the invention is in one embodiment an oligopeptide comprising repeats of (G2S) selected from the group consisting of GGSGGS (SEQ ID NO: l); GGSGGSGGSGGS (SEQ ID NO: 2); GGSGGSGGSGGSGGSGGS (SEQ ID NO: 3);
GGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 4);
GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 5);
GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 6); and GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 7) (lane 30, pages 25-26). The specification envisions fusion of the recombinases with a linker comprising (G2S) repeats resulted in some cases in a decrease of recombination activity compared to the non-linked recombinases. Further experiments have shown that the length of the linker plays a significant role on the activity of the recombinases. Good results were achieved with a linker comprising eight (G2S) repeats. A
further increase of the linker length did not improve recombination efficacy, however, decreased the specificity as the obtained fusion proteins showed activity on the symmetric sites of the recombinase target sites, which is not desired. Accordingly, in a preferred embodiment, the linker comprised in the fusion protein of the invention is most preferably an oligopeptide
consisting of 24 amino acids (e.g., lane 10, page 26). The specification envisions further embodiment a fusion protein, wherein said linker
comprises or consists of an oligopeptide selected from the group consisting of AEATSEGGSGGSGGSGGSNGARRT (SEQ ID NO: 8);
AGTTARGGSGGSGGSGGSGRRGAK (SEQ ID NO: 9);
KNGRGRGGSGGSGGSGGSRTKRET (SEQ ID NO: 10);
GGSGGSTAAKEGAASSASGGSGGS (SEQ ID NO: 11);
GGSGGSNSRSNTENSDKGGGSGGS (SEQ ID NO: 12);
GGSGGSNGEEGTERGKATGGSGGS (SEQ ID NO: 13);
GGSGGSTTKANRAKGGRGGGSGGS (SEQ ID NO: 14);
GANEDTNTEAAGSEGNEKTGTNSA (SEQ ID NO: 15); and
GESRAEDGAKGNGRGKGEATAGAA (SEQ ID NO: 16) (e.g., lane 1, page 32).
The working examples teach different linkers for fusing the F8 recombinase heterodimer were designed and tested. Flexible linkers with sequences (Gly-Gly-Ser)n and number of repeats (n) 2, 4, 6 and 8 were designed; showing from the results of the fusion with (G2S)2-s linkers, the length of the linker influences the recombination activity (e.g., lane 20, page 66; Example 3).
As such, the claim encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claim 22.
Claims 6-9, 11-12, 17, 22-32 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The dependent claim 11 is drawn to a genus of fusion protein wherein the first recombinase enzyme is a protein. The rejected claim comprise a genus consisted of an amino acid sequence with at least 70% sequence identity to the amino acid sequence as shown in with a sequence according to SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 93 or SEQ ID NO: 99. Dependent claim 12 is drawn to a genus of fusion protein consisting of a first recombinase enzyme and a second recombinase enzyme, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and/or a downstream target site of a recombinase, wherein said first recombinase enzyme is a polypeptide, which has an amino acid sequence with at least 70 % sequence identity with a sequence according to SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 93 or SEQ ID NO: 99 and/or wherein said second recombinase enzyme is a polypeptide, which has an amino acid sequence with at least 70 %, sequence identity with a sequence according to SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 94 or SEQ ID NO: 100. Dependent claim 28 is drawn to a genus of the fusion protein of claim 6, wherein said fusion protein has an amino acid sequence with at least 70 % sequence identity with a sequence according to SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 97 or SEQ ID NO: 103, wherein SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 97 and SEQ ID NO: 103 each include a linker with the amino acid sequence of SEQ ID NO: 14.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification describes the DNA recombinase, which consists of a heterodimer, wherein said heterodimer comprises or consists of a first recombinase enzyme and a second recombinase enzyme, wherein said first recombinase enzyme is a
polypeptide, which has an amino acid sequence with at least 70 %, preferably 80 %, more preferably 90 %, sequence identity with a sequence according to SEQ ID NO: 30 (recombinase 28-L) and wherein said second recombinase enzyme is a polypeptide, which has an amino acid sequence with at least 70 %, preferably 80 %, more preferably 90 %, sequence identity with a sequence according to SEQ ID NO: 31 (recombinase 28-R) (e.g., lane 6, page 36). The specification envisions the recombinase of SEQ ID NO: 30 has, compared to the Cre recombinase
protein of SEQ ID NO: 68, one or more, preferably two, three, for, five, six, eight, nine, ten or more mutations selected from the group consisting of N3D, L5Q, V7L, Pl2S, Pl5L, V23A, M30V, H40A, K43N, M44T, L58S, K62E, Y77H, A80V, K86N, Q90R, G93A, Q94L, Sl08G, Al31V, Kl32R, El50G, Nl51S, Cl55R, Q156N, 1166V, A175S, V182I, I195V, K219R,
D232G, T253S, S257T, R259D, A260V, E262R, E266V, T268A, I272V, Y273H, K276R, A285R, P307 A, N317T, N319E, l320S, N323S and l325L. In a most preferred embodiment, the recombinase of SEQ ID NO: 30 has all of the aforementioned mutations (e.g., lane 12, page 36).
The specification envisions a DNA recombinase, which is a
heterodimer, wherein the first monomer is recombinase enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 % sequence identity with a sequence according to SEQ ID NO. 32 recombinase D7-L) and wherein the second monomer is a recombinase
enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 %, sequence identity with a sequence according to SEQ ID NO. 33 (recombinase D7-R). Preferably, the DNA recombinase of SEQ ID NO: 32 comprises one or more mutations compared to the Cre recombinase protein of SEQ ID NO: 68. Exemplary mutations include 15 L5Q, V7L, Pl2S, Pl5L, Vl6A, Dl 7N, V23A, M30V, Q35R, H40A, M44T, S51 T, Y77H, A80V, K86N, Q90R, G93A, Q94L, Sl08G, NlllS, A131V, Kl32R, Ql44K, El50G, 1166V, Al 75S, Vl82I, K219R, E222G, D232G, R259D, A260V, E262R, I264V, E266A, T268A, I272V, A275T, R282G, A285T, P307A, N317T, N319E, l320S, N323S and l325L (e.g., lane 7, page 38).
The specification envisions a DNA recombinase, which is a
heterodimer, wherein the first monomer is recombinase enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 % sequence identity with a sequence according to SEQ ID NO. 93 (recombinase A4-L) and wherein the second monomer is a recombinase
enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 %, sequence identity with a sequence according to SEQ ID NO. 94 (recombinase A4-R). Preferably, the DNA recombinase of SEQ ID NO: 93 comprises one or more mutations compared to the Cre recombinase protein of SEQ ID NO: 68. Exemplary mutations include
N3S, L5Q, V7L, Pl2S, Pl5L, V23A, K25E, M28I, D29G, M30G, H40A, M44T, N60S, Y77H, A80V, K86N, Q90R, G93A, Q94L, Sl08G, A131V, Ql44R, 1166V, 1174V, Al75S, K211E, K219R, D232G, N257T, R259D, A260V, E262R, E266V, T268A, K276R, P307A, N317T, N319E, l320S, N323S and l325L. In a more preferred embodiment, the recombinase of SEQ ID NO: 93 has, compared to the Cre recombinase protein of SEQ ID NO: 68, one or more preferably two, three, for, five, six, eight, nine, ten or more mutations selected from the group consisting ofN3S, L5Q, V7L, Pl2S, Pl5L, V23A, K25E, M28I, D29G, M30G, H40A, M44T, N60S, Y77H, A80V, K86N, Q90R, G93A, Q94L, Sl08G, A131V, Ql44R, 1166V, 1174V, A175S, K211E, K219R, D232G, N257T, R259D, A260V, E262R, E266V, T268A, K276R, P307 A, N3 l 7T, N3 l 9E, l320S, N323S and l325L. In a most preferred embodiment, the recombinase of SEQ ID NO: 93 has all of the aforementioned mutations (e.g., lane 21, pages 40-41). The specification envisions a DNA recombinase, which is a heterodimer, wherein the first monomer is recombinase enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 % sequence identity with a sequence according to SEQ ID NO. 99 (recombinase Dll-L) and wherein the second monomer is a recombinase enzyme which has a sequence with at least 70 %, preferably 80 %, more preferably 90 %, sequence identity with a sequence according to SEQ ID NO. 100 (recombinase Dl 1-R).
Preferably, the DNA recombinase of SEQ ID NO: 99 comprises one or more mutations compared to the Cre recombinase protein of SEQ ID NO: 68. Exemplary mutations include L5Q, V71, Pl2T, Ll4S, Pl5L, Vl6A, V23A, M30V, F31L, H40A, M44T, S51T, L58S, Y77H, A80V, K86N, Q90R, G93A, Q94L, S108G, A131V, Kl32R, Fl42L, El50G, Nl51D, Ql56K, L164P, 1166V, A175S, V182I, K219R, A249V, R259D, A260V, E262R, E266A, A267T,
T268A, I272V, K276R, D278G, Y283F, A285T, P307A, N317T, N319G and l320S. In a more preferred embodiment, the recombinase of SEQ ID NO: 99 has, compared to the Cre recombinase protein of SEQ ID NO: 68, one or more, preferably two, three, for, five, six, eight, nine, ten or more mutations selected from the group consisting ofL5Q, V71, Pl2T, Ll4S, Pl5L,
Vl6A, V23A, M30V, F31L, H40A, M44T, S51T, L58S, Y77H, A80V, K86N, Q90R, G93A, Q94L, Sl08G, Al31V, Kl32R, Fl42L, El50G, Nl51D, Ql56K, Ll64P, 1166V, Al75S, Vl82I, K219R, A249V, R259D, A260V, E262R, E266A, A267T, T268A, I272V, K276R, D278G, Y283F, A285T, P307A, N317T, N319G and l320S. In a most preferred embodiment, the recombinase of SEQ ID NO: 99 has all of the aforementioned mutations (e.g., lane 31, pages 42-43).
No description is provided of any modifications of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 93, SEQ ID NO: 99, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 94, SEQ ID NO: 100, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 97 or SEQ ID NO: 103, with at least 70%, homology that retain the ability to perform recombination activity.
The examples described in the specification does not meet the limitation of the rejected claim (at least 70%, identity), the examples are only representative of SED ID NO 32 and 33. “At least 70 % identity” encompasses a very large number of different amino acid sequences (i.e., every sequence at least 70% identical to SEQ ID NO: 32), but there is insufficient guidance provided indicating any of the elements that are critical to the functioning of the polypeptide, thus it cannot be determined which amino acids can be changed without disrupting the function of the polypeptide; thus further experimentation would be required to determine which variants of SEQ ID NO: 32 are functional and which are not.
As such, the independent and dependent claims encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 6-9, 11-12, 17, 22-32.
The claims listed in the statement of rejection but not otherwise discussed are rejected because they are similarly not limited to particular polynucleotides that are considered to be adequately described by the specification.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 6, 8-9, 17, 21-23, 26-27, 30, 32 are rejected under 35 U.S.C. 103 as being unpatentable over Buchholz et al. (“Buchholz”, WO 2018/229226 A1) in view of Havranek et al. (“Havranek”, US 2017 /0058297 A1, cited as reference 5 on IDS filed 02/15/2023) and Raymond et al. (“Raymond”, WO 2014/093330 A1, cited as reference 2 on IDS filed 07/08/2022).
Regarding claims 1, 8-9, 30, 32, Buchholz teaches methods and means for specifically altering the DNA sequence in a genome. Provided are vectors and methods to generate designer DNA recombining enzymes. The invention is useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material from cells or tissue and to cure diseases (e.g., paragraph 1st, page 1; Fig. 1 [see below]).
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Buchholz teaches naturally occurring DNA recombining enzymes, in particular site-specific recombinase (SSR) systems (such tyrosine-type SSRs), generally consist of four identical monomers. They recognize two identical and symmetric, palindromic target sites, which consist each of two 13 nucleotide long half sites separated by an asymmetric frequently 8 nucleotide long spacer. Depending on the number and relative orientation of the target sites the DNA recombining enzyme either causes a deletion, an insertion, an inversion or a replacement of genetic content (e.g., paragraph 1st, page 2). Buchholz teaches designer DNA-recombining enzymes according to the invention comprises one or two or up to four different monomers. Buchholz teaches a method know in the state of the art to obtain a tailored recombinase that works one target sites by substrate-linked protein evolution (SLiPE) (e.g., paragraph 4th, page 54; Fig. 8). Buchholz teaches a method to obtain a DNA Recombining enzyme for DRiGD by applying a novel directed evolution strategy (QuSLiDE DRiGD)) that delivers two pairs of monomers of DNA recombining enzymes that work in conjunction to recombine two different target sites. Each monomer is directed to one half site of one target site (white triangle lox 1 and black triangle lox 2) present in the genome starting with a source vector with four libraries of DNA recombining enzymes (e.g., paragraph 3rd, page 54; Fig. 7 [see below]).
Fig. 7
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Regarding claim 17, Buchholz teaches pharmaceutical composition comprising a DNA recombining enzyme according to the invention, a nucleic acid or vector encoding for a designer DNA-recombining enzyme according to the invention or the set according to the invention as well as suitable carriers (e.g., paragraph 1st, page 27). Buchholz teaches that delivery of the DNA recombining enzyme according to the invention, a nucleic acid or vector encoding it and a donor sequence carrying a desired sequence, a wild type or other functional variant of the gene (or a part thereof - like an exon) the mutant part of the gene is replaced by the desired (functional) sequence. To enable site-specific recombination the desired sequences in the donor sequence is flanked by the target sites for the DNA recombining enzyme (e.g., paragraph 6th, page 26).
Regarding claim 22, Buchholz teaches naturally occurring DNA recombining enzymes, in particular site-specific recombinase (SSR)
systems (such tyrosine-type SSRs), generally consist of four identical monomers. Depending on the number and relative orientation of the target sites the DNA recombining enzyme either causes a deletion, an insertion, an inversion or a replacement of genetic content (e.g., paragraph 1st, page 2).
Regarding claim 30, Buchholz teaches DNA recombining enzymes, in particular site-specific recombinase (SSR) systems, allow precise manipulation of DNA without triggering endogenous DNA repair pathways and possess the unique ability to fulfill both cleavage and immediate resealing of the processed DNA in vivo. Furthermore, SSR systems, such as Cre/loxP (e.g., paragraph 3rd, page 1). Buchholz teaches other SSR systems know in the art include the Flp/FRT system and the Dre/rox system, the VCreNloxP system and the sCre/SloxP system, as well as the Vika/vox, the Nigri/nox and the Panto/pox systems (e.g., paragraph 4th, page 1).
Buchholz does not teach the linker comprising an oligopeptide as required by instant claims 6, 8, 23, 26, 27, 32. However, this is cured by Havranek.
Buchholz does not teach SEQ ID NOs 17-18 as required by instant claims 6, 21, 26, 32. However, this is cured by Raymond.
Havranek teaches enzymes, compositions and methods for catalyzing site specific recombination at asymmetric sites (e.g., paragraph 0003). Havranek teaches a method for mediating asymmetric site-specific recombination in a nucleic acid. The method comprises contacting an isolated enzyme of the disclosure with a nucleic acid. The
isolated enzyme recognizes asymmetric sites on the nucleic acid and cleaves the asymmetric sites. The asymmetric site-specific recombination is selected from the group consisting of inversion, excision, insertion and translocation (e.g., paragraph 0009). Havranek teaches an isolated polypeptide may comprise subunit A attached to subunit B via a linker stretching between the C-terminus of subunit A to the N-terminus of subunit B. Alternatively, an isolated polypeptide may comprise subunit A attached to subunit B via a linker stretching between the C-terminus of subunit B to the N-terminus of subunit A (e.g., paragraph 0034). Havranek teaches genomic applications of Cre recombinase. Depending on the number and relative orientation of the
loxP sites, Cre recombinase can perform deletion, inversion, insertion or exchange of genetic content (e.g., paragraph 0012; Fig. 1 [see below]).
Fig. 1:
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Raymond teaches a method for generating a tagged DNA library comprising treating fragmented DNA with end-repair enzymes to generate
fragmented end-repaired DNA; and ligating a random nucleic acid tag sequence, and optionally a sample code sequence and/or a PCR primer sequence to the fragmented end-repaired DNA to generate the tagged DNA library (e.g., paragraph 3rd, column 2). Raymond teaches SEQ ID NO 146 that has 100% homology with SEQ ID NO 17 and SEQ ID NO 18 of the instant claims (it reads on LoxF8 target site) (e.g., Table 27, column 85).
Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the DNA Recombining enzyme that delivers two pairs of monomers of DNA recombining enzymes that work in conjunction to recombine two different target sites, where each monomer is directed to one half site of one target site as taught by Buchholz, with the teachings of Havranek – a CRE recombinase polypeptide comprising subunit A attached to subunit B via a linker stretching between the C-terminus of subunit A to the N-terminus of subunit B and target the SEQ ID NO 146 taught by Raymond; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of DNA Recombining enzyme comprising two pairs of monomers of DNA recombining enzymes that work in conjunction to target sequence corresponding to LoxF8 target sites, where each monomer is directed to one half site of one target site.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to do so in order to develop a method for specifically altering the DNA sequence in a genome using site-specific naturally occurring DNA recombining enzymes, in particular tyrosine-type. Delivering of the DNA recombining enzyme, a nucleic acid or vector encoding it and a donor sequence carrying a desired sequence, a wild type or other functional variant of the gene the mutant part of the gene is replaced by the desired (functional) sequence in the subject to suffer from a disease.
Claims 7 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Buchholz et al. (“Buchholz”, WO 2018/229226 A1), Havranek et al. (“Havranek”, US 2017/0058297 A1, cited as reference 5 on IDS filed 02/15/2023) and Raymond et al. (“Raymond”, WO 2014/093330 A1, cited as reference 2 on IDS filed 07/08/2022) as applied to claims 6, 8-9, 17, 21-23, 26-27, 30, 32 above, and further in view of Seehra et al. (“Seehra”, WO 2019/217715 A1).
Buchholz, Havranek and Raymond do not teach the linker comprising formula 1-3, as required by instant claims 7 and 24. However, this is cured by Seehra.
Seehra teaches pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions involving low red blood cell levels, e.g., anemia or blood loss; fibrosis; or pulmonary hypertension (e.g., abstract). Seehra teaches polypeptide described herein may further include an Fc domain monomer fused to the C-terminus of the polypeptide by way of a linker (e.g., lane 38, page 3). Seehra teaches SEQ ID NO 144 (GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG) (e.g., lane 35, page 4).
Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the DNA Recombining enzyme that delivers two pairs of monomers of DNA recombining enzymes that work in conjunction to recombine two different target sites, where each monomer is directed to one half site of one target site as taught by Buchholz, with the teachings of Havranek – a CRE recombinase polypeptide comprising subunit A attached to subunit B via a linker stretching between the C-terminus of subunit A to the N-terminus of subunit B and target the SEQ ID NO 146 taught by Raymond and using the linker of SEQ ID NO 144 taught by Seehra; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of DNA Recombining enzyme comprising two DNA recombining enzymes linked by SEQ ID NO 144, that work in conjunction to target sequence corresponding to LoxF8 target sites, where each monomer is directed to one half site of one target site.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to use peptide linker to ensure the proper folding and function of both DNA recombinases and avoid steric hindrance in order to develop a method for specifically altering the DNA sequence in a genome using site-specific naturally occurring DNA recombining enzymes, in particular tyrosine-type.
Claims 11-12, 31 are rejected under 35 U.S.C. 103 as being unpatentable over Buchholz et al. (“Buchholz”, WO 2018/229226 A1), Havranek et al. (“Havranek”, US 2017 /0058297 A1, cited as reference 5 on IDS filed 02/15/2023) and Raymond et al. (“Raymond”, WO 2014/093330 A1, cited as reference 2 on IDS filed 07/08/2022) as applied to claims 6, 8-9, 17, 21-23, 26-27, 30, 32 above, and further in view of Hauber et al. (“Hauber”, US 2013/0164271 A1, cited as reference 2 on IDS filed 10/17/2025) and Sauer et al. (“Sauer”, US 2006/0014264 A1).
Buchholz, Havranek and Raymond do not teach SEQ ID NO 93, 100 as required by instant claims 11-12, 31. However, this is cured by Hauber and Sauer.
Hauber teaches method for generating an expression vector encoding a tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains of one species inserted into the genome of a host cell. Recombinases have been tailored to recognize asymmetric target sites different from their native symmetric target sites by splitting up the substrate into a number of new subsets with smaller differences from the original target and stepwise tailoring recombinases to recognize these subsets (e.g., paragraph 0012). Hauber teaches a Cre recombinase SEQ ID NO 57 that has 90% homology with SEQ ID NO 93 of the instant claims.
Sauer teaches Cre/lox system having a lox site with additional nucleotide base pairs within the spacer region. The invention provides novel Cre mutant polypeptides that can catalyze site specific recombination or excision at a mutant lox site having additional nucleotide base pairs in the spacer region (e.g., paragraph 0007). Sauer teaches SEQ ID NO 150 with 90.5% homology with SEQ ID NO 100 of the instant claims.
Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the DNA Recombining enzyme that delivers two pairs of monomers of DNA recombining enzymes that work in conjunction to recombine two different target sites, where each monomer is directed to one half site of one target site as taught by Buchholz, with the teachings of Havranek – a CRE recombinase polypeptide comprising subunit A attached to subunit B via a linker stretching between the C-terminus of subunit A to the N-terminus of subunit B and target the SEQ ID NO 146 taught by Raymond and replace the first recombinase with Cre recombinase SEQ ID NO 57 taught by Hauber and the second recombinase with SEQ ID NO 150 taught by Sauer; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of DNA Recombining enzyme comprising two Cre recombinase SEQ ID NO 57 and linked to SEQ ID NO 150, both DNA recombining enzymes work in conjunction to target sequence corresponding to LoxF8 target sites, where each monomer is directed to one half site of one target site.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to do so in order to develop a method for specifically altering the DNA sequence in a genome using two site-specific naturally occurring Cre DNA recombining enzymes, in particular tyrosine-type, directed to one half site of one target site.
Conclusion
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/J. E. ANGELL, Ph.D./